Bio Hydrometallurgy

Lecture 11: Biohydrometallurgy Of Copper Dump And Heap Leaching NPTEL Web Course
Lecture 11
Biohydrometallurgy Of Copper Dump And Heap Leaching
Keywords: Heap Leaching, Dump Leaching
Dump (heap) bioleaching
Thin layer leaching was developed and modified to process copper oxides and sulfides and
include acid curing and agglomeration of fines. The prepared ore materials are stacked in
properly designed heaps, ensuring good permeability for oxygen and leach solutions. Irrigation
through the top and sides can be done through sprinklers. Engineering, control and optimisation
of heap operations have been adequately established. Optimisation of heaps as bioreactors to
enhance copper extraction has also become possible. Optimal microbial activity in the heap need
be ensured through adequate aeration, monitoring of microbial activity and control of growth
parameters. Recycle solutions containing indigenous microbes can be utilized. The BIOPRO TM
process involves heap inoculation during acid curing and agglomeration.
Efforts have been made to develop predictive models to facilitate heap design and control.
Several models have been proposed. Dixon has used an extensive matrix of heap subprocesses.
Some industrial heap bioleaching operations for copper are illustrated in table 11.1.
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Course Title: Metals Biotechnology
Course Co-ordinator: Prof. K. A. Natarajan, IISc Bangalore
Table 11.1: Some industrial heap bioleaching operations for copper
Heap bioleach plant Copper, t / year
Western Metals Ltd. Australia/Mount Gordon, (insitu) 1991 - Chalcocite, bornite 33 000
Lince II, Chile, 1991 - Oxides / Sulfides 27 000
Cerro Colorado, Chile, 1993 - Chalcocite, covellite 1.0 lakh
Quebrada Blanca, Chile, 1994 - Chalcocite 75 000
Ivan-Zar, Chile, 1994 - Oxides / Sulfides 10 000 12 000
Punta del Cobre, SA/Chile, 1994 Oxides / Sulfides 7 000-8 000
Escondida, Chile, 1995 - Oxides / Sulfides 200 000
Hellenic Copper Mines/Cyprus, 1996 - Oxides / Sulfides 8 000
Dos Amigos, Chile, 1996 - Chalcocite 21 000
Cerro Verde, Peru, 1977 - Oxides / Sulfides 54 200
Lomas Bayas, Chile, 1998 - Oxides / Sulfides 60 000
Zaldivar, Chile, 1998 - Chalcocite 150 000
Nifty Copper, Australia, 1998 - Chalcocite, Oxides 16 000
S & K Copper, Monywa, Myannar, 1999 - Chalcocite 40 000
Morenci, Arizona, 2001 - Chalcocite, Pyrite 380 000
Jinchuan Copper, China, 2006 - Chalcocite, Enargite, Covellite 10 000
Constellation Copper Corporation/Lisbon Valley, Utah, 2006 - Chalcocite, Oxides 27 000
Whim Creek and Mons Cupri, Australia, 2006 - Oxides, Sulfides 17 000
BHP Billiton/Chile, Spence, 2007 Sulfides, Oxides 200 000
1. M.Gericke, J.W.Neale and P. J. Van Staden, A Mintek perspective of the past 25 years in
minerals bioleaching, J.S.Aftican Inst. Min. Met, 109, (2009) 567 585.
2. H.R. Watling, The bioleaching of sulfide minerals with emphasis on copper sulfides A
review, Hydrometallurgy, 84, (2006) 81-108.
3. C. L. Brierley, How will biomining be applied in future? Trans. Nonferrous met. Soc. China,
18, (2008), 1302 1310.
2
Factors affecting leaching rates

Dump / Heap very slow 20 40% Cu recovery in years.
Leaching rates depend on biological, physico-chemical, technological factors.
Number and activity of bacteria-critical factor-bacteria attached and in solution.
Ferric hydroxide / Jarosite / Elemental sulfur limit leaching rates
Easily leachable ores have the following characteristics
Oxide copper or easily oxidizable secondary sulphides.
Low acid consumption of host gangue (not exceeding 20 Kg/ton).
Fine mineral dissemination.
Good permeability and porosity
Increased biomass availability.
Rates of copper leaching from run-of-mine ores classified as:
Refractory chalcopyrite / Bornite-about 5 6% (10% maximum)
Mixed ores with primary sulphides 10 20%
Mixed ores with secondary sulphides 20- 25%
Mixed ores with 10 20% sulphides 40 50%
Microflora in dumps
Acidithiobacillus ferrooxidans
Acidithiobacillus thiooxidans
Fungi
Thermophilic organisms (Sulfolobus)
Cell population 105 - 108
Temperature inside 400 800C
Solution analysis g / I
System pH
Cu++ Fe++ Fe+++ AI Mg SO4- -
Dump 0.6-1.5 0.5- 1.0 1.5-2.0 2-2.5 1-1.8 15-20 2.3-2.4
In situ 1.4 1.4 3.2 1.5 1.6 26 2.1
Large column 1.8 2.0 20.0 1.2 0.7 66 2-2.2
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Lecture 12: Biohydrometallurgy Of Copper Modern Developments NPTEL Web Course
Lecture 12
Biohydrometallurgy Of Copper Modern Developments
Keywords: Developments In Heap, Thermophile Leaching, Chalcopyrite Leaching.
Bioleaching of chalcopyrite
Most of the research has been done using chalcopyrite concentrates and little information is
available on its heap bioleaching aspects. As mentioned before, chalcopyrite is a refractory
mineral not readily amenable to bioleaching using acidophilic mesophiles. The leaching rate can
be significantly enhanced at higher temperatures at controlled redox potentials. Appropriate
control of iron chemistry and ore preparation are essential to achieve desirable copper extraction
from chalcopyrite ores. Beneficial role of fine grinding of chalcopyrite concentrates before
bioleaching has been assessed.
Heap bioleaching of chalcopyrite ores need several controls such as
Knowledge of indigenous microflora and factors that promote their growth and activity.
If the heap need be operated at high temperatures, inoculation with a consortia of
thermophiles.
Proper irrigation methods and nutrient addition
Agglomeration of fines using inoculum.
A number of methods have been suggested for generation and maintenance of heat in heaps.
Use of hot water while acid curing and also heap heating.
Promoting exothermic reactions through external addition of sulfides.
Proper heap insulation and prevention of evaporation.
Control heat transfer Humidity and aeration control.
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Heap (bio) leaching of copper ores is a practical technology.

Robust and proven under different climatic conditions for oxides and secondary
sulphides.
Flexible-heap engineering and management can accommodate site peculiarities in remote
localities; suited to small deposits
Simple-a technology that can be communicated to non-scientific personnel.
Low cost-stacking, irrigation, aeration, solution collection are all basic.
Efficient heap bioleaching requires the indigenous presence of effective and appropriate bacterial
population. Bacterial inoculation with the leach liquor cannot be effective, since many organisms
are naturally adherent to the ore particle surfaces and do not penetrate to the interior depths.
The StickBugsTM process, currently being developed, is capable of generation of sufficient

population of desirable bacteria and to facilitate attachment to inner depths of heaps. Bacterial
activity can thus be enhanced following effective inoculation.
The above StickBugsTM process prevents the desirable bacterial population from remaining at the
top of the heap and facilitate their penetration into the heap. Bacteria lose their adhesion
tendency if deprived of an essential nutrient. Generation of micro non-adherent bacteria find
application in plugging of permeable subterranean strata, in oil recovery. Ultra-micro-bacteria
(UMB) are generated through nutrient starvation. UMBs do not attach to mineral surfaces in the
absence of nutrients, but can be revived by re-introduction of the missing nutrient.
Assaying methods for microbial population (both planktonic and sessile) in bioheaps have been
developed.
Qualitative and quantitative estimation of the microorganisms that colonise the ore surfaces and
as available in the liquid phase within a heap can be performed using newly developed molecular
biology techniques.
Attached organisms can be dislocated from the solid phase through successive washes with
detergents and acidified water and the cells then enumerated. Genetic DNA can be isolated.
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Microbial identification can be achieved through restriction endonuclease analysis of the 16S
rRNA genes, and also as 16S rRNA gene sequencing.
Thermophile bioleach status
Successful batch bioleaching of chalcopyrite (98%), enargite (98%), and sphalerite

(95%) achieved in laboratory.
Established A 13 bioleach pilot plant in January 1998 for continuous thermophile bioleaching
of a Chilean chalcopyrite concentrate,
Successful bioleaching of copper concentrate achieved on continuous basis yielding > 99%
copper recovery at economic residence times.
Continuous operation of bioleaching mini plant treating copper/cobalt concentrate yielded
98% copper and cobalt recovery.
Limited dissolution of chalcopyrite owing to mineral surface passivation.
Slow kinetics for enargite.
Require high redox potentials and high temperatures.
Require long residence times for metal dissolution.
Difficult to attain complete sulphide oxidation.
Precious metals recovery may be problematic.
Bac Tech/Mintek process (using moderate thermophiles)
Successful bioleaching of Mount Lyell copper concentrate in 1997.

Fully integrated circuit consisting of 200 litre mini plant with return raffinate from SX.
Achieved 96% copper dissolution on Mount Lyell concentrate.
Geocoat Process
The GEOCOAT process incorporates two successful and commercially amenable technological
concepts heap leaching and biooxidation. Sulfide ores can be concentrated by flotation and
thickened. The resulting slurry coated onto crushed, screened support rock, stacked on a lined
pad, and bioleached. Coating achieved by spraying concentrated slurry onto the support rock.
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The support rock is uniform sized, in the range of 6 to 30 millimeters and the concentrate coating
is less than one millimeter in thickness. The weight ratio of support rock to concentrate is in the
range of 5 10 : 1.
The hydrophobic nature of the concentrate assists in the coating and no binding agents required.
Depending on the desired temperature of operation, the heap is inoculated with naturally
occurring sulfide oxidizing bacteria, such as the mesophiles; Acidithiobacillus ferrooxidans,
Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans or moderate thermophiles;
Acidithiobacillus caldus, Sulfobacillus thermosulfidooxidans or extreme thermophiles; Acidianus
brierleyi, Acidianus infernus, Metallosphaera sedula, Sulfolobus acidocaldarius, Sulfolobus
shibatae and sulfolobus metallicus.
Nutrients added to the heap through recirculating solution. As biooxidation progresses, the
sulfide minerals in the concentrate are oxidized and the solubilized metal ions, iron and sulphate
are recovered from the heap and stripped solution recirculated.
Chalcopyrite ores or flotation concentrates can be bioleached using thermophiles using the
Geocoat process. (fig. 12.1)
Fig.12.1: Schematic representation of chalcopyrite coating on a support rock in a heap configuration.
Typical pilot plant operations for chalcopyrite concentrate reactor bioleaching and for high
temperature heap bioleaching are illustrated in figs 12.2 and 12.3.
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Fig 12.2: Pilot Plant consisting of Regrinding, Agitated Bioleaching, Iron Removal, Copper-SXEW and Zinc
Precipitation, to treat Polymetallic Chalcopyrite Concentrate.
(Kind courtesy from MINTEK, P.J.van Staden, manager, Biotechnology Divn.MINTEK)
(Permission from MINTEK thankfully acknowledged)
Fig. 12.3: Pilot Plant for High Temperature Heap Bioleaching

Showing the first of three 20,000 t cells stacked.
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Heap bioleaching tests on Iranian low grade chalcopyrite ores

The Darehzar Copper Mine located in the Kerman province of Iran, belongs to the National
Iranian Copper Industries Company (NICICO).
Temperatures, higher than those normally attained in the heap bioleaching of secondary sulphide
copper ores, are required to obtain faster copper leach kinetics from the hypogene ore of the
Darehzar Mine in Iran. Through mathematical modeling, it could be established that certain
blends of the hypogene and supergene ores could become amenable to heap bioleaching.
Three independent pilot heaps, each of about 25,000 tones, were constructed on site at the
Sarcheshmeh copper mine. Copper extraction, bacterial activity and acid consumption were
monitored as a function of time. Mintek developed the control strategy.
The Darehzar ore deposit has been estimated at around 50 to 80 million tonnes assaying 0.6%
copper, with up to 143 million tonnes assaying 0.44% copper. Chalcopyrite amounts to 79% of
the copper content in the hypogene, for 24% of the copper in the supergene, and for 54% of the
copper in a 70:30 blend. The pyrite content is about 4%.
About 0.6% copper as average could be achieved through a 70:30 blend.
Laboratory test work and analysis were carried out to establish the mineralogy and chemistry of
ore for a period of an year.
Effect of particle size, temperature, pH and redox potential on copper and iron recovery as
well as acid consumption.
Agglomeration characteristics with various binders and admixtures.
Establishment of conditions amenable to bacterial activity and sulfide oxidation.
Bacterial action and sustainability in the heap. Effect of temperature, oxygen, carbon source,
nutrients and solution chemistry on bacterial growth and activity.
Control of iron precipitation in the form of jarosites.
Control of segregation and migration of fines during stacking and leaching.
Initial test runs were carried out in columns
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References (Lectures 10-12):
42. H. R. Watling, The bioleaching of sulphide minerals with emphasis on copper sulphides A
review, Hydrometallurgy 84 (2006), 81 108.
43. N.Pradhan, K.C. Nathsarma, K.Srinivasa Rao, L.B. Sukla, B.K. Mishra, Heap bioleaching of
chalcopyrite: A review; Minerals Engineering, 21 (2008), 355 365.
44. M.Gericke, J.W.Neale and P. J. Van Staden, A Mintek perspective of the past 25 years in
45. C. L. Brierley, How will biomining be applied in future? Trans. Nonferrous met. Soc. China,
18, (2008), 1302 1310.
46. Anon, Penoles bio-leach demonstration plant. Bateman press release (2001).
47. J.A. Brierley, Heap leaching of gold-bearing deposits: theory and operational description. In:
Rawlings, D.E. (Ed.), Biomining: Theory, Microbes and Industrial Processes. Springer,
Berlin, (1997) 103 115.
48. M.E. Clark, J. Batty, C. Van Buuren, D. Dew, M. Eamonn, Biotechnology in Minerals
processing: Technological Break throughs Creating Value. In: Harrison, S. T.L, Rawlings,
D.E., Petersen. J (Eds.) Proc. 16th Int. Biohydrometallurgy Symposium (Cape Town). IBS,
Cape Town, (2005) 17 24.
49. D.G. Dixon, Heap leach modelling the current state of the art. In:Young, C.A, Alfantazi,
A.M, Anderson, C.G, Dreisinger, D.B., Harris, B., James,A. (Eds.), Hydrometallurgy
Volume 1: Leaching and Solution Purification. TMS. Warrendale. (2003), 289-314.
50. D. G. Dixon, J.Petersen, Comprehensive modelling study of chalcocite column and heap
bioleaching. In: Riveros, P.A., Dixon, D., Dreisinger, D.B., Menacho,T. (Eds.), Copper-
Cobro (Santiago) Volum VI-Hydrometallurgy of Copper (Book 2) Modeling, Impurity
Control and Solvent Extraction. Canadian Instiute of Mining, Metallurgy and Petroleum,
Montreal, (2003), 493-516.
51. D.G. Dixion, J.Petersen, Modelling the dynamics of heap bioleaching for process
improvement and innovation. Hydro-Sulfides, Intl. Colloquium on Hydrometallurgical
Processing of Copper Sulfides (Santiago). University of Chile, Santiago, (2004), 13-45.
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52. C. du Plessis, Delivery system for heap bioleaching. World Patent WO 2,003, 068, 999
(2003), 21 August.
53. T. Gehrke, R.Hallman, K.Kinzler, W.Sand, The EPS of Acidithiobacillus ferrooxidans a
model for structure function relationships of attached bacteria and their physiology. Water
Science and Technology 43, (2001), 159-167.
54. M. Gericke, A.Pinches, Bioleaching of copper sulphide concentrate using extreme
thermophilic bacteria. Minerals Engineering 12, (1999), 893-904.
55. M. Gericke, A.Pinches, J.V. Van Rooyen, Bioleaching of chalcopyrite concentrate using an
extremely thermophilic culture. International Journal of Mineral Processing 62, (2001), 243-
255.
56. M.Gericke, H.H. Muller, J.W. Neale, A.E. Norton, F.K.Crundwell, Inoculation of heap
leaching operations. In: Harrison, S.T.L., Rawlings, D.E., Petersen, J. (Eds.), Proc. Intl.
Biohydrometallurgy Symposium (Cap Town). IBS, Cape Town, (2005), 255-264.
57. C. Hunter, Bioheap leaching of a primary nickel-copper sulphide ore. ALTA Nickel/Cobalt
2002 (Perth, WA). ALTA. Melbourne. (2002).
58. C.J. Hunter, T.L Williams, Adaptation of bacteria for leaching, world patent WO 02,066,689,
(2002), 29 August.
59. D.E. Rawlings, The molecular genetics of mesophilic, acidophilic, chemolithotrophic, iron
or sulfur-oxidizing micro-organisms. In: Amils, R., Ballester, A. (Eds.), Proc. Intl. Bio
hydrometallurgy Symposium (Madrid, Spain), Part B. Elsevier, Amsterdam, (1999), 3-20.
60. D.E. Rawlings, The molecular genetics of Thiobacillus ferrooxidans and other mesophilic,
acidophilic, chemolithotrophic, iron- or sulfur-oxidizing bacteria. Hydrometallurgy 59,
(2001), 187-201.
61. A.I.M. Ritchie, Optimization of biooxidation heaps, In: Rawlings, D.E (Ed.), Biomining:
Theory, microbes and Industrial Processes. Springer Verlag, Berlin, (1997), 201-226.
62. P.J.van Staden, B.Shaisaee, M.Yazdani, A collaborative plan towards the heap bioleaching of
low grade chalcopyritic ore from a new Iranian mine. In: Harrison, S.T.L., Rawlings, D.E.,
Petersen J. (Eds.), Proc, 16th Intl. Biohydrometallurgical Symposium (Cape Town). IBS,
Cape Town, (2005), 115-123.
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63. M.Vasquez, R.T. Espejo, Chemolithotrophic bacteria in copper ores leached at high sulfuric
acid concentration. Applied and Environmental Microbiology 63, (1997), 332-334.
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Lecture 13: Bioleaching Of Nickel From Sulfides And Laterites NPTEL Web Course
Lecture 13
Bioleaching Of Nickel From Sulfides And Laterites
Keywords: Bioleaching Of Nickel, Nickel Sulfides, Laterites
In lectures 13, bioleaching of nickel from sulfide ores and concentrates as well as from
lateritic ores is discussed. Heap bioleaching of nickel along with other metals such as zinc
and copper from a schist belt as practised in Talvivaara, Finland is illustrated in lecture 14
[64 86].
Bioleaching of nickel ores and concentrates

Nickel occurs in nature mainly as its sulfides such as pentlandite, (Ni Fe) 9 S8 along with pyrite
and pyrrhotite or interlocked with chalcopyrite-cubanite and pyrrhotite. Another form of nickel is
as its oxide in laterites. Both the above forms are amenable for nickel biodissolution; either
through heap bioleaching or reactor leaching of bulk concentrates. [64 80].
Several microorganisms inhabiting nickel ore deposits develop natural tolerance to nickel.
Nickel tolerance is important in judging bacterial nickel dissolution. Nickel tolerance can also
be imparted to mine-isolated organisms through serial subculturing in the presence of increasing
nickel ion concentrations. Bioleaching of nickel sulfides is temperature sensitive and use of
thermophiles in place of mesophiles significantly enhances leaching kinetics.
Typical bioleaching reactions involving nickel sulfides are illustrated below:
(Ni Fe)9 S8 = 9Ni++ + 9Fe++ + 8S + 18e

Pentlandite
Fe++ = Fe+++ + e
2S + 2H2O + 3O2 = 2H2SO4
NiS + H2SO4 = NiSO4 + H2S

Millerite
NiS + 2Fe+++ = Ni++ + 2Fe++ + S

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Pyrite or pyrrhotite present in the nickel sulfide ores also get biodissolved similarly.
Pyrrhotite (Fe(1-x) S) is commonly associated with nickel sulfide ores. It is easily dissociated and
oxidized under acidic conditions.
FeS + H2SO4 = FeSO4 + H2S
On the otherhand, pyrite is a nobler mineral compared to electrochemically active pentlandite or
millerite. Presence of pyrite can galvanically enhance anodic dissolution of pentlandite, so also
the presence of chalcopyrite.
The role of galvanic interactions in the bioleaching of pentlandite containing complex sulfides
has been studied.
Rest potential measurements in a bioleaching medium at pH-2.5 in the absence of iron, indicated
the following values.
Mineral Eh, mV
Chalcopyrite +500mV
Pentlandite +350 420 mV
Pyrrhotite + 350 370 mV
In a pyrite-chalcopyrite-pentlandite-pyrrhotite combination, galvanic dissolution of pentlandite

and pyrrhotite will be promoted. In a pentlandite-pyrrhotite combination, higher dissolution of
pyrrhotite can be expected. Similarly, in a chalcopyrite-pentlandite galvanic contact, enhanced
selective dissolution of pentlandite can be expected. (fig. 13.1).
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A B
Fig 13.1: Scanning electron micrographs depicting (A) exfoliation and corrosion of pentalandite in contact with
chalcopyrite (B) Attached At.ferrooxidans on corroded pentalandite.
In laboratory bioleaching tests using a copper-nickel-iron sulfide ore in the presence of

At.ferrooxidans, nickel dissolution was found to be faster and in all cases, occurred before
copper extraction was initiated. Nickel dissolution (from pentlandite) was almost 2-3 times
higher than that of copper (from chalcopyrite)
Thermophiles such as Sulfobacillus isolated from nickel ore deposits and tailings can develop
nickel tolerance. General observations with regard to nickel tolerance, especially in
At.ferrooxidans are given below:
Strains isolated from nickel-rich zones exhibit higher tolerance compared to other normal
strains.
Mesophiles exhibit relatively higher nickel tolerance compared to thermophilic organisms.
Higher nickel tolerance to normal strains can be imparted through serial subculturing in the
medium containing successively increasing metal concentrations.
It is also essential to maintain nickel-tolerant strains under the developed nickel stress to
retain the acquired metal tolerance.
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Several laboratory research studies have been carried out on the bioleaching of pentlandite ores
and concentrates.
Nickel-copper-iron sulfide concentrate grown adapted At.ferrooxidans, could extract about

55% nickel after 18 weeks of adaptation.
Selective bioleaching of a pentlandite-chalcopyrite concentrate in the presence of
At.ferrooxidans in a bioreactor established preferential nickel extraction (55%).
Bioleaching of a nickel-rich pyrrhotite concentrate in the presence of unadapted and adapted
strains of At.ferrooxidans yielded 87% and 91% recovery in 16 days, respectively.
Bioleaching of a pentlandite-pyrrhotite-chalcopyrite concentrate resulted in about 60% nickel
recovery after about a month.
Thermophiles (Acidimicrobium, Sulfobacillus, At.caldus) at higher temperatures of about 50-
800C were found to be more efficient in nickel extraction. For example, at about 70 0C, 98%
nickel could be extracted with a retention period of four days using adapted cultures from
nickel-copper concentrates.
In the presence of Acidianus brierleyi, a nickel-copper concentrate was bioleached at 680C and
pH 1.6 to recover 99.8% if nickel and about 86% of copper in about 4 days.
Several studies have been made using pentlandite ores in different countries such as Finland
and China. The black schist ore of Talvivaara in Finland has been studied in detail for the
bioextraction of nickel, zinc, copper and cobalt. Based on laboratory results, pilot trials on
heap bioleaching were carried out and ultimately commercial scale heap bioleaching is being
attempted.
Nickel-copper ores in the Jinchuan mines of China were also studied using Acidithiobacillus
bacteria. Role of adapted bacteria was assessed. In an airlift reactor at 15% pulp density,
more than 95% of nickel and 48% of copper and 82% of cobalt could be bioleached after 20
days.
BioheapTM technology for nickel-copper sulfides uses moderate thermophiles and tested on an
W.Australian ore. Two 5000 tonnes heaps were erected at Radio Hill and inoculated at 50-550C
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at pH 1.8 with mixed cultures of Sulfobacillus and Thermoplasma. 90% nickel recovery was
achieved in less than an year with 50% copper.
Heap leaching trials for nickel-copper sulfide ores of Jinchuan in China have been reported.
Pilot tests were also undertaken at a nickel mine in Mojiang county of S.China. Six test heaps
were arranged (6-8 meters high) having 10,000 tonnes of crushed ore in each heap and
inoculated with a mixed mesophilic culture and aerated. In an year, nickel and cobalt recoveries
higher than 60% were achieved.
A salt-tolerant mixed culture has also been developed for application in Bioheap TM technology in
W.Australia.
The BioNic is a bioleaching process conceived in early 1990s in stages form batch tests to
demonstration level for 300 Kg/day of pentlandite concentrate. Mixed cultures including
At.ferrooxidans, At.thiooxidans and L.ferrooxidans were adapted to the concentrate. In the
temperature range of 30-400C, higher than 90% nickel recoveries in 8 days were achieved.
Validity of the BioNic process for commercial applications has been confirmed. Modification
of the above process using thermophiles at 65-800C has also been reported.
Bioleaching of nickel from laterites
Recently, renewed interest has been generated to establish nickel bioleaching processes not only
from nickel sulfide ores and concentrates, but also for nickel laterites.[81 84].
Applicability of bioleaching has also been tested to extraction of nickel from the more abundant
lateritic ore reserves. Almost 72% of worlds cobalt and nickel is contained in lateritic ores.
Unlike in sulfide mineralization, the nickel and cobalt in the laterites are not discrete and
separate, but are invariably interlocked with other oxides such as those of iron. Such
interlocking of nickel and cobalt in laterites makes their recovery more difficult due to poor
liberation even in smaller sizes. Prior reduction of iron oxides (hematite, magnetite, goethite,
limonite) becomes essential to liberate the encapsulated nickel and cobalt to facilitate their easy
dissolution in a lixiviant. Reductive dissolution can be brought about either by bacterial
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reduction or through use of reducing agents secreted by microorganisms. The following

biological approaches are possible:
Use of biologically-derived reducing and metal-complexing organic acids.

Use of fungi and their metabolites (organic acids)
Use of Acidithiobacillus type bacteria which produces sulfite and thiosulfate reducers,
and also mineral acids.
Use of acidophilic organisms capable of dissimilatory ferric iron reduction-iron reducing
bacteria and bacterially-catalyzed reductive dissolution.
Bioprocessing of laterite ores has been studied using fungi such as Aspergillus and Penicillium.
Bacteria of the genus Acidithiobacillus have been shown to solubilise nickel through production
of sulfuric acid and reducing thiosulphate and thionate species.
A mixed culture containing At.ferrooxidans, At.caldus and L.ferrooxidans was used to extract
nickel from laterites.
In oxidative dissolution, the mineral substrate provides the energy (as reduced sulfur compounds
or ferrous iron) to the microbes, while in reductive dissolution, an external electron donor
becomes necessary.
Reductive dissolution of goethite coupled to sulfur oxidation is given below
6 - Fe O.OH + S + 10H+ = 6 Fe++ + SO4 - - + 8H2O
Acidophiles such as At.ferrooxidans and Acd.organivorans were found to catalyse reductive

dissolution of nickel laterite, demonstrating nickel release under atmospheric conditions in acid
pH (1.5 2).
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A new process, titled, Ferredox process is reported recently to extract nickel and cobalt from
limonitic (goethite) laterites through reductive dissolution. Iron dissolution and precipitation
occur separately.
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Lecture 14: Heap Bioleaching Technology For Nickel NPTEL Web Course
Lecture 14
Heap Bioleaching Technology For Nickel
Keywords: Polymetallic Schist, Heap Bioleaching, Talvivaara Experience
Heap bioleaching of a polymetallic nickel ore at Talvivaara, Finland.
Talvivaara deposits located 350 Km south of the Arctic circle in Finland comprise of one of the
largest nickel sulfide resources. The deposits are situated in the southern part of the early
Proterozoic schist belt and the Ni-Cu-Co-Zn mineralizations are part of a high-grade, meta
morphosed black schist, consisting of micas, quartz, graphite and sulfides. Sulfide minerals such
as pyrite, pyrrhotite, sphalerite, pentlandite, chalcopyrite and violarite are present with an
average composition of 0.23% Ni, 0.50% Zn, 0.13% Cu, 0.02% Co, 10.3% Fe and 8.4% S.
Bioheap leaching was considered as the most economic option. In May 2005, a 17,000 tonnes
on-site pilot heap was started with initial heap bioleaching in August 2005. In April 2008, ore
mining started at open pit and in July 2008, heap bioleaching was started. Metal sulfides through
bioleaching were produced in October 2008.
The ore is crushed and screened in four stages into p80 < 8 mm. Materials less than 10 mm were
agglomerated for heap bioleaching in the presence of sulfuric acid. After agglomeration, ore is
stacked eight meters high on primary pad for about 18 months for bioleaching. The heaps were
aerated and irrigated from the top. After this period, the leached ore was restacked on secondary
heap to leach metals further through a secondary heap leach cycle.
Indigenous microorganisms are used and viable conditions for bacterial growth created in the
heaps.[85 86]
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Heap bioleaching conditions are summarized below:
Ore supply 25 Mtpa

Particle size P 80 8 mm
Temperature 20 800C
Duration 13 14 months for primary
3.5 years for secondary.
Acid consumption 15 Kg / tonne and 2 Kg / tonne for primary and secondary heaps.
Heap dimensions 8m high (dynamic) for primary.

Secondary permanent pad (4 x 15m).
Bioleaching tests with black schist samples were carried out with iron and sulfur oxidizing
acidophilic bacteria which included both mesophiles and thermophiles. Water samples and
bioleach solutions were found to contain many of the above microbes.
The following organisms were detected in column tests at acidic pH.
At. thiooxidans
A.caldus
Leptospirillum ferrooxidans
Ferrimicrobium
Sulfobacillus
L.ferriphilum
Ferroplasma
Bacterial inoculum for the pilot heap was from near-by metal-rich ponds and the enrichment was
grown on sulfur, ferrous ions as well as the ore. Enrichment culture contained At.ferrooxidans,
2
L. ferrooxidans and At. caldus. During some periods of leaching, moderate thermophiles like
At.caldus and Sulfobacillus thermosulfidooxidans were observed to be dominant. Photographs
illustrating heap bioleaching at Talvivaara are shown in fig. 14.1.
A B
C D
Fig. 14.1: Photographs of Talvivaara heap bioleaching (A) General outline of heap bioleaching(B-C)
Actual heaps (D) Irrigation
(Photographs kind courtesy from Dr. Marja Riekkola-Vanhanen, Senior Biotechnology Adviser,
Talvivaara Mining Company Plc, Espoo, Finland).
(Permission from Talvivaara Mining Company Plc, Finland thankfully acknowledged)
3
Metals recovery flowsheet is illustrated in fig. 14.2.
Fig. 14.2: Metals recovery flowsheet.

(Kind courtesy from Dr. Marja Riekkola-Vanhanen, Senior Biotechnology Adviser, Talvivaara
Mining Company Plc, Espoo, Finland).
(Permission from Talvivaara Mining Company Plc, Finland thankfully acknowledged)
4
Demonstration leaching resulted in good recoveries. 80% nickel could be recovered within 400
days with 80% zinc in about 480 days. Copper and cobalt recoveries were lower for 500 days.
Secondary leaching was continued till November 2008.
Mineralogical data with respect to original heap material and the ore after secondary leaching are
shown in table 14.1:
Table 14.1: Heap leaching data for various minerals

Mineral Before leaching After leaching
Pentlandite 0.57 % 0.07 %

Chalcopyrite 0.45 % 0.14 %
Sphalerite 1.05 % 0.13 %
Sulfur --- 0.98 %
Anticipated total recoveries from both primary and secondary heap bioleach systems are 85% Ni,
80% Zn and about 50% for Cu and Co. The leached metals are precipitated using hydrogen
sulfide.
5
64. H.R. Watling, The bioleaching of nickel-copper sulfides, Hydrometallurgy, 91 (2008) 70-
88.
65. K.A. Natarajan and I. Iwasaki, Role of galvanic interactions in the bioleaching of Duluth
gabbro copper-nickel sulfides, Separation Science and Technology, 18 (1983) 1095-1111.
66. K. Bosecker, Leaching of lateritic nickel ores with heterotrophic microorganisms, in
Biohydrometallurgy Int. Symp. Proc. CANMET, OTTAWA, Canada, (1986) p 15-24.
67. Q. Chen, Z.Fang, Bioleaching of Ni, Cu and Co from a low grade Ni-Cu sulfide ore. The
Chinese Journal of Process Engineering 1, (2001) 369-373.
68. D.W. Dew, C. Van Buuren, K.McEwan, C. Bowker, Bioleaching of base metal sulphide
concentrates: A comparison of mesophile and thermophile bacterial cultures. In:
R.Amils, A.Ballester (Eds), Biohydrometallurgy and the Environment Toward the
Mining of the 21st Century (Madrid, Spain) Part A. Elsevier, Amsterdam, (1999) pp 229-
238.
69. T. Heinzle, D. Miller, V. Nagel, Results of an integrated pilot plant operation using the
BioNIC process to produce nickel. Proceedings Biomine99 and Water Management in
Metallurgical Operations. AMF, Glenside, S.A., (1999) pp 16-25.
70. C. Hunter, BioHeapTM leaching of a primary nickel-copper sulphide ore. Nickel/Cobalt-
8 Technical Proceedings (Perth). ALTA Metallurgical Services, Melbourne. (2002) 11p.
71. L.J Mason, N.M. Rice, The adaptation of Thiobacillus ferrooxidans for the treatment of
nickel-iron sulphide concentrates. Minerals Engineering 15, (2002) 795-808.
72. R. Pogaku, B. Kodali, Optimization of bacterial oxidation process parameters for
selective leaching of nickel by Thiobacillus ferrooxidans. International Journal of
Chemical Reactor Engineering, Vol. 4 . Article A1, (2006) 14pp.
73. J. Puhakka, O.H Tuovinen, Microbiological solubilisation of metals from complex ore
material in aerated column reactors. Acta Biotechnologica 6, (1986a) 233-238.
74. J. Puhakka, O.H. Tuovinen, Biological leaching of sulfide minerals with the use of shake
flasks, aerated columns, air-lift reactor and percolation techniques. Acta Biotechnologica
6, (1986b) 345-6, 354.
6
75. M. Riekkola-Vanhanen, S., Heimala, Study of the bioleaching of a nickel containing

black-schist ore. In: Amils, R, Ballester, A (Eds.), Biohydrometallurgy and the
Environment toward the Mining of the 21st Century (Madrid, Spain) Part A. Elsevier,
Amsterdam, (1999) 533-554.
76. M. Riekkola-Vanhanen, Talvivaara black schist bioheapleaching demonstration plant.
Advanced materials Research, (2007) 20-21. 30-33.
77. L.R.G. Santos, A.F. Barbosa, A.D. Souza, V.A. Leao, Bioleaching of a complex nickel-
iron concentrate by mesophile bacteria. Minerals Engineering 19 (2006) 1251-1258.
78. Z.Wang, L.Zhao, The acidic oxidizing pressure leaching of Jinchuan pyrrhotite
concentrates. In: Fu, C., He, H., Zhang, C. (Eds.), proceedings of the International
Conference on Mining and Metallurgy of Complex Nickel Ores (Jinchang, China).
International Academic Publishers, Jinchang, (1993), 267-273.
79. T. Williams, BIOHEAPTM bacterial leaching of the Sherlock Bay Nickel Mine primary
nickel-sulphide ore in saline water. Nickel/Cobalt Conference (Perth). ALTA
Metallurgical Services, Melbourne. (2006) 13p.
80. Y. Zhao, Z. Fang, Bioleaching of Ni-Cu sulfide with acidophilic thermophile Acidianus
brierleyi. The Chinese Journal of Process Engineering 3, (2003) 161-164.
81. C.A. du Plessis, W. Slabbert, K.B Hallberg and D.B. Johnson, Ferredox: A
Biohydrometallurgical processing concept for limonitic nickel laterites, Hydrometallurgy,
109 (2011), 221 229.
82. K. B. Hallberg, B.M. Grail, C.A. du Plessis and D.B. Johnson, Reductive dissolution of
ferric iron minerals: A new approach for bioprocessing nickel laterites, Minerals
Engineering, 24 (2011), 620 624.
83. K.Bosecker, Leaching of lateritic nickel ores with heterotrophic microorganisms. In:
Sallely, J., McCready, R.G.L., Wichlacz P. (Eds.), Biohydrometallurgy International
Symposium Proceedings. CANMET, Ottawa, (1986), 15-24.
84. G.S. Simate, S. Ndlovu, Bacterial leaching of nickel laterites using chemoautotrophic
microorganisms: identifying influential factors using statistical design of experiments.
International Journal of Mineral Processing 88 (2008), 31-36.
7
85. Riekkola-Vanhanen M., Talvivaara Sotkamo Mine bioleaching of a polymetallic nickel

ore in subarctic conditions. Nova Biotechnologica 10-1 (2010) pp 7-14.
86. Riekkola-Vanhanen M., Talvivaara mining company from a project to a mine. Paper
presented at Biohydrometallurgy-12, Falmouth, Cornwall, UK (June 2012).
8
Lecture 15: Bioleaching Of Zinc Sulfide Ores And Concentrates NPTEL Web Course
Lecture 15
Bioleaching Of Zinc Sulfide Ores And Concentrates
Keywords: Zinc Bioleaching, Sphalerite, Zinc Concentrates
The leaching rate of zinc with mixed cultures of Acidithiobacillus ferrooxidans and
Acidithiobacillus thiooxidans is higher than that of Acidithiobacillus ferrooxidans or
Acidithiobacillus thiooxidans alone [87]. Moreover, the addition of an appropriate concentration
of ferric iron to the leaching systems is beneficial to zinc dissolution. Too high Fe 3+
concentration reduces the rate of zinc dissolution due to the formation of jarosites. In addition,
XRD, SEM and EDS analyses of the residues also indicate that elemental sulfur layers exist on
the surface of sphalerite leached without bacteria or only with Acidithiobacillus ferrooxidans.
These layers block the mineral surface from being attacked by the bacteria. The residues
bioleached by Acidithiobacillus thiooxidans, however, show no sulphur existed.
The mechanism of accelerating the zinc dissolution rate through the mixed culture is given
below: Acidithiobacillus ferrooxidans regenerate the oxidative ferric iron that is consumed in
the leaching process and keep a high redox potential. The role of Acidithiobacillus thiooxidans
is to oxidize elemental sulfur layers to sulfuric acid, which is beneficial to chemical leaching of
the sphalerite:
ZnS + ZH+ = Zn++ + ZH2S

2H2S + O2 = 2S0 + H2O
2So + 3O2 + 2H2O = 2H2SO4
ZnS + 2Fe+3 = Zn2+ + 2Fe+2 + S
Mechanisms in the bioleaching of sphalerite in the presence of Acidithiobacillus spp. and a

leaching model are illustrated in fig. 15.1.
1
O2 ZnS + 2H+ + O2 = Zn++ + S0 + H2O
Acidithiobacillus
ZnS 4Fe++ + O2 + 4H+ = 4Fe+++ + 2H2O
A ZnS + 2Fe+++ = Zn++ + S0 +2Fe++

Sulfur layer
Acidithiobacillus
2S0 + 3O2 + 2H2O = 2H2SO4

O2
Chemical
leaching
B Sulfur layer
Bacterial
leaching
No Sulfur layer
Fig. 15.1: (A) Electrochemical and (B) Shrinking core models for sphalerite oxidation
2
Biooxidation of a fine-grained, complex zinc and gold-containing sulphide ore has been
performed in a series of experiments at bench scale with 20 1 leaching volume in a series of three
continuously stirred reactors. A mixed culture of moderate thermophilic bacteria was used for
bioleaching at 45C and a mixed culture of extreme thermophilic archea were used for
bioleaching at 65C [88]. The leaching rates for zinc were in the range 80-87% with the
moderate thermophilic bacteria and 96-98% with the extremely thermophilic microorganisms. It
was found that, to obtain a high zinc recovery with a low degree of pyrite oxidation, a fine
particle size was essential. Changes in retention time did not influence zinc solubilisation to any
greater extent. Fig.15.2 shows the advantage of bioleaching of sphalerite using thermophilic
bacteria in place of mesophiles.
100
A. Thermophile A
B. Mesophile
80
B
60
Percent Zn
40
20
0
0 5 10 15 20 25 30
Days
Fig 15.2: Bioleaching of sphalerite using mesophilic and thermophilic strains
Heap bioleaching of low grade zinc sulfide ores offers great promise. A HydroZinc TM process
was developed by Teck Cominco Metals Ltd, which incorporates heap bioleaching of zinc
sulfide ores along with subsequent neutralization, solvent extraction and electrowinning. Column
leaching tests as well as demonstration bioheap tests were carried out. About 82% zinc recovery
after 740 days was attained [89].
3
A comprehensive modeling study of the above heap bioleach process using Heapsim modeling
was also undertaken. The model took into consideration various sub-processes such as mineral
and sulfur oxidation, ferric ion reduction, topological effects, particle size distribution, microbial
growth, attachment and activity, as well as solution flow and heat balance. Zinc sulfide (as
marmatite) and iron sulfide (as pyrite) were used in reaction kinetics and electrochemical
models. Optimized parameters were
Heap height 6-8 m
Irrigation rate 10-15 Kg/m2/h
Side length 0.20 0.15 m
Acid 15 g/L
400day simulation has projected an optimized scenario at 78% zinc extraction [90].
GeoBiotics, LLC has developed a proprietary heap bioleaching technology for the processing of
sulphide base metal concentrates [91]. In this process, known as GEOCOAT , thickened
flotation concentrate is contacted during heap stacking with gravel-sized support rock, forming a
thin adherent concentrate coating on the support rock particles. The stacked heap has an open
structure and is highly permeable to the flows of solution and air. Acid solution, which contains
acidophilic bacteria, is circulated through the heap to biooxidise and leach the contained metals.
The heat produced by the exothermic oxidation reactions causes the internal temperature of the
heap to rise. Heat is transferred to the percolating solution and to the air blown up through the
heap. Rates of solution application and aeration can be varied to control the heap temperature
and maintain it within the optimum range for bacterial activity. GeoBiotics and Kumba
Resources have investigated the feasibility of the above process to the leaching and recovery of
zinc from a low-grade sphalerite concentrate produced from accumulated flotation tailings at
Kumbas Rosh Pinah zinc mine in Namibia. To confirm that a GEOCOAT heap to bioleach
this concentrate would operate autothermally, a large engineering column test was conducted.
The column was filled to a 6 m height with concentrate coated support rock. After acid
stabilization the column was inoculated with an adapted mixed mesophilic bacterial culture. The
temperature in the column was increased from ambient to a maximum of 49 C. The solution
4
application rate and the aeration rate were adjusted to control the temperature and to prevent it
from rising beyond the maximum tolerable by mesophiles. Final zinc dissolution after 90 days
was 91% with a corresponding sulphide sulphur oxidation of 89%. Concentrations of in excess
of 90 g/l Zn in the column effluent did not appear to inhibit the microbial oxidation. The
operation of the large diameter column was successful in demonstrating that the GEOCOAT
process can be operated autothermally at mesophilic temperatures treating a low-grade sphalerite
concentrate. Zinc dissolution in excess of 90% can be achieved in a leach time of 60 days, while
results of small diameter column testing indicate zinc dissolutions in excess of 95% in 60100
days.
BioZincTM process developed by BHP Billiton Ltd uses tank oxidation and leaching of zinc
sulfides.
Yet another process has been developed for zinc production from zinc concentrates by
integrating zinc bioleaching with zinc solvent extraction and electrowinning. The zinc
bioleaching stage was developed from small scale continuous trials through to the construction
and operation of a 1300L scale pilot plant. Using both a commercial zinc concentrate, and a
mixed lead-zinc concentrate as feedstocks, zinc extraction of 95-99% were obtained, depending
on the process conditions. The integration of zinc bioleaching with solvent extraction and
electrowinning has significant advantages including a simplified flowsheet resulting in low
capital costs, high zinc recoveries and the supply of the key reagent requirements (such as
sulphuric acid and CO2) for the bioleach. A fully integrated zinc pilot plant based on the
integrated process was operated over a 12 month period, treating both the zinc concentrate and
mixed lead-zinc concentrate feeds. Overall zinc recoveries of 96% (for the zinc concentrate) and
93-96% (mixed lead-zinc) were obtained, with routine production of SHG zinc cathode [92].
5
References
87. Le-xian XIA., Jian-she LIU., Li XIAO., Jia ZENG., Ban-mei LI., Mei-mei GENG. and
Guan-zhou QIU., Single and cooperative bioleaching of sphalerite by two kinds of bacteria
Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. Transaction of Nonferrous
Metals Society of China, (18) 2008, pp. 190
88. Sandstrom A. and Peterson S., Bioleaching of a complex sulphide ore with moderate
thermophilic and extreme thermophilic microorganisms. Hydrometallurgy, (46) 1997, pp.
181.
89. Lizama H.M, Harlamovs J.R, Belanger S and Brienne S. H, The Teck Cominco HydroZincTM
process Proc. 5th Int. Symp. Electrometallurgy and Environmental Hydrometallurgy TMS,
Warrendale, PA (2003).
90. Petersen J and Dixon D.G, Modelling Zinc heap bioleaching, Hydrometallurgy, 85 (2007),
127 143.
91. Sampson M.I., Van der Merwe J.W., Harvey T.J. and Bath M.D., Testing the ability of low
grade sphalerite concentrate to achieve autothermality during biooxidation heap leaching,
Minerals Engineering, (18) 2005, pp. 427.
92. Steemson M.L., Wong F.S. and Goebel B., 1997. In: Proceedings of IBS-Biomine 97, The
integration of zinc bioleaching with solvent extraction for the production of zinc metal from
zinc concentrates. (Sydney).
6
Lecture 16: Biohydrometallurgy Of Uranium Microorganisms And Mechanisms NPTEL Web Course
Lecture 16
Biohydrometallurgy Of Uranium Microorganisms And
Mechanisms
Keywords: Uranium Leaching, At.ferrooxidans, Mechanisms
In lectures 16-18, biohydrometallurgy of uranium is discussed in the light of useful

microorganisms, leaching mechanisms, dump, heap and insitu leaching techniques,
industrial examples as well as bioremediation [93-114]
Basic electrochemical principles governing uranium leaching in an acid solution are given
below: In naturally occurring uranium ores such as uraninite, uranium occurs in the tetravalent
state.
Tetravalent U is insoluble in dilute sulfuric acid and must be oxidized to hexavalent state for
easy dissolution. Molecular O2 is slow / inefficient as an oxidant. Ferric ion accelerates
oxidative dissolution of U(IV). Ferric ion must be regenerated to maintain oxidising conditions
during leaching. NaCIO3 / MnO2 has been used as Fe+2 oxidising agents.
Use of Acidithiobacillus ferrooxidans or Leptospirillum ferrooxidans as Fe++ oxidant.
Ferric ion is an oxidant (via) electrochemical surface reaction and a concentration of about 1-2
g/L is usually adequate.
UO2 + 2Fe+++ = UO2 ++ + 2Fe++
Oxidation of U(IV) occurs at > 400mV
U-bioleaching studies have extensively used enrichment or pure cultures of iron and sulfur
oxidising At.ferrooxidans or mixed cultures of At.ferrooxidans and At.thiooxidans or mixture of
Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans [93-114]
Major microorganisms relevant in the bioleaching of uranium ores are listed below along with
their major characteristics. (Table 16.1).
1
Table 16.1: Major microorganisms relevant in the bioleaching of uranium ores
Oxidize Fe++, So, Aerobe Obligate 1.2 6. 28 350C

Acidithiobacillus
U4+, Cu+, Se++, chemolithotroph
ferrooxidans
Thiosulphate,
Tetrathionate, S--
Acidithiobacillus Oxidize So, Aerobe Obligate 0.5 6.0 28 - 300C

thiooxidans Thiosulphate, Strict chemolithotroph
Tetrathionate
Thiobacillus Oxidize So organic Aerobe Facultative 1.5 6.0 25 - 300C

acidophilus compounds autotroph,
Leptospirillum Oxidize Fe++, Aerobe Obligate 1.5 4.5 20 400C

ferrooxidans Pyrite chemolithotroph
Sulfolobus Oxidize Fe++, So, Aerobe Facultative 1.9 3.0 20 600C

thermosulfidooxidans Sm autotroph
Sulfolobus Oxidize Fe++, So Facultative 2.0 7.0 55 850C

acidocaldarius autotroph
Oxidize So, yeast Aerobe Facultative 1.0 6.0 45 750C

Acidianus brierleyi
extract Strict chemolithotroph
Pseudomonas Accumulate U, Cu, Aerobe Heterotroph 7 8.5 300C

Pb intracellular Strict
Penicillium Accumulate U, Ra, Aerobe Heterotroph 57 15 300C

Rb in the cell wall Strict
Accumulate U, Th, Aerobe Heterotroph 46 24 - 270C

Rhizopus
Ra in the cell wall Strict
Desulfovibrio Remove dissolved Anaerobe Heterotroph 47 25 300C

desulfuricans U from effluents
2
Microbial habitat in uranium deposits:
Moderately acidophilic Acidithiobacillus (T.thioparus, T.neopolitanus, T.intermedius and

T.novellus) are found in ore samples and mine waters at near pH values.
Consortia of acidophilic Acidithiobacillus (Acidithiobacillus ferrooxidans and Acidithiobacillus
thiooxidans) and Leptospirilli are believed to be superior. Microbial diversity is extensive in acid
mine drainage including uranium mine leach solutions.
Thermophilic archaea resembling sulfolobus and acidianus Spp. have also been isolated from U-
mines.
Numerous heterotrophic bacteria are also present in U-mines besides yeasts and fungi.
Various types of uranium minerals along with their amenability for bioleaching are illustrated in
table 16.2.
Table 16.2: Various types of uranium minerals along with their amenability for bioleaching
Amenability
Uraninite UO2 Easy
Brannerite (U,Ca,Ce)(Ti,Fe)2O6 Easy
Davidite (Fe, Ce, U)2 (Ti, Fe, V, Cr)5 O2 Easy
Coffinite U(SiO4)1-x(OH)4x Hard
Uranophane Ca(UO2)2(SiO3OH)25H2O Variable
Autunite Ca(UO2)2(PO4)2 12H2O Easy
Torbernite Cu(UO2)2(PO4)2 12 H2O Easy
Carnotite K2(UO2)2(VO4)23H2O Variable
Uranopilite (UO2)6SO4(OH)6O212H2O Easy
Johannite Cu[UO2(OH)SO4]28H2O Easy
Schroeckingerite NaCa3(UO2)(CO3)3(SO4)F.10(H2O) Easy
3
Basic requirements to be borne in mind when considering the suitability of uranium ores for
bioleaching are the following:
Uranium mineralogy sensitive to bacterial leaching;
Permeability of rock and compactness.
Sufficiently high pyrites content to promote Acidithiobacillus activity
Absence of basic compounds in the ore (acid consumption)
Presence of mineral salts which act as essential nutrients for the microorganisms.
Schematic presentation of uranium oxidation coupled with Fe+++ and O2 reduction is given
below:
URANIUM IV (UO2)
Acid insoluble
2 Fe+ ++ 2 Fe++
+ +
0.5 O2 H2O
2H+
URANIUM VI (UO2++)
Soluble at acid pH
4
Schematic of bacterial reoxidation of Fe++ in uranium leaching is illustrated below
Oxidation of FeS2 and other iron sulfides

by iron and sulfur - oxidizing chemolithotrophs
Fe+++
Bacterial reoxidation Iron reduction

during U- (IV) leaching
Fe++
Reactions in bioleaching of uranium ores containing pyrite are given below with respect to stages
in the anodic and cathodic reactions through the indirect mechanisms. Acidophiles such as
At.ferrooxidans, At.thiooxidans and L.ferrooxidans can efficiently bring about acidic dissolution
of uranium in the ore, through bacterial oxidation of ferrous iron. Pyrite present in various
uranium containing ores serve as energy source for the above microorganisms.
5
FeS2 + H2O + 3.5 O2 = Fe++ + 2 SO4- - + 2H+ UO2 + 2Fe+++ = (UO2)++ + 2Fe++
Indirect solubilization of metal sulfide Indirect solubilization of uraninite
Bioleaching Variables
Formation of solid leach products such as sulfur, ferric precipitates, jarosites retard leaching rates
and need to be controlled.
Factors influencing rate of bioleaching are
Morphology and structure of mineral
Particle size and surface area
pH and redox potential
Temperature, oxygen, presence of catalytic reagents
Water and humidity levels
Chemical/mineralogical composition of ore
Climatic conditions (seasons, ambient temperature, rainfall)
Siliceous / carbonaceous gangue (calcite/dolomite) acid consumption.
6
Acidithiobacllus ferrooxidans in leach solutions are found to be tolerant to larger concentrations

of dissolved uranium (12 g/L U3O8 highly metal tolerant development of uranium tolerance.)
Regeneration of Fe+++ containing leach solution by bacterial oxidation considered as a separate

process as it could be controlled to maximize oxidation kinetics through nutrient addition,
aeration and pH control. Continuous oxidation of ferrous sulfate leach solutions from U-
processing plants can be achieved.
Bacfox solution saturated with air over a biofilm of Acidithiobacilli adhering to a solid
corrugated surface rate of Fe++ oxidation 7.5g/m2/h.
Other fixed film systems for biological conversion of ferrous to ferric sulfate such as packed-
bed and fluidized-bed reactor designs can also be used.
Generally, bioleaching of uranium has been carried out on ores that contain iron and other metal
sulfides.
However, amenability studies have been reported for bacterial leaching of U-containing sand
stone deposits utility of heterotrophic bacteria / fungi for neutral leaching need be established
for ores not containing sulfides, iron / sulfur can be supplied from outside for Acidithiobacillus
Eg: pyrite tailings.
7
Lecture 17: Biohydrometallurgy Of Uranium Dump, Heap and Insitu Leaching NPTEL Web Course
Lecture 17
Biohydrometallurgy Of Uranium Dump, Heap and Insitu
Leaching
Keywords: Dump, Heaps, In-situ Leaching
Dump, Heap, in situ leaching

Dump millions of tons of over burden / waste rock Dumps often built in a valley to
take advantage of natural slope not inoculated- flooding / sprinkling the top surface.
Heap-rocks are often crushed to avoid solution contact problems - built up on
impermeable pads to prevent loss of solution to underground - aeration systems installed
to increase flow of air.
Inplace leaching for low grade ores present in inaccessible sites abandoned
workings depleted mine working leach solution directly applied to walls and roofs of
an intact stope (an underground excavation from which ore has been removed ) or to the
rubble of fractured workings.
Percolation bacterial leaching methods for the treatment of uranium ores

In-situ leaching: Submarginal ores obtained by blasting; ore treated for 5 25 years with an
operational capacity of 4 million tons of ore.
Fig. 17.1 illustrates design aspects of heap bioleaching for uranium.
1
Fig 17.1: Design aspects (A-B) of heap and (C) shallow stope leaching for uranium.
2
Surface ore heaps and underground mined stopes can be efficiently bioleached as shown in fig.
17.2
Fig. 17.2: Bioleaching of surface ore heaps and underground mined stopes.
In situ leaching concepts are illustrated in fig. 17.3.
3
Fig. 17.3: In situ leaching concepts for uranium from underground deposits.
4
Dump leaching: Submarginal ores obtained by bulldozing; ore treated for 3 20 years with an
operational capacity of 5 million tons of ore.
Heap leaching: Low grade ores obtained by crushing - ore treated for 1-2 years with an
operational capacity of 3 x 105 t of ore.
Vat leaching: Similar to leaching in heaps but for 10 30 days and with an operational capacity
of 5 x 103 t of ore.
Mine waters: Water collected after rain in open pit mines.
In situ ore leaching from injection wells and producing wells

There may be difficulties connected with ecology since it is necessary to collect the leach liquor
after it has passed through the ore beds. Unsuitable silting may lead to large amounts of the
leaching fluid escaping underground.
There are some bacterial leaching processes which in a broader sense can be termed in situ
leaching. For example, percolating of a worked-out mine with residual ore. In Canadian
uranium mines after they were worked-out, the walls, roofs and floors were hosed down at
intervals of several months. The water was collected and the uranium extracted.
Bioleaching of uranium ores depends wholly on indirect oxidation in presence of ferric/ferrous

iron system. Leaching of uranium ores containing pyrite as an iron source is most economical.
Otherwise one has to add pyrite or another source of iron to facilitate bioleaching. Heap or basin
leaching can be applied. The ore is set up in basins. The mode of operation is preferably a two
stage leaching: the out-flowing liquor, in which the iron is largely in the ferrous form, is treated
in an oxidation pond. The leach systems are aerated to enable Acidithiobacillus ferrooxidans to
oxidize ferrous iron and to obtain the ferric iron required for oxidation of uranium. The oxidized
liquor is then pumped back to the dump or basin.
5
Microbially mediated uranium recovery from low-grade ores dates back to the 1950s.
Initial work in early 1950s directed towards preventing solubilization

Urgeiric Uranium ore (Portugal) Problem due to U-solubilisation by Fe and S bacteria and
washout by rain water.
During 1952 53, U-commercial bioleaching at Urgeirica (Portugal).
Subsequently, bioleaching processes for commercial extraction of U by heap, dump and stope
leaching of mine waste rocks and worked out stopes in the Elliot lake Area, Ontario, Canada in
the early 1960s.
Leach solutions containing acidic ferric sulfate circulated through surface heaps and
underground stopes.
Laboratory, pilot and commercial scale bioleaching have been evaluated in many uranium
producing countries. Potential of this biotechnology to low-grade U-ores containing < 0.50 %
U3O8 recognized.
6
NPTEL Web Course
Lecture 19: Biotechnology For Gold Biogenesis, Microorganisms And Gold Nanobiotechnology
Lecture 19
Biotechnology For Gold Biogenesis, Microorganisms And Gold
Nanobiotechnology
Keywords: Gold Biogenesis, Microorganisms, Mechanisms
In lectures 19-21, biotechnological aspects of gold production are illustrated with respect to
biomineralization, involved microorganisms, principles and mechanisms involved in
biooxidation of refractory sulfide gold concentrates, bioreactor practices as well as
bioremediation of cyanides and newer developments in gold biotechnology [115 126].
Different microorganisms occur indigenously associated with gold ore deposits. Bacteria and
archaea are known to be involved in the biogeochemical cycles of gold involving gold
mineralization, dissolution, precipitation and mobilization. Microorganisms can be used to
achieve the following beneficial consequences.
Biooxidation of gold-encapsulated pyrite and arsenopyrite concentrates to liberate metallic

gold (Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum
ferrooxidans and thermophiles.)
Cyanide-free reductive dissolution of gold (Anaerobes such as sulfate reducing bacteria)
Chelation of gold (Amino acids produced by Bacillus sp.)
Precipitation of gold nanoparticles (Bacillus subtilis, Shewanella, Rhodococcus
and Chlorella vulgaris)
Bioaccumulation of gold (Fungi, Yeasts and bacterial biomass)
Biodegradation of cyanides (Pseudomonas spp.)
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Biogenesis [115]
Microorganisms play a significant role in the geochemical cycling of gold. Though gold and its
complexes are generally toxic to microbial growth, many bacteria, archaea and fungi are capable
of solubilising and precipitating gold under natural conditions. Bacteriform structures of
secondary gold grains have been taken as a pointer towards microbial gold precipitation and
biomineralization. Biogeochemical cycling of gold can be linked to biological processes of
concentration. Both abiotic and biological mechanisms are involved in natural gold-bearing ore
mineralization. Due to its insolubility in aqueous solutions, various biologically-derived
complexing agents are needed to solubilise gold. Weathering of base metal sulfides due to geo -
and biochemical forces can promote chemical mobility of gold which can complex with
thiosulfate, bisulfides and chlorides. Organic gold complexes are found in soils. Organic acids
such as humic acids, amino and carboxylic acids are generated by soil microorganisms, leading
to gold solubilisation and precipitation .
Sorption of gold complexes and colloidal gold to organic matter and clays need also be
considered. Bioaccumulation and biomineralization can lead to the formation of secondary gold
particles. Bacteria and archea attach to ore particles under aerobic and anaerobic conditions.
Anaerobes such as sulfate reducing bacteria (SRB) and thermophiles can be associated with
natural gold mineralization. Pyrite and arsenopyrite mineralization encapsulating gold particles
is a typical example.
Microbial gold solubilisation is based on the ability of microorganisms to generate gold

solubilising complexing reagents. Chemolithotrophic iron-sulfur-oxidising bacteria such as
Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans can oxidize gold containing
pyrite and arsenopyrite. Such biooxidation of sulfide minerals leads to liberation of associated
gold. Biologically generated thiosulfate can complex with gold leading to its solubilisation.
Thiosulfate and bisulfides are produced by anaerobes such a sulfate reducing bacteria which
participate in gold mobilization under natural conditions.
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Heterotrophic bacteria such as Bacillus subtilis, B.megaterium, Pseudomonas and

B.mesentericus are capable of gold solubilisation. A close correlation between gold
solubilisation and microbial production of aminoacids in soils under natural conditions exist.
Oxidation and complexation of gold with microbially-generated cyanides are also possible.
Cyanogenic microbes inhabiting ore deposits generate cyanide compounds.
The above observations, clearly establishes the biological aspects of gold occurrence and its
extraction. Bacteria serve as biological indicators for gold occurrences. Both direct and indirect
gold solubilsation using microbiological processes then, become possible.
Microbial precipitation of gold nanoparticles [117-118]
Many aerobic, anaerobic, photosynthetic and cyano-bacteria are capable of producing hydrogen.
Biohydrogen technologies can be used to produce gold nanoparticles under controlled
conditions. Nanoparticles of gold can be biopreapitated by controlled growth with respect to
shape and size. Gold nanoparticles can be synthesized using mesophilic bacteria or algae from
gold chloride solutions. Suspension of dried cells of an alga, chlorella vulgaris in HAuCl4
solution promote accumulation of elemental gold in the cells. Different microorganisms useful
in the synthesis of gold nanoparticles include Bacillus subtilis, Shewanella algae, Rhodococcus,
Chlorella vulgaris and fungi such as Verticillium.. It is possible to harvest the metal
nanoparticles formed with in the fungal or bacterial biomass.
Microorganisms in gold processing
Some important microorganisms relevant to gold processing are illustrated in Figure 19.1.
Among them Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans are used in
enhanced gold recovery from refractory pyrite-arsenopyrite concentrates containing entrapped
gold. Sulphate Reducing Bacteria (SRB) can be used to solublise gold through bisulfide
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generation. Bacillus subtilis is known to solubilise gold directly. Fungi and yeasts are used as
good biosorbents for gold from effluents and leach liquors. Pseudomonas spp. can biodegrade
cyanides while other groups of bacteria are used in the production of gold nanoparticles and
colloids. Cyanobacteria can produce cyanide solvents which can complex gold.
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Thermophiles Fungi Bacillus subtilis Leptospirillum

ferrooxidans
Acidithiobacillus Sulphate Reducing Pseudomonas

ferrooxidans Bacteria Yeast
Fig. 19.1: Microrganisms relevant to gold processing
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Lecture 1: Biology Materials Interface And Biomaterials Processing NPTEL Web Course
Lecture 1
Biology Materials Interface And Biomaterials Processing
Keywords: Biomaterials Processing, Biomimetics, Nanosynthesis
History of the biology-materials interface is several billion years old. Nucleosynthesis of

materials in the stellar level goes back almost 20 billion years. Nonbiotic materials processing
by mother earth dates back to almost 4.5 billion years after which comes biologically-mediated
processing of materials (about 3.5 billion years ago).
In this cosmic scale, human efforts to synthesis of materials is relatively new, coming only after
stellar and microbiological contributions. In this respect, we have to learn a lot from the tiny
microorganisms inhabiting earth and ocean floors. Microorganisms, which can be termed,
extremophiles bringing about biogenesis biomineralization and biomaterials processing since
more than billion years ago have undergone mutational and genetic changes to survive in
extreme environments with respect to acidity, oxygen availability, temperature and metal
toxicity.[1]
Biotechnology-materials cycle in nature involves biogenesis of ore minerals, biooxidation and

dissolution of metals from ores, biosynthesis of bulk and engineering materials, biodegradation,
biofouling and biocorrosion of materials, and lastly, bioaccumulation and bioremediation of
polluted environments. What had been produced and processed through biogenic processes
ultimately assimilates back to mother earth completing the biological cycle.
In this respect, the microbial cell functions as a fully- automated bioreactor capable of different
functions such as production of bioreagents and catalysts, inter-and intracellular metal
accumulation and sorption, dissolution and corrosion of metals, as well as degradation and
remediation of environment. Plasmid genes have evolved over a period of time for defence
against stress and metal toxicity. Most of the toxic metal resistance may be plasmid-mediated.
New populations possessing higher tolerance to metal ion concentrations, pH, temperature,
pressure differences and oxygen concentration gradients emerge as microbial evolution
progresses with time. Genetic engineering of such mining and metal-specific microorganisms
has now become possible. Coding genes from plasmids can be cut by restriction enzymes,
1
classified within different fractions and reinserted into a vector plasmid through enzymatic
action. The recombinant DNA can then be located within a host cell for replication. Such
genetic engineering procedures can lead to mass production of super bugs capable of
performing a desired engineering feat due to their newly acquired capabilities such as metal and
high temperature resistance and synthesis of useful bioreagents.
Materials processing capabilities of a microbial cell are illustrated below.(Fig.1.1)
Fig.1.1: Microbial cell with its potential functions.
Biology-materials interface include several materials processing steps and cycles such as
Biogenesis, Biomineralization
Biomaterials processing
Bioextraction of metals (Biohydrometallurgy)
Biobeneficiation to produce acceptable quality raw materials for process engineering.
Biofouling, Biodeterioration and Biocorrosion
Environmental degradation
Bioremediation and environmental protection.
2
Many of the above biological functions and processes are welcome developments in modern
biotechnology. For example, an understanding of biogenesis and biomineralization of earthly
resources such as fossil fuels and ore minerals will lead to isolation and industrial use of many
natural organisms to extract metals and synthesize modern bulk materials and nano materials.
This would also pave the way for developments in nanobiotechnology and use of
environmentally-benign, cost-effective and energy-efficient bioreagents in place of toxic
petroleum-based chemicals. Biohydrometallurgy and biobeneficiation processes are potential
routes for environmentally-acceptable production of raw materials and finished metals. Not so
advantageous and often destructive aspects of this interface include biofouling, biodeterioration,
biocorrosion of metals and alloys and environmental pollution brought about by microbial
activities. Thanks to positive developments in biotechnology, all the above deleterious activities
can be controlled and minimized to a greater extent. While several microorganisms are
implicated in environmental pollution, there exists in the same polluted environments, potential
bioremediating organisms that can be harvested and utilized for detoxification of contaminated
soils and waters.
Biomaterials and biomimetics
There are several minerals and metals which are classified as biogenic. Some very common
examples are limestone (CaCO3) and silica (SiO2) which are formed through participation of
several microorganisms. Iron oxides such as magnetite (Fe 3O4) are formed by the action of
several anaerobes and magnetotactic bacteria. Many sulfide, sulfate and oxide minerals also
have associated bacterial cycles in nature. Among the metals gold, silver, selenium and
tellurium are biological products, so also sulfur and volatile mercury. Bacteria, algae, fungi and
plant forms participate in mineral generation, conversion and speciation. Most of the metals
have a bacteria-involved cycle involved and mineral-metal-bacteria cycles in nature are
important in geomicrobiology and biogeochemistry.[2-3]
Simpler organisms such as bacteria and higher organisms like mollusks and plants bring about
biogenesis of minerals and metals. Bacterial activity involves surface binding of metal ions,
oxidation-reduction, bioaccumulation and precipitation. It is through the intervention of higher
organisms, more structurally-ordered materials are generated-structural polymer-mineral
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composites such as sea shells, conches and other silica-calcite based architectures. Growth of
polymer CaCO3 composites on mollusk shells is a classical example. Involvement of ferritin -
the protein shell that contains channels to permit permeation of metal cations (such as iron and
manganese) and anions such as carbonates, sulfates and chlorides in the formation of
biomaterials such as magnetite, apatite and manganic compounds is known.
Significant progress has been achieved in the bioprocessing of several engineering materials
utilizing the principles of biomineralization. Biomimetic materials processing are governed by
the following principles:
Mineral specificity
Incremental net shape formation
Compartmentalized processing
Micro-emulsions and reversed micelles are used to synthesize inorganic nanoparticles with
controlled sizes and shapes. By sequential deposition procedures, high density ceramics can be
formed.
Biomineralization presents enough scope for newer developments in materials science.

Relevance of organic supramolecular assemblies in biomaterial systems is significant.
Interaction between molecular architecture and materials science through biomineralization and
biomimetic strategies is very important. Many metallic elements such as copper, iron, lead and
zinc can be deposited on bacterial cell walls in the form of sulfides, oxides or carbonates.
Biominerals are formed in well defined sites-intra or inter-cellular. Close association of organic
and inorganic phases occurs in biomineralization.
Biogenic iron oxides include magnetite, goethite and ferrihydrite. Both biologically- controlled
and - induced processes are common with participation of bacterial proteins and enzymes. In
nano-scale, biomineralization consists of discrete, self assembled supramolecular parts such as
vesicles and micelles. Mann classifies four constructional processes of biomineralization,
namely, supramolecular preorganization, interfacial molecular recognition, vectorial regulation
and cellular processing.
4
Bioconcepts, biomolecules and biosystems form the major divisions of biomimetic materials
science. Nano-scale synthesis of materials and composites involve strategies such as host-guest
and ligand capping. Crystal engineering of materials involve templating, directed growth and
micro - structural fabrication.
Biogenesis is mimicked to construct special materials. Artificial polymerized vesicles can be

designed to mimick biological functions. For example, colloidal gold, silver and platinum and
compounds such as metal sulfides, metal oxides and chlorides can be synthesized in different
shapes, sizes and in crystalline or amorphous states.
Metal cycles in nature are driven by microorganisms since they provide microbial nutrition (Mg,
Fe, Ni, Zn, Co, Cu etc). Number of redox couples, such Fe(II / III), As (III / V) and Mn (II/IV)
provide energy for bacterial metabolism. Metal ions such as silver, mercury, copper, uranium,
lead, cobalt, molybdenum and zinc can also impart toxicity to microbial growth in varying
concentration levels.
For example, the sulfur-bacteria cycle in nature is very important with respect to bacterial
oxidation of mineral sulfides and bacterial reduction of sulfate to sulfides. Aerobic acidophiles
such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans participate in mineral
sulfide oxidation to sulfur, tetrathionates and ultimately to sulfate.
Both At.ferrooxidans and Leptospirillum ferrooxidans are capable of oxidising iron in the sulfide
minerals, resulting in the formation of acidic ferric sulfate. Anaerobic heterotrophs such as
Sulfate Reducing Bacteria (SRB) reduce the biogenic sulfates back to sulfides. Biogenic
formation of elemental sulfur, and metal sulfides in nature becomes thus possible.
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Lecture 20: Biotechnology For Gold Biooxidation Of Refractory Sulfidic Concentrates
Lecture 20
Biotechnology For Gold Biooxidation Of Refractory Sulfidic
Concentrates
Keywords: Refractory Sulfides, Biooxidation, Bioreactors
Biotechnology could be effectively used to extract gold from the following unconventional and
waste resources. [116-123]
a) Tailing dumps accumulated at the mine sites over a period of several years amounting to
several millions of tonnes, which in few locations contain as high as 1 gram / tonne of
gold
b) Lean grade sulfidic gold ores containing less than 1-2 gram/tonne of gold, which cannot
be economically processed through conventional processes.
c) Refractory ores-sulphidic or carbonaceous, where finely disseminated gold particles are
locked up, making them uneconomical for direct cyanidation
Gold occurs in nature in its native state in three different types of ores, namely, free milling,
base metal and refractory. Free-milling ores are those from which the precious metal can be
efficiently liberated from the host rock (quartz) through size reduction. Most of the gold-
processing plants around the world utilize free milling ores to recover the metal through the
cyanidation process. Base metal ores containing copper, lead, zinc and iron as their sulphides
often contain gold which is recovered as a by-product metal. The third type, namely, refractory
ores, is becoming commercially important as a potential primary source of gold. Among the
refractory gold ore occurrences, two types are important, namely, sulphidic and carbonaceous. In
sulfide deposits, the precious metal is finely disseminated within sulphide minerals such as pyrite
and arsenopyrite and the encapsulated gold particles cannot be efficiently liberated even after
fine grinding and direct cyanidation would be inefficient for metal recovery. In the case of
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refractory gold bearing carbonaceous ores, gold particles are locked-up, making recovery by
gravity or cyanidation methods unacceptably low. Another problem with carbonaceous materials
is preg-robbing which means even if liberated gold particles are cyanided, they get reabsorbed
onto carbon matter making recovery extremely difficult.
The use of mixed strains of bacteria containing Acidithiobacillus ferrooxidans, Acidithiobacillus

thiooxidans and Leptospirillum ferrooxidans may prove to be more efficient in sulphide mineral
oxidation due to synergistic mechanisms. The use of thermophilies such as Sulfolobus which
have optimum activity at about 60-80oC would ensure enhanced leaching kinetics in gold
liberation.
Biooxidation reactions to liberate gold entrapped in the sulfide matrix are given below:
2FeAsS + 7O2 + H2SO4 + 2H2O = 2H3AsO4 + Fe2(SO4)3

4FeS2 + 15O2 + 2H2O = 2Fe2(SO4)3 + 2H2SO4
4FeS + 9O2 + 2H2O = 2Fe2 (SO4)3 + 2H2SO4
FeS2 + Fe2(SO4)3 = 3FeSO4 + 2S
2S + 2H2O + 3O2 = 2H2SO4
4FeSO4 + O2 + 2H2SO4 = 2Fe2(SO4)3 + 2H2O
Both direct and indirect biooxidation mechanisms may be operative. Bacteria require nutrients,
O2 and CO2 for growth which are provided through aeration and supplementing growth media.
The following substances can be toxic to bacterial growth.
Cyanides and thiocyanates even in low levels

Grease, oily matter
Chlorides
Dissolved arsenic [As (III) more toxic than As (V)], ferric ions, copper and other metals
depending on concentrations.
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It may be reiterated that biooxidation of sulphidic gold-bearing concentrates is essentially a

pretreatment process for liberation of locked-up gold particles. The acid bioleached residues
need be lime-treated before subsequent cyanidation. In the absence of prior biotreatment, even
finer grinding of such refractory ores would result in very poor gold recoveries, even as little as
6-10% and at any rate never exceeds 30-40% even under optimum processing conditions. The
improvement in gold and silver recovery after prior biooxidation would be significant, often
exceeding 95%.
There is a direct relationship between the degree of sulphide bio-oxidation and percent gold
recovery. Complete oxidation of sulphides is not always essential to achieve acceptable gold
recovery. Depending on the sulphide entity, high gold recoveries can be obtained with even as
low as 50% oxidation of the total sulphides. The entire biooxidation process is agitation leaching
for the concentrates carried out in stirred bioreactors or Pachuca type reactors. The percent
oxidation of sulfide biooxidation depends on the type of sulphide involved, whether pyrite or
arsenopyrite.
Though gold bearing sulphides can be directly bioleached, it is often preferable to treat a
flotation concentrate of the ore since it enables easy handling of a reduced tonnage of enriched
material. The following variables need be closely controlled to achieve optimum efficiency.
Appropriate preadapted bacterial culture (adapted to sulfide concentrates and arsenic)

Particle size finer size means faster oxidation
Pulp density 20-30% optimum
Oxygen and carbon dioxide availability (aeration)
Agitation and homogeneous mixing of pulp
Temperature, 30oC to 45oC (use of moderately thermophilic strains)
pH 1 to 2
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Residence period 2 7 days

Eh 700-900 mV
Availability of nutrients
Removal of toxic and interfering reaction products
Large-scale industrial applications of biotechnology for refractory gold ores and concentrates
have shown great promise.
There are several stages in the feed preparation for biooxidation in reactors.
Thickening and remilling (if necessary of a flotation concentrate (pyrite, arsenopyrite or
pyrite-arsenopyrite) size about 70 - 80m.
Adjustment of pup density at about 20% solids (pulp density can be optimized depending on
the nature of concentrates).
Nutrient addition
Use of primary and secondary reactors (series or parallel arrangement).
Typical residence period between 3-6 days.
Oxygen requirement-supply of air and dispersal through mechanical agitators to ensure

acceptable oxygen transfer.
Impellers-different designs.
Slurry temperature to be maintained 340 450C Due to exothermic reactions, slurry

temperature can shoot up- Provision for cooling essential.
pH maintained between 1 -2.
Counter-current decantation before cyanidation of oxidized residues.
Neutralization by lime (or CaCO3). Removal of arsenic and iron from neutralized and
alkaline slurry.
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2H3AsO4 + Fe2(SO4)3 + 3CaCO3 = 2FeAsO4 + 3CaSO4 + CO2 + H2O
Fe2(SO4)3 + 3CaCO3 + 3H2O = 2Fe(OH)3 + 3CaSO4 + 3CO2
H2SO4 + CaO = CaSO4 + H2O (lime neutralization)
A list of industrial bioreactor operations for gold operations in different parts of the world is
given in Table 20.1.
Typical industrial bioreactors for gold biooxidation and cyanidation are illustrated in Figs 20.1
and 20.2.
Bioreactor engineering for refractory sulphidic gold concentrates: an Indian

experience
India is the largest consumer of gold in the world while in terms of production of the precious
metal, India produces only about 4 tons of the metal per year. The Hutti Gold Mines Company
Limited (HGML) in Karnataka is the only primary gold producer in the country today. Most of
the ore is mined by underground mining and gold is extracted from free milling ores by
cyanidation using carbon-in-pulp method. The typical grade of the ore is about 4-5 gm/ton.
In recent years, HGML has identified several newer deposits for extracting gold and silver.
G.R.Halli and Anjanahalli deposits are sulphidic ore deposits containing about 3-4 gm of gold
per ton of ore. As expected, extraction of gold from such ore or its flotation concentrate by
conventional cyanidation has not been encouraging. The concentrate needs to be biooxidized
before it is cyanided to enhance gold recovery. It is in this regard that HGML and Indian
Institute of Science undertook a joint project to develop bioreactor technology for biooxidation
of the refractory G.R.Halli concentrate with financial support from the Department of
Biotechnology, Government of India during 19972002.
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Table 20.1: Gold Bioreactor operations
Concentrate,
tonnes/day
Fairview, South Africa, 1986 - Biox 65 - 80
Sao Bento, Brazil, 1991 - Biox 380
Harbour Lights, Australia, 1991 94 Biox 40
Wiluna, W. Australia, 1993 - Biox 158
Ashanti, Ghana, 1994 - Biox 960
Yauanmi, W.Australia, 1994-98 BacTech 120
Tamboraque, Peru, 1998 - Biox 60
Beaconsfield, Tasmania, Australia, 2000 - BacTech 70
Laizhou, China, 2001 - BacTech 100
Olympiada, Krasnoyark, Russia, 2001 - 1000
Suzdal, Kazakhsan, 2005 - Biox (Gold field) 196
Fosterville, Victoria, Australia, 2005 - Biox (Gold field) 211
Bogoso, Ghana, 2006 - Bac Tech - Bacox 750
Jinfeng, China, 2006 - Biox 790
Kokpatas, Uzbekistan, 2008 - Biox 1089
Ref: M.Gericke, J.W.Neale and P. J. Van Staden, A Mintek perspective of the past 25 years in
H.R. Watling, The bioleaching of sulfide minerals with emphasis on copper sulfides A
review, Hydrometallurgy, 84, (2006) 81-108.
C. L. Brierley, How will biomining be applied in future? Trans. Nonferrous met. Soc.
China, 18, (2008), 1302 1310.
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Fig. 20.1: Agitated Bioleach Reactors to Oxidise Refractory Gold Concentrate

Fig. 20.2: Agitated Bioleach Reactors and Cyanidation Plant to treat Refractory Gold Concentrate
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Initial agitation leaching tests were carried in laboratory scale bioreactors using At.ferrooxidans
preadapted to concentrates (fig. 20.3).
Fig. 20.3: Laboratory bioreactor set-up
Flowsheet for pilot scale biooxidation and typical photographs of pilot bioreactors are
shown in figs.20.4 and 20.5.
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Fig. 20.4: Flowsheet for pilot bioreactor set-up for gold-silver bioprocessing
Fig. 20.5: Views of pilot bioreactors.
Gold and silver recovery data after biooxidation and cyanidation are given in fig. 20.6.
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120
100
80
60 Direct cyanidation
Bacterial oxidation
40
20
0
Gold Recovery Silver Recovery
Fig. 20.6: Percent extraction of gold and silver after cyanidation
Biooxidation tests were carried out on a continuous mode, the reactor was first run in batch mode
to bring the pulp density upto 10%. Initial the experiments were carried out with 1% pulp
density and the iron leached was estimated. When the iron leached out approximately attained
the theoretical amount in the concentrate, more concentrate was added and such step leaching
continued until the pulp density reached 10-15%. Further increase in pulp density did not
enhance the leaching rate. The high iron and sulphur content in the concentrate introduced a lag
period, slowing the leaching rate. The above method of step leaching is more effective than
starting with 10% pulp density at the beginning itself. Most of the iron leached was in the form
of ferric ions while very little ferrous was present. The leaching rate of iron was consistent with
reported results for pyrite and arsenopyrite bearing gold ores. The reactor was then operated in a
continuous mode with a feed rate of 1.5L /day and 4 days residence time. The slurry was pumped
using a peristaltic pump. On an average, 90% of gold and 95% of silver could be extracted from
biooxidised concentrate. The bioreactor was operated for about 15 days in this mode and it was
observed that consistently good recoveries could be achieved over the period of study.
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Biooxidation as a pretreatment can be used effectively for treating G. R. Halli sulphidic

concentrate at the HGML.
After intensive laboratory testing for nearly a year, it was decided to test the technology on-site
at HGML. For this purpose, a demonstration pilot facility was designed with a capacity to
process 100Kg of concentrate per day. Three bioreactors with a total capacity of 6 m3 was used.
The concentrate was fed from the feed tank to the first two bioreactors, which were in parallel,
through peristaltic pumps. Outputs from reactors 1 and 2 were fed to reactor 3 from where the
treated slurry was sent to a settling tank. The solids were drawn from the settling tank for
cyanidation. The reactors were provided with water jackets for controlling the temperature. The
start-up strategy was to get all the three bioreactors running in batch mode before the continuous
operation started. The bacteria (At.ferrooxidans) were cultured in the bioreactors itself in the
presence of the concentrate by the step leaching technique with all the necessary nutrients for
bacterial growth without ferrous sulphate. The feed was acid stabilized in the feed tank before it
was fed to the bioreactors. The important process variables such as Eh, pH, temperature pulp
density, iron concentration and bacterial population were monitored at regular intervals.
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Lecture 21:Biotechnology For Gold Recent Developments NPTEL Web Course
Lecture 21
Biotechnology For Gold Recent Developments
Keywords: Biosorption Of Gold, Cyanide Degradation, Cyanide- Free Leaching
Biosorption of gold and biodegradation of cyanides [124 126]

One of the most recent developments in environmental biotechnology is the use of microbe-
based sorbents for the removal and recovery of precious metals from industrial waste
effluents. Such biotreatment processes are of interest for the detoxification of industrial
wastes. Moreover, the subsequent recovery of accumulated metals is possible after suitable
treatment of the metal-loaded biomass. Leaching in a cyanide solution followed by
cementation of the dissolved gold with zinc or aborption in carbon is being used by a number
of gold-processing plants around the world. The cyanide effluents discharged from the plants
contain small amounts of unrecovered gold and varying concentrations of base metals such as
zinc, copper, iron and lead. These base metals enter the process stream through many unit
operations. The recovery of gold from such effluents is of great economic significance, and
the removal of heavy metals is environmentally beneficial. The removal of heavy metals
would also enable efficient recycling of the used cyanide solutions within the process circuit.
The use of microorganisms for the recovery of gold and for the removal of heavy-metal ions
from gold-plant cyanide effluents is of great commercial and environmental significance.
Microorganisms are known to be active participants in the formation, conversion and

accumulation of gold in mineral deposits. They are known to be capable of dissolving gold,
reducing it to an elemental state and accumulating it within their biomass. The activity of
Aspergillus sp. biomass often exceeds that of activated carbon by a factor of 10 to 12 in the
recovery of gold from colloidal solutions. Microscopic fungi are capable of absorbing gold
from aqueous solutions with the formation of complex compounds. Gold content of bacterial
cells associated with gold ore deposits are 15 to 600 times higher than that of the mine
waters. Almost complete removal of gold from synthetic solutions by algae could be
achieved. Fungal biomass as well waste industrial biomass are good gold biosorbents from
cyanide solutions. Degradation of cyanides by microorganisms from tailings solutions and
other process effluents is an attractive alternative to conventional chemical processes.
Degradation of cyanides by microorganisms is a natural process that can be readily exploited
1
and engineered. Cyanide degradation by microorganisms was first commercially

demonstrated in the gold mining industry at the Homestake Gold Mining company in USA in
the middle 1980s. Since then, several other studies on the microbial destruction of cyanide
have been reported including laboratory, pilot and full-scale facilities.
Various processes have been developed using both aerobic and anaerobic methods in full-
scale active, passive, and in-situ treatment facilities. Aerobic and anaerobic biotechnological
applications include active treatment of cyanide containing solutions from tailing ponds and
barren solutions to passive treatment of gold heap leach pad drain using in-situ anaerobic
systems. At the Homestake Gold Mine, full-scale operations have been in use treating high
volumes of tailings pond solution for nearly three decades. The aerobic attached growth fix
film biological facility consists of five stages of forty-eight rotating biological contactors
(RBCs) for the removal of thiocyanate, cyanide, ammonia, and metals. [125]
Microbial action, either naturally occurring or as a cyanide detoxification technique, causes

transformation of cyanide to ammonia. Metal ions released from metal cyanides will be
absorbed by the biomass and thiocyanates are converted to sulfate:
Cu2CN + 2H2O + O2 = Cu-biofilm = HCO3 + NH3

SCN + 2H2O + 2 O2 = SO4- - + HCO3 - + NH3
Treatment of cyanide heap leaches and tailings
Further microbial action will convert the ammonia to nitrate:
NH4+ + 1 O2 = NO2- + 2H+ + H2O

NO2- + O2 = NO3-
The aim of biological treatment processes is to greatly increase the rate at which these natral
transformations occur.
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Homestake cyanide biodegradation

Full scale operation 1984
21000 m3 of water per day discharge (4 million gallons of waste water per day)
Pseudomonas sp. -48 Rotating biological contactors
Near complete degradation of thiocyanate, free cyanide and metal complexed cyanides
(98 100%)
Total cost of waste water treatment plant was 2/3 cost of similar sized H 2O2 plant.
Concentration, mg/L
Total Thiocyanate WAD
Cyanide Cyanide,
Inflow 4.0 63 2.4
Outflow 0.08 <0.1 0.03
Permissible 0.15 1 0.14
Cyanide-free biodissolution of gold [126]

The development of a novel bioprocess for direct gold solubilization is based on the
following anaerobic reaction.
Au + H2S + HS- = Au(HS)-2 + H2
The use of naturally-occurring sulfate-reducing bacteria (SRB) to biocatalyze the following

hydrogen gas-consuming/bisulfide-producing reaction is of great commercial significance.
SO4- - + 4H2 + H+ = HS- + 4H2O
The microorganisms can also neutralize the solution by consuming hydrogen ions. Such a
bioprocess for gold extraction is cost-effective and environment-friendly. The process is
novel and innovative in precious-metals extraction for exploitation of ore deposits that are not
amenable to conventional leaching processes. It would also eliminate the need to impound
waste tailings containing residual cyanide.
3
In the above bioprocess, sulfidic ores are initially aerobically oxidized to mobilize gold
before bisulfide leaching through the use of mesophilic or thermophilic chemolithotrophs. In
bio-oxidation, aerobic, acidophilic bacteria, such as Acidithiobacillus ferrooxidans,
Leptospirillum ferrooxidans and Sulfolobus, can be used to oxidize iron and sulfur minerals
to liberate entrapped gold particles.
The second step uses anaerobic bacteria. Bisulfide ions can be generated biologically at very
low cost using an acidic sulfate waste product. Microbial sulfate reduction is brought about
by indigenous sulfate-reducing bacteria (SRB). In dissimilatory sulfate reduction, sulfate is
used as the electron acceptor for the oxidation of an electron donor, such as an organic
compound or hydrogen and the end product is hydrogen sulfide, which is produced by
anaerobic bacteria. Desulfovibrio, Desulfomonas and Desulfotomaculum genera of SRB can
be used to achieve the above reactions.
A soil bacterium, Bacillus subtilis is capable of dissolving gold through the generation of
amino acids that can complex with the precious metal.
Heap bioleaching of refractory sulfidic ores
The concept of biooxidation for pretreatment of sulfidic refractory gold-bearing ores in heaps
has commercial potentials. Fine ores or tailings containing gold-entrapped sulfides (pyrite-
arsenopyrite) can be agglomerated and arranged in heaps. Acidophilic organisms such as
At.ferrooxidans, L.ferrooxidans and At.thiooxidans can be inoculated into the heap initially.
Efforts can be made to promote indigenous growth of the above organisms through sprinkling
and irrigation of acidic liquor and nutrients. Such biooxidation is a pretreatment step to
liberate entrapped gold from sulfide minerals and may take several months for completion.
The bioleached residues need be lime-treated and cyanided for recovery of liberated gold.
4
In fig.21.1 conceptual bioprocessing applications in various facets of gold mining and

processing are illustrated.
BELTS OF GOLD MINERALIZATION
REFRACTORY FREE MILLING HIGH GRADE ORES LEAN GRADE SULFIDE

SULPHIDIC ORES ORES
SIZE REDUCTION SIZE REDUCTION

LIME.
LIME NaCN BIOHEAP LEACH
LEACHED MASS
GROUND MICROBIALLY CYANIDATION
ORE AIDED FLOTATION
CONCENTRATE
REACTOR GOLD LADDEN INSOLUBLE

BIOLEACH SOLUTION RESIDUE
ACIDS
BIOLEACHED ZINC BOXES

RESIDUE (Carbon-in-pulp) TAILING DUMPS
WASTE
CYANIDE
GOLD CYANIDE BIODEGRADATION
SOLUTION
EFFLUENT OF CYANIDE &
TOXIC ELEMENTS
METAL-LOADED BIOSORPTION & METAL-FREE CYANIDE

BIOMASS BIOACCUMULATION
Fig. 21.1: Integrated bioprocessing alternatives for gold
5
115. F. Reith, M.F. Lengke, D. Falconer, D. Craw and G. Southam, The geomicrobiology
of gold, The ISME Journal, 1-18, 2007.
116. F. Reith, S.L .Rogers, K.C. McPhail and J. Brugger, Potential for the utilization of
microorganisms in gold processing, World Gold Conference, October 2007.
117. K. Kashefi, J.M. Tor, K.P. Nevin and D.R. Loveley, Reductive precipitation of gold
by dissimilatory Fe(III) reducing bacteria and archaea; App.Environ.Microbiology,
67(2001), pp.3275-3279.
118. M. Gericke and A. Pinches, Microbial production of gold nanoparticles, Gold

Bulletin, 2006, 39/1
119. K.A. Natarajan, Bioprocessing for enhanced gold recovery, Mineral Processing and
Extractive Metallurgy Review 8(1992), pp 143-153.
120. K.A. Natarajan, Biotechnology in gold processing, Bull. Mat. Sci., 16 (1993) pp.
501-508.
121. Natarajan K.A. Microbes, Minerals and Environment, Geological Survey of India
(Bangalore) 1998.
122. K.A.Natarajan, J.M.Modak and A.M.Raichur, Bioreactor engineering for treating

refractory gold-bearing concentrates: An Indian Experience, V.S.T.Ciminelli and
O.Garcia Jr(Eds), BIOHYDROMETALLURGY: Fundamentals, Technology and
Sustainable Development, Elsevier(Amsterdam), 2001, pp 183-190.
123. M. N. Chandraprabha, Jayant M Modak, K. A. Natarajan, Ashok M Raichur,

Strategies For Efficient Start-Up of Continuous Biooxidation Process For Refractory
Gold Ores, MINERALS ENGINEERING, 15(2002), pp. 751-753.
124. M. Balakrishnan, J.M. Modak, K.A.Natarajan and J.S. Gururaj Naik, Biological
uptake of precious and base metals from gold-process cyanide effluents, Minerals &
Met. Processing, vol 11, No. 4, Nov. 1994, pp. 197-202.
125. Ata Akcil and T. Mudder, Microbial destruction of cyanide wastes in gold mining,
Biotechnology letters, 25 (2003), pp. 445-450
126. R.M. Hunter, F.M. Stewart, T. Darsow, M.L. Fogelsong, D.W. Mogk, E.H. Abbott
and C.A. Young, New alternative to cyanidation, Biocatalysed bisulfide leaching,
Min.Process. Ext. Met. Rev. 19 (1998), pp. 183-197.
6
Lecture 22: Biomineralization and Bioprocessing of Ocean ferromanganese nodules NPTEL Web Course
Lecture 22
Biomineralization and Bioprocessing of Ocean ferromanganese
nodules
Keywords: Ocean Nodules, Biomineralisation, Bioprocessing
What are ocean nodules?
Manganese and ferromanganese deposits occur at different depths as nodules and crusts in
oceans such as the Pacific and Indian oceans.
They can be Hydrogeneous, Halmyrolytic, Hydrothermal and Diagenetic deposits
A large reservoir of strategic and valuable metals such as Cu, Ni and Co are incorporated into
the ocean nodules.
MnO2, Fe2O3, CuO, Ni3O4 and Co2O3 are the most stable species in the ocean environment.
What nodules?
Mineral resources recoverable, conditional and hypothetical.

Recoverable land based resources of Cu and Mn 420 Mt and 170 Mt in India.
No recoverable primary resources of Co and Ni (strategic metals)
Indian Ocean nodules: A potential reservoir of Co and Ni (0.6 Mt of Co and 3 Mt of Ni)
Morphology of the ocean ferromanganese nodules is illustrated in fig. 22.1
Fig. 22.1: Morphology of ocean ferromanganese nodules
1
Why bioprocessing?
Chemical leaching Bioleaching
Requires high temperature and pressure Generally ambient temperature

Fast kinetics Kinetics relatively slow in most of the
cases (but higher leaching kinetics can be
attained through use of thermophiles)
Costly and non recyclable reagents Low operating costs
Byproducts often environmentally unsafe Environment friendly
Ocean nodules harbor a consortia of microorganisms, a significant part of them are Mn (IV)
reducing in nature. Use of isolates inhabiting ocean nodules would be beneficial for recovering
the valuable metals such as copper, nickel and cobalt.
Average chemical composition (percent) of the nodules as a function of size fractions is

illustrated below:
Size (m) Cu Co Ni Fe Mn
-300 + 150 1.3 0.13 1.2 5.6 23

-150 + 100 1.1 0.12 1.0 5.5 20
-100 + 75 10 0.12 1.0 5.6 17
-75 + 50 1.1 0.15 1.2 6.8 22
-50 +36 1.1 0.18 1.2 6.4 23
Indian Ocean 0.1- 0.1- 0.3- 4.5- 11.8-

nodules 1 0.3 1.4 11.2 20.2
Core 0.3 0.02 0.2 2.6 4.3

Outer Layer 0.9 0.1 0.7 4.4 10.3
2
Bioreduction of Manganese
Manganese nodules are essentially a conglomerate of transition metal oxides having Mn (IV) and
/ or Fe (III) oxide as major phases. Manganese-reducing heterotrophs can be used as potential
bioleaching agents. A variety of organisms have the ability to reduce Mn (IV). Some bacteria
can reduce Mn(IV) oxide either aerobically or anaerobically, while others reduce it only
anaerobically. [127].
Ehrlich has proposed a model to explain how bacteria (Bacillus) reduce MnO2 either aerobically
or anaerobically. Direct physical contact between bacteria and the MnO 2 particle surface is
essential.
Mn++ present on the cell surface reacts with MnO2 raising the oxidation state from +2 to +3.
Electron transport from bacterial electron donors through enzymes, reducing to Mn++.
Most of the reduced Mn++is released into environment; some bound to the cell envelope
Some bacteria and many of the Mn-reducing fungi reduce it nonenzymatically. Metabolic
products can act as strong reductants for manganic oxides.
3
Microorganisms useful in reduction of manganic oxides are given table 22.1.
Table 22.1: Microorganisms useful in reduction of manganic oxides
Acidithiobacillus thiooxidans Reduction by S0 or thiosulfate S2O3- -

Shewanella putrefaciens Reduction by biogenic species, H2S, sulfide and
Fe+ +.
Reduction by partially reduced sulfur species,
thiosulfate or sulfite.
Reduction by SO3 - - produced from S0
Geobacter metallireducens Reduction by acetate produced biologically
Vibrio Mn (IV) used as terminal electron acceptor.
Desulphovibrio desulfuricans, Mn (IV) acts as electron acceptor. Reduction by

Desulfobacterium autotrophicum, H2S and sulfides.
Desulfuromonas acetoxidans,
Geobacter metallireducens
3H+ + HCOO - + MnO2 = Mn+ + + CO2 + 2H2O
A bacterial metabolic product such as pyruvate can also react nonenzymatically with Mn oxide at
acidic pH. Sulfate reducers produce H2S from sulfate anaerobically and Shewanella putrefaciens
can produce H2S and sulfite from thiosulfate and Fe++ from Fe+++ . Biogenic H2S, sulfite, and
Fe+ + can reduce MnO2.
Microbial mobilization of manganese [127]
Since manganic oxides are insoluble at neutral pH range, manganese is not available directly as
micronutrients to soil organisms. Manganese in higher oxidation states must be reduced to
soluble Mn (II) to be used microbially. Mn (IV) in soils can be solubilized by elemental sulfur or
4
thiosulfate produced by At.thiooxidans. Ferrous iron and H2S produced in microbial iron (III)
and sulfate reduction can chemically reduce Mn (IV) oxides in ores and sediments At.thiooxidans
can thus be used to extract Mn from the manganiferrous ores. The reducing activity of partially
reduced sulfur species such as thiosulfate and sulfite, generated by the bacteria can lead to
reductive dissolution of Mn (IV).
Apart from autotrophs, heterotrophic organisms can also dissolve MnO 2.
Mn-reducing bacteria have also been isolated from seawater, marine sediment, and
ferromanganese nodules.
H2S produced anerobically by sulfate-reducing bacteria present in seawater can reduce manganic
oxides nonenzymatically.
MnO2 + HS- + 3H+ = Mn+ + + 2H2O + S0
In the presence of MnO2, H2S is rapidly oxidized at S0
MnO2 + H2S = S0 + Mn+ + + 2OH-
Biohydrometallurgy of manganese nodules [128-131]
Konishi et al. (1997) studied bioleaching of Pacific ocean nodules using thermophilic and
mesophilic sulfur-oxidizing bacteria. Leachablity of various S-oxidizing bacteria such as
At.ferrooxidans, At. thiooxidans and A. brierleyi under similar conditions were compared.
Leaching kinetics as well as recovery of metals was much higher in presence of a thermophile
such as A. brierleyi at 650C. At optimum conditions, using A.brierleyi, both copper and zinc
were totally leached out within 4 days while in 10 days 85% nickel, 70% cobalt and 55%
manganese were solubilised.
Mukherjee et. al. (2004) isolated heterotrophic Gam-positive Mn-reducing organisms from
Indian ocean nodules and recovered 30% Cu and Ni, 50% Co at room temperature at neutral pH
within a time period of 4h using the spent liquor of the fully grown culture as lixiviant. Leaching
is due to combination of two separate processes growth of the isolate under sterile conditions
5
and chemical leaching by the spent liquor. To accelerate the leaching process and to improve the
recovery of major metals, organic reagents (were added) to the spent liquor of the marine isolate.
With the addition of 10- 3
M concentration of ascorbic acid to the spent liquor of the marine
isolate, 70% Co, 60% Cu, and 60% Ni could b e extracted.
Some experimental details of a novel bioleaching process using a marine bacterium isolated from
the nodules and the spent growth medium from its fully grown culture are detailed below:
Isolation of marine organism from Indian Ocean nodules
Nodules boiled in water for 30 minutes.

Aseptically crushed in sterilized mortar and pestle.
1-2 g added to 20 mL of Artificial sea water (ASW)
nutrient broth.
Incubated at 300C for 24 hours.
Streaked on ASW nutrient agar and incubated
at 370C for 24 hours.
Round, smooth surface colonies grow.
The marine bacterial isolate was used in the bioleaching of Indian ocean nodules.
45% Co dissolution at pH 8.20 in two hours, comparable to that in 2.5M HCI Solutions.
Similar metal recovery trend in growing culture and the spent liquor of the isolate.
Metabolic products of the marine isolate may be responsible for leaching.
Metal dissolution was independent of pulp density upto 10%.
Percent metal leached with modified spent liquor and synthetic catechol is shown in table 22.2.
6
Table 22.2: Percent metal leached with modified spent liquor and synthetic catechol
(pH 8.2, time period 4 h, pulp density 1% size fraction -74, +50 m)
Leaching
Co Cu Ni Mn Fe
agent
Spent liquor 45 23 26 20 40
concentrate
Protein free 40 27 24 23 42
spent liquor
Synthetic 43 20 20 24 45
Catechol
Spent liquor 45 25 25 20 40
The spent liquor of the marine isolate contains siderophore like compounds, which are
capable of complexing transition metals at near neutral pH.
These organic complexants form soluble Fe(III) complexes dissolving Co species associated
with Fe (III) oxide phase .
Process modification to enhance metal recovery
Multistep leaching:
Leaching in spent liquor of marine isolate followed by
Spent liquor of the marine isolate
pH 2 sulfuric acid solutions
Spent liquor of Acidithiobacillus thiooxidans
7
Leaching with organic acids in spent liquor

Ascorbic acid
Salicylic acid
Oxalic acid
Na Lactate
Leaching by starch solutions

Leaching with aqueous solutions of starch
0.5% to 10% starch in spent liquor
Results from a four-step leaching process are given table 22.3.
Table 22.3: Percent metal leaching in multiple stage bioleaching process

(pH 8.2, each cycle for 4 h, pulp density 1%, size fraction -74, + 50 m.)
Cycles Co Cu Ni Fe Mn
A 45 25 23 50 27
B 23 17 16 24 18
C 12 12 12 11 13
D 5 9 8 5 12
A + B +C + D 85 63 59 90 70
Cycle A : Leaching in spent liquor of marine isolate

Cycle B : Releaching the residue from cycle A
Cycle C : Releaching the residue from cycle B
Cycle D : Releaching the residue from cycle C
8
About 85% Co, 90% Cu and 60% Ni could be dissolved in two-stage leaching where the
bioleached residue was further treated with the acidic metabolites from the growth of
Acidithiobacillus thiooxidans in the second cycle.
Effect of addition of 0.1 10% of an organic reductant such as starch to the spent growth
medium was also studied. About 80-85% extraction of copper, cobalt and nickel could be
achieved at a starch concentration of 3%. (table 22.4).
Table 22.4: Percent leaching with spent liquor in presence of starch

(Pulp density 1%, size fraction -74, +50 m, time period 4 h, pH 8.2)
Starch in spent liquor Co Cu Ni Fe Mn
0.1 % 65 35 35 30 50
1.0 % 80 80 65 85 85
3% 80 83 83 85 80
5% 84 85 86 89 87
10% 85 87 88 85 88
Control 45 20 28 25 50
Four-stage leaching yielded 80-85% Cu, Co and Ni dissolution in spent liquor.

Sodium salicylate and ascorbic acid added to the spent liquor of marine isolate had an
additive effect on metal recovery while Na-lactate and oxalic acid had no such effect.
Metal dissolution increased with increase in concentration of starch in spent liquor upto 3%,
thereafter the dissolution stabilized.
With 3% starch in the spent liquor, 80-85% Cu, Co and Ni were dissolved.
9
References:
127. H. L. Ehrlich, Geomicrobiology Marcel Dekker, N.Y., (1996).

128.Y. Konishi, S. Asai, and Y. Sawada, Leaching of marine manganese nodules by acidophilic
bacteria growing on elemental sulfur, Metall. Mater. Trans., 28B, (1997), pp. 25-32.
129.Mukherjee, A.M. Raichur, J.M. Modak and K.A. Natarajan, Bioprocessing of Indian
Ocean nodules using a marine isolate-effect of organics, Minerals engg. 16, (2003), pp.
651-657.
130.Mukherjee, A.M. Raichur, J.M. Modak and K.A. Natarajan, Recent developments in
processing ocean manganese nodules A critical Review, Mineral Proc. Ext. Met. Rev. 25,
(2004), pp. 91-127.
131.Mukherjee, A.M. Raichur, J.M. Modak and K.A. Natarajan, Exploring process options to
enhance metal dissolution in bioleaching of India Ocean nodules, J. Chemical Technology
and Biotechnology, 79, (2004), pp. 512 517.
10
Lecture 23:Bioprocessing Of Industrial Wastes NPTEL Web Course
Lecture 23
Bioprocessing Of Industrial Wastes
Keywords: Industrial Wastes, Bioprocessing, Leaching Bacteria
Applications of biohydrometallurgy for extraction of metals from primary ore deposits

have already been discussed. Yet another promising application of bioleaching is
bioprocessing of industrial and other wastes to recover valuable metals and also for value
addition. [132 147]
There are several types of hazardous and nonhazardous wastes generated by industries.
Petroleum processing Organic wastes, spent catalysts and waste

sludges.
Electronics and software Electronic scrap, computer circuits, waste and
discarded batteries.
Mining and metals production Slags and tailings, mined wastes, Flue dust,
organic and inorganic containing solid-liquid
wastes.
Metals processing Scraps and sludges, cyanides, paint waste
Leather processing Organics, Chromium-based chemicals and
effluents.
Printing Waste solvents, metal containing solutions
Chemicals Acids and bases, organic and inorganic
solvents.
Numerous industries such as metal-finishing, electronic, Nonferrous, steel and coal production,
petrochemicals, pharmaceuticals and electroplating generate huge quantities of heavy metal
1
containing toxic wastes. Any developed process to treat such wastes should be cost-effective,
energy-efficient and environment-friendly.
Bioprocessing possesses all the above attributes. Another beneficial aspect of bioprocessing for
treatment of wastes is that valuable metals can be efficiently recovered with simultaneous
detoxification of the processed wastes. For example, most of industrial wastes contain metals
such as gold, platinum, copper, nickel, molybdenum, vanadium, cobalt, chromium, zinc and
cadmium. Used catalysts from chemical and petroleum refineries contain valuable metals such
as nickel, vanadium, cobalt and molybdenum. Treatment of fly ash generated from coal-based
thermal plants is yet another serious problem. Discarded electronic components contain precious
metals such as gold and silver, besides copper and cobalt. [136 147]
Both solid and liquid wastes can be bioprocessed. As discussed before, microbial cells and their
biomass possess capabilities to accumulate, concentrate, detoxify and transport various metal and
other organic and inorganic pollutants. Various biologically promoted functions useful in waste
processing include
Biosorption and Bioaccumulation

Biodissolution through generation of lixiviants
Biotransformation (oxidation-reduction, precipitation)
All the above can be characterized as green technology.
Microorganisms relevant to bioleaching and bioremediation have been discussed already. Many
of the organisms used in biohydrometallurgy find application also in waste processing.
Acidophilic bacteria Acidithiobacillus ferrooxidans

Archea Sulfolobus and other thermophiles
Fungi Aspergillus sp
Penicillium sp
2
Reported studies dealing with bioleaching of some industrial wastes are summarized below:
1. Used batteries Acidithiobacillus Cadmium, nickel, lithium,

cobalt
2. Spent catalysts Acidithiobacillus Cobalt, nickel, vanadium,
molybdenum
3. Tannery wastes Acidithiobacillus, Chromium
Heterotrophs
4. Electronic scrap Acidithiobacillus, Copper, nickel, zinc
Thermophilic archea, Fungi
5. Fly ash Acidithiobacillus, Fungi such Copper, cadmium, zinc,
as Aspergillus aluminium
6. Automobile parts, Bacillus spp. Fungi Precious metals, copper,
Jewellery wastes nickel, chromium
Similarly, there are reported laboratory studies on the bioleaching of nonferrous, blast furnace
and steel slags using Acidithiobacillus spp, archaea and Aspergillus spp to recover valuable
metals such as copper and zinc. Industrial waste sludge, tailings and aluminium-industry red
mud also could be bioleached using acidophilic bacteria and heterotrophs to recovery metals and
to render the wastes environmentally acceptable.
Other than bioleaching, biosorption using biosorbents can be used to remover (or recover) metals
from wastes. Waste biomass of fungi, algae and bacteria could be used for biosorption of
precious metals, such as gold, platinum, palladium and silver from liquid wastes.
3
Bacteria Bacillus subtilis

Desulfovibrio spp.
Streptomyces spp.
Fungi / Yeasts Aspergillus sp

Fusarium sp
Rhizopus arrhizus
Saccharomyces cerevisiae
Cladosporium sp.
Algae Chlorella vulgaris

Sargassum natans
Alginates
Bioproteins from biomass
Biologically
derived bioreagents
Use of immobilized and preconditioned biomass would be more reactive, having larger surface
area. Column and stirred bioreactors could be used in series or parallel to achieve biosorption in
counter-current circuits. Adsorbed metals could be desorbed and recovered through elution.
Used solutions and biomass could be recovered and recycled with economical benefits.
4
References:
132. H. Eccles, Removal of heavy metals from effluent streams- Why select a biological
process? International Biodeterioration and Biodegradation, (1995), 35, 5-16.
133. A.Seidel, Zimmels, R. Armon, Mechanism of bioleaching of coal fly ash by Thiobacillus
thiooxidans. Chemical Engineering Journal, (2001), 83, 123-130
134.T. J. Xu, Y.P Ting, Fungi bioleaching of incineration fly ash: metal extraction and modeling
growth kinetics. Enzyme and Microbial Technology, (2009), 44, 323-328.
135.D. Fang, L.Zhao, Z.Q. Yang, H.X. Shan, Y. Gao and Q.Yang, Effect of sulfur concentration
on bioleaching of heavy metals from contaminated dredged sediments. Environmental
Technology, (2007), 147, 319-324.
136.S.Ilyas, C. Ruan, H.N. Bhatti, M.A Ghauri, M.A. Anwar, Column bioleaching of metals
from electronic scrap, Hydrometallurgy, (2010, 101, 135-140.
137.H.Brandl, R. Bosshard, M.Wegmann, Computer-munching microbes: Metal leaching from
electronic scrap by bacteria and fungi, Hydrometallurgy. (2001), 59, 319-326.
138.T. Yang, Z. Xu, J. Wen, L. Yang, Factors influencing bioleaching copper from waste printed
circuit board by Acidithiobacillus ferrooxidans, Hydrometallurgy, (2009), 97, 29-32.
139.D. Mishra, D.J Kim, D.E. Ralph, J.G Ahn and J.G Rhee, Bioleaching of metals from spent
lithium ion secondary batteries using Acidithiobacillus ferrooxidans, Waste Management,
(2008), 28, 333-338.
140.B.Xin, D. Zhang, X. Zhang, Y. Xia, F. Wu, S.Chen and L. Li, Bioleaching mechanism of
Co and Li from spent lithium-ion battery by the mixed culture of acidophilic sulfur-
oxidizing and iron-oxidizing bacteria. Bioresource Technology, (2009), 100, 6163-6169.
141.D. Mishra, D.J Kim, D.E. Ralph, J.G Ahn and Y.H Rhee, Bioleaching of vanadium rich
spent refinery catalysts using sulfur oxidizing lithotrophs, Hydrometallurgy, (2005), 88,
202-209.
142.V. Bosio, M. Viera, E. Donati, Integrated bacterial process for the treatment of a spent
nickel catalyst. Journal of Hazardous Materials, (2008), 154, 804810.
5
143.D. Mishra, D.J. Kim, D.E. Ralph, J.G. Ahn and Y.H Rhee, Bioleaching of spent hydro-
processing catalyst using acidophilic bacteria and its kinetics aspect. Journal of Hazardous
Materials, (2008), 152, 1082-1091.
144.D. Saanthiya and Y.P Ting, Bioleaching of spent refinery processing catalyst using
Aspergillus niger with high-yield oxalic acid. Journal of Biotechnology, (2005), 116, 171-
184.
145.D. Mishra and Y.H Rhee, Current research trends of microbiological leaching for metal
recovery from industrial wastes, Current Research, Technology and Education, Topics in
Applied Microbiology and Microbial Biotechnology, (2010).
146.J.C. Lee and B.D Pandey, Bioprocessing of solid wastes and secondary resources for metal
extraction-A review, Waste Management 32, (2012), 3-18.
147.J. Cui and L. Zhang, Metallurgical recovery of metals from electronic waste: A review, J.
Hazardous Materials, 158 (2008) 228-256.
6
Lecture 24: Electrochemistry Of Sulfide Minerals Will Respect To Bioleaching NPTEL Web Course
Lecture 24
Electrochemistry Of Sulfide Minerals With Respect To Bioleaching
Keywords: Electrochemistry, Galvanic Series Of Sulfides, Eh-pH diagrams
Many countries in the world are utilizing bioleaching processes to recover metals from a variety
of ores. Of the numerous potential applications for microbial processing, only a few, notably
heap leaching of copper and uranium and the bioreactor processing of gold and cobaltic sulfide
concentrates are currently used industrially. Heap and insitu leaching are being practiced for
copper, and uranium. About 20-25% of copper, 15% of uranium and 10-15% of gold production
around the world are attributed to bioleaching.
The most important organism for the leaching of sulfide minerals is Acidithiobacillus
ferrooxidans. It is a Gram-negative, acidophilic, mesophilic, chemolithotroph which has the
ability to oxidize complex sulfide minerals. Since it has a special ability to utilize electrons
released in the oxidation of iron or sulphur, it can oxidize virtually any sulfide mineral such as
that of iron, copper, nickel and zinc. It is also reported that At. ferrooxidans oxidize a number of
metal sulfides namely arsenopyrite, bornite, Chalcocite, chalcopyrite, covellite, galena enargite
(3Cu2S, As2S5), millerite (NiS), orpiment (As2S3), pyrite, marcasite, sphalerite (ZnS),
stibnite(Sb2S3) and tetrahedrite (Cu8Sb2S7).
However, the most common practical difficulty associated with bioleaching processes in the
presence of mesophilic acidophiles such as At.ferrooxidans, is the slower rate of biodissolution.
The slowness of the biooxidation reactions and also the practical difficulties associated with the
harvesting of sufficient biomass of the bacteria (At.ferrooxidans), have limited the scope and
wider commercial utilization of biohydrometallurgy. Bacterial activity and cell biomass are the
two most important parameters influencing biodissolution rates. Electrobioleaching, a new
concept in biohydrometallurgy could prove to be an efficient method to speed up bioleaching
rates in the presence of At.ferrooxidans. Electrobioleaching can be defined as a biodissolution
process carried out in the simultaneous presence of both microorganisms and applied potentials
(currents). In this process, a number of mutually complementary as well as divergent
1
mechanisms, such as chemical, biochemical, electrochemical and galvanic interactions come into
play. Galvanic interactions in the bioleaching of mixed multisulfides could be taken advantage
of in achieving selective dissolution of the desired mineral.
Electrochemistry of sulfide minerals
Before discussing the role of applied DC potentials on the dissolution behaviour of different
sulfide minerals, it is essential to understand their basic electrochemical behavior in a leaching
medium.
Significance of measured rest potentials in assessing galvanic interactions between different

sulfide mineral combinations present in a leaching system has been recognized. Various sulfide
minerals such as pyrite, chalcopyrite, galena, pentlandite, sphalerite and pyrrhotite can be
arranged in the form of a galvanic series in an acid medium with reference to their relative
activities as shown in table 24.1.
Table 24.1. Galvanic series of some metal sulphides in bioleaching

medium
Pyrite Noble
Chalcopyrite
Pentlandite
Galena
Pyrrhotite
Sphalerite Active
Based on such a galvanic series, the electrochemical behaviour of one sulfide mineral in contact
with another can readily be predicted. Such galvanic interactions can accelerate the oxidation
(dissolution) of one or more members of an electrochemically coupled system, while cathodic
protection (reduction) would be conferred on other nobler minerals. For example, in a
chalcopyrite-pyrite couple, chalcopyrite has the lower rest potential and becomes the anode,
2
while pyrite behaves as a cathode. As a result, chalcopyrite (anodic mineral) undergoes rapid
dissolution while the nobler pyrite remains essentially unaffected. Electrochemical dissolution
behaviour of the sulfide minerals having intermediate rest potential values such as pentlandite,
galena, sphalerite and pyrrhotite depends on the relative activity of the mineral with which they
are in close contact. For example, chalcopyrite while behaving anodically in contact with pyrite,
acts as a cathode, if placed in the vicinity of pentlandite, galena or sphalerite. Minerals such as
pyrite and sphalerite occuping the top and bottom positions in the galvanic series, will always
behave cathodic and anodic, respectively, irrespective of the mineral combination. Pyrite is
cathodic and sphalerite is anodic under all types of combinations (Fig. 24.1).
Pyrite Chalcopyrite Galena Sphalerite
Cathode Anode Anode Anode
Cathode Cathode
Fig.24.1 Multisulfide mineral electrode galvanic cells consisting of pyrite, chalcopyrite, galena and Sphalerite.
Anode and cathode direction of arrow indicate relative anodic and cathodic behaviour of intermediate
minerals with respect to the mineral in the immediate vicinity.
The following major factors influence the rate of galvanic interactions among sulfide minerals in
a bioleaching medium.
Relative surface areas between anode and cathode (area effect)

Interelectrode distance (distance effect)
Nature and duration of contact.
Conductivity of the mineral and electrolyte.
3
Solution properties such as pH, presence or absence of O2 (oxidising agents) and presence of
other cations and anions.
Presence or absence of microorganisms.
Rest potential of the sulfide minerals as well as Eh (redox potential) of the leaching medium are
the two important electrochemical parameters. A large difference between Eh of the medium
and the rest potential of the sulfides must exist for efficient mineral dissolution to proceed.
Optimum Eh for bacterial activity and oxidation of the desired sulfide has to be established.
During the growth of At.ferrooxidans Eh values in the range of 700 to 800V are attained at a pH
of about 2 in aerated bioleaching solutions.
Fe++ = Fe+++ + e
Eh = 0.771 + 0.059 log
The presence of the bacteria further increases the Eh of the system containing ferrous and ferric
ions.
Eh-pH diagrams can readily be constructed to portray the growth and stability regions for
At.ferrooxidans, with respect to different ferric-ferrous ratios, and thus optimum Eh values
promoting bacterial leaching could be established. It has been observed that during growth of
At.ferrooxidans, Eh is in the range of 600-800mV in the pH region of 1.5 to 3.0. The stability
limits for the important phases involved in a chalcopyrite bioleaching system are represented in
the simplified Eh-pH diagram in Fig 24.2. Measured Eh values from a chalcopyrite bioleaching
flask in the presence and absence of At.ferrooxidans are also indicated in the diagram. The Eh
values are higher for chalcopyrite leaching in the presence of bacteria indicating the contribution
of microorganisms towards maintaining a higher oxidation potential to facilitate faster mineral
dissolution. Eh values are lower in the absence of bacteria due to lack of ferrous ion oxidation.
4
Fig. 24.2 Measured rest potentials in the presence (closed squares) and absence (open squares) of
At.ferrooxidans in the bioleaching of chalcopyrite.
Measured rest potential values for pyrite, chalcopyrite, galena and sphalerite in a bioleaching
medium in the presence and absence of At.ferrooxidans are given in table 24.2. Increase in
measured potentials in the presence of At.ferrooxidans could be seen due to enhanced oxidation
of ferrous iron.
5
Table 24.2. Measured rest potentials for some mineral sulphides in 0.9K medium
Rest potential, Eh, mV
Mineral Uninoculated Inoculated iron-free Inoculated iron-
medium containing medium
Pyrite 545 600 700

Chalcopyrite 450 500 570
Galena 250 260 420
Sphalerite 200 220 230
6
Lecture 25: Galvanic And Electrobioleaching Of Sulfide Minerals NPTEL Web Course
Lecture 25
Galvanic And Electrobioleaching Of Sulfide Minerals
Keywords: Galvanic Interactions, Multimetal Sulfides, Electrobioreactors
Effect of galvanic interactions in multi-sulfide mineral dissolution. [149 150]

Types of mineral contacts play an important role in the process of natural mineral oxidation. The
rate of oxidation of many natural sulfide minerals is greatly increased by the presence of pyrite
under environmental conditions. The rate of chalcopyrite dissolution was found to be
accelerated in the presence of pyrite, molybdenite and stibnite, but was retarded in the presence
of galena, which were present as impurities along with chalcopyrite. Galvanic interaction
between chalcopyrite and copper can lead to rapid conversion of chalcopyrite to chalcocite and
covellite.
The measured rest potentials of sulfide minerals could vary depending on the type of solution,
pH, nature of the impurities in the mineral as well as solution purity. For example, the rest
potential of sphalerite will be influenced by its iron content and other impurity contents.
The presence of At.ferrooxidans further catalyses such galvanic reactions in bioleaching systems
and the combined effect of such bio-and electrochemical reactions could be taken advantage of
in the selective bioleaching of multisulfides. For example, in a chalcopyrite-pyrite combination,
galvanic interactions will facilitate chalcopyrite to dissolve more rapidly than pyrite, which will
be passivated and the presence of At.ferrooxidans will catalyse the above reaction by
continuously oxidising the film of elemental sulphur formed on anodic mineral surfaces. In the
absence of bacterial oxidation, the elemental sulfur would hinder efficient electron transfer. In
the presence of pyrite and microorganisms, the dissolution rate of chalcopyrite and sphalerite
would be significantly enhanced. The bacteria were found to attack sphalerite and chalcopyrite
in preference to pyrite and the microbes did not randomly attack the sulfide minerals. Similarly,
more of zinc was found to be leached out in the bacterial leaching of a bulk flotation concentrate
1
containing Pb-Zn-Cu sulfides, due to the differences in the electrochemical properties of the
sulfide minerals involved (Fig. 25.1).
100 5
80 4
60 3
% Zinc
% Cu
40 2
20 1
0 0
0 20 40 60
Period, d
Fig. 25.1: Bacterial leaching of a Lead-Zinc-Copper Sulfide Ore
Arsenopyrite which was found to be more active than chalcopyrite could be selectively leached
out using At.ferrooxidans from an ore or concentrate containing the above two sulfide
combinations. Break down of arsenopyrite takes place first and that of chalcopyrite takes place
only after all the arsenopyrite has been oxidized.
Contribution of galvanic interactions to the acid bacterial leaching of mixed metal sulfides has
been studied in the presence and absence of At.ferrooxidans. Coupled chalcopyrite/pyrite,
sphalerite/pyrite and chalcopyrite/pyrite/sphalerite systems showed galvanic behavior unlike
leaching of single mineral systems. Dissolution of copper was observed to increase significantly
in the galvanic leaching of chalcopyrite/pyrite mixtures as compared to individual chalcopyrite
leaching. When bacteria were present, copper dissolution further increased by an additional
factor of 2 in the chalcopyrite/pyrite system. Similarly, from a complex ore containing about 8%
zinc, 6% lead and 1% copper, almost 99.5% of the zinc could be selectively bioleached, while
only 2% of the copper was solubilized (Fig. 25.1). Enhanced selective dissolution of pyrrhotite
2
after contacting with chalcopyrite could also be observed. As the proportion of pyrrhotite
content in the mixture with chalcopyrite was increased, copper dissolution decreased
significantly. From an ore containing 0.8% copper, 0.2% nickel, 11.9% iron and 2% sulphur, as
chalcopyrite-cubanite, pentlandite and pyrrhotite, only less than about 2% copper was
solubilized, while about 25% nickel was bioleached after 40 days. Nickel releases into the
solution were rapid and in all cases took place, before copper appeared in solution and also
nickel concentrations in solutions were several times higher than those of copper. Copper
concentration in solution was observed to increase only after most of the nickel has been
solubilized. Rest potential measurements in the bioleaching medium indicated that pentlandite
and pyrrhotite are anodic to chalcopyrite-cubanite; and selective nickel dissolution in preference
to copper could be expected. The bioleaching of copper from different binary and ternary
combinations were also studied. Since chalcopyrite is anodic to nobler pyrite, selective leaching
of chalcopyrite when contacted with pyrite could occur. On the otherhand, dissolution of copper
from chalcopyrite when contacted with either galena or sphalerite was found to be significantly
lower than that obtained from a chalcopyrite-pyrite contact. Larger potential differences among
the sulfide minerals promote enhanced selective dissolution of the anodic mineral (Fig. 25.2).
Fig. 25.2 Copper bioleached from chalcopyrite when contacted with equal weight of pyrite, galena and
sphalerite
3
Influence of sulfide mineral ratios
Studies have also indicated that At.ferrooxidans can utilize sulfides even in the absence of iron.
Dissolution studies in the presence of the bacteria with chemically prepared ZnS and CuS
indicated that the dissolution of zinc was enhanced in the presence of CuS, while copper
dissolution decreased. Another important factor on which the rate of selective anodic sulfide
mineral dissolution is dependent on is the ratio of mixing of two or more minerals. Galvanic
dissolution is dependent on the relative surface areas of the anodic and cathodic surfaces exposed
to bacterial leaching.
To prove the above relative surface area concept, chalcopyrite concentrates were mixed in
different ratios by weight with a sphalerite concentrate and the copper and zinc dissolution were
monitored in the presence and absence of At.ferrooxidans (Fig. 25.3). The results are illustrated
in table 25.1. Zinc dissolution increase with increase in chalcopyrite in the mixture, while
copper dissolution decreased with increase in sphalerite. Although the presence of bacteria
enhances the dissolution of both copper and zinc, zinc dissolution is found to be selectively
preferred galvanically. There is an optimum ratio of mixing of the above two concentrates when
zinc dissolution is the highest with negligible copper dissolution.
4
80
60
e
rit
Py
ith
W
% Zn
40
With Chalcopyrite
20
With Galena
0
0 20 40 60 80 100
Period, d
Fig. 25.3: Percent zinc bioleached from sphalerite when contacted with equal amount of pyrite,
chalcopyrite and galena.
Table 25.1: Copper and zinc leaching from chalcopyrite (Cp) sphalerite (Sp) concentrates
(At.ferrooxidans)
Percent Zn Percent Cu
Cp (g) Sp (g) Control Inoculated Control Inoculated
9 1 10 91 7 19
8 2 90 17
6 4 89 12
5 5 10.8 89 7 5
2 8 88 5
The significance of the ratio of mixing on the rate of leaching of a desirable mineral in a mixture
containing various sulfide minerals assumes industrial relevance. In a mixture of chalcopyrite
and pyrrhotite, the amount of copper dissolved was found to decrease with increase in pyrrhotite.
5
With increase in pyrrhotite-to-chalcopyrite ratio, copper recovery was found to increase initially,
reaching a maximum and then steadily decreased to attain practically zero, for ratios higher than
20-to-1.
Copper dissolution from pyrite-chalcopyrite mixtures was faster than that from pyrrhotite-
chalcopyrite or from pure chalcopyrite since the galvanic effect is favourable for copper
dissolution in the presence of pyrite. The dissolution behaviour of sphalerite in binary contact
with pyrite, chalcopyrite and galena at two different ratios of mixing of the pure mineral
samples, namely 5:5 and 5:3 by weight was studied (Fig. 25.3). Zinc dissolution from sphalerite
in various binary combinations follows the galvanic mechanism. The percent zinc dissolved was
the highest from a sphalerite-pyrite couple followed by that from a sphalerite-chalcopyrite
combination. About 70% of zinc could be dissolved in the presence of At.ferrooxidans, from a
1:1 mixture of sphalerite and pyrite, as opposed to only about 20 25% zinc dissolution from
sphalerite contacted with chalcopyrite under similar conditions. Only about 5% of zinc was
solubilized from the sphalerite-galena couple. The presence of bacteria and the ratio of mixing
(relative surface areas) influenced the galvanic dissolution of active sphalerite. Also, zinc
dissolution from sphalerite was higher from a 1:1 mixture compared to a 5:3 mixture in different
binary combinations.
For maintaining the galvanic interactions in a multisulfide leaching system, intimate and
continuous mineral-mineral contacts need be ensured. In stirred tank leaching of fine particles,
making and breaking of such contacts take place continuously.
Role of applied potentials in the presence of microorganisms [160, 162]
The dissolution behaviour of different sulfide minerals in an acid medium can be controlled
through application of appropriate DC potentials or currents. For example, sphalerite can be
selectively dissolved from a mixture of chalcopyrite and sphalerite under an applied potential in
a leaching medium.
Electroleaching of metal sulfides, such as nickel sulfide, copper sulfides, lead sulfides and zinc
sulfides has been studied.
6
Biomass of At.ferrooxidans can be significantly enhanced through electrochemical means. For

example, At.ferrooxidans can be grown in an electrolytic bioreactor containing ferrous sulphate
medium. When a cathodic current is passed through the reactor, the bacterially generated ferric
iron would be reduced to the ferrous state, which provides the energy source for the bacteria in
situ. In such an electrochemical bioreactor, continuous external addition of ferrous sulfate thus
becomes unnecessary. Under electrolytic growth conditions, the protein concentration was
found to increase by about 4 times and the protein (cell) production rate by a factor of 7. From
the results illustrated in tables 25.2 and 25.3, advantages of electrolytic growth of
At.ferrooxidans become clear.
Table 25.2: A comparison of electrolytic growth parameters of At.ferrooxidans
Parameters Electrolytic adaptation Unadapted
Iron oxidation rate (mg/l h) 140 130

Protein concentration (mg/l) 30 25
Protein production rate (mg/l h) 3 2
Table 25.3: Minimum generation times for growth of At.ferrooxidans
Conditions Generation time (hours)
Non-electrolytic, unadapted 21
Non-electrolytic, adapted 23
Electrolytic, unadapted 8
Electrolytic, adapted 6
Prior bacterial adaptation to electrolytic conditions increased the maximum iron concentration,
protein production and the yield coefficient. The generation time of the bacteria was also found
to be affected by electrolytic growth conditions. Minimum generation time for various bacterial
7
growth conditions are shown in table 25.3. Adaptation to electrolytic growth conditions
significantly decreased the generation time.
Typical electrochemical reactor system used for this purpose is shown in Fig 25.4. The catholyte
containing ferrous sulfate and bacterial cells is pumped into the cathode compartment. The
acidic anolyte is contained in the anode compartment.
Fig. 25.4: Electrochemical bioreactor system for enhanced growth of At.ferrooxidans
Electrochemical aspects of such a reactor is illustrated in fig.25.5.
Fig. 25.5: Electrochemical reactions in an Electro-bioreactor.
8
Electrobioleaching of sulfide minerals [161-162]
Typical laboratory arrangement for an Electrobioleaching setup is illustrated in fig. 25.6.
Fig 25.6: Electrochemical cell arrangement used in bioleaching under applied potentials
Application of such an in-situ electrolysis resulted in a significant increase in cell mass unlike
that could be achieved in a batch culture without electrolysis. The effect of applied DC
potentials on the activity and growth of At.ferrooxidans has also been investigated. The
application of positive potential upto +1000mV in an acid bioleaching medium was found to be
detrimental to bacterial activity; whereas the application of negative potentials enhanced both the
activity and growth through electrochemical regeneration of ferrous iron (Fig. 25.7 and 25.8).
9
100
A. Metabolite in the absence of cells
B. Metabolite + cells
B
80
A
60
++
% Fe
40
20
0
0 20 40 60 80
Period, min
Fig. 25.7: Ferric iron reduction as a function of time in a culture fluid in the presence and absence
of At.ferrooxidans under an applied potential of 800mV.
A. - 500 mV applied potential

B. 9K Medium
6
A
Cell density / mL (10 )
8
2
B
0
0 40 80 120 160
Time, h
Fig 25.8: Variation in cell number with time in a bacterial culture under normal and electrolytic
growth conditions.
10
Lecture 27: Principles Governing Microbe-Mineral Interfacial Phenomena NPTEL Web Course
Lecture 27
Principles Governing Microbe-Mineral Interfacial Phenomena
Keywords: Biomineral Beneficiation, Bioreagents, Cell Surface Hydrophobicity
Utility of various microorganisms in bioleaching was discussed so far. Yet another

application of mining microorganisms is mineral beneficiation. Principles governing
microbe-mineral interfacial phenomena and applicability of microbially-induced mineral
beneficiation are discussed in lectures 27-33. Use of autotrophic and heterotrophic
microorganisms including bacteria, fungi and yeasts is illustrated with respect to
beneficiation of sulfide and oxide minerals [171 226].
Depletion of richer ore deposits coupled with higher demand and consumption for valuable
metals have necessitated the development of alternative efficient beneficiation processes.
Commercial applications of Microbially-mediated mineral extraction processes have been
demonstrated in the extraction of copper, uranium, cobalt and gold. Several microorganisms
have been identified to be involved in natural mineral formation and conversion processes. The
divergent role of microbes in the field of mineral processing starting from mining, beneficiation,
metal extraction to efficient waste disposal has been well recognized. Increased environmental
awareness and stringent regulations have promoted the applicability of biotechnology in mineral
beneficiation.
Utility of microorganisms and bioreagents in many metallurgical applications has become
possible. For example, microbially induced flocculation or flotation of minerals, remediation of
toxic chemicals discharged from mineral processing operations, degradation of cyanide, etc. As
different from bioleaching, biobeneficiation, by definition, refers to selective removal of
undesirable mineral constituents from an ore through interaction with microorganisms or
bioreagents thus enriching it with respect to the desired valuable minerals. Unlike
conventional techniques, microbially induced flotation and flocculation would be more energy
efficient, economically viable and an environmentally benign process for the effective separation
1
of various ore minerals. Conventional beneficiation techniques involve use of various toxic
reagents, which could be safely replaced by environment friendly bioreagents.
Some consequences of microbe-mineral interactions relevant to microbially-induced mineral
beneficiation are
Adhesion to mineral substrates resulting in biofilm formation,
Bio-catalised oxidation and reduction reactions, and
Interaction of metabolic products.
Such biogenic reactions result in surface modification, dissolution of mineral constituents as well
as bioaccumulation and remediation.
Microbially-induced beneficiation is thus brought about by microorganisms and bioreagents,

resulting in surface chemical and physico-chemical changes, such as
Changes in surface chemistry of minerals and surface hydrophobicity.
Generation of surface active chemicals and bioreagents.
Selective dissolution of mineral phases in an ore matrix and
Sorption, accumulation and precipitation of metal ions and products.
Several types of autotrophic and heterotrophic bacteria, fungi, yeasts and algae can be
used in mineral beneficiation processes. Selective dissolution, flotation and flocculation are
some of the processes involved in biobeneficiation.
Microbiological, surface-chemical as well as physico-chemical factors governing microbially-
induced beneficiation are discussed. [179]
Microbially induced mineral beneficiation processes are influenced by surface properties of
bacterial cells, their adhesion behaviour towards minerals and the surface chemical changes of
the minerals consequent to microbe-mineral interaction.
Bacterial cell wall architecture

Structure of the bacterial cell wall plays a significant role in adhesion to mineral surfaces and
resulting consequences.
2
Bacteria are classified into two major categories with respect to cell wall structure, namely,
Gram-positive and Gram-negative. The chemical composition and structural format of cell walls
are different in these two types of bacteria. Gram-positive bacteria usually possess a well
defined, rigid outer cell wall 15-30 nm thick, and an inner, closely applied, cell-limiting plasma
membrane. The wall constitutes 15 to 30% dry weight of the cell and the rigidity is due to major
polymeric component, peptidoglycan. Cell wall may also contain one or more accessory /
secondary polymers such as teichoic acids and teichuronic acids.
The cell walls of Gram-negative bacteria contain a more general structural format. They have an
outer membrane placed above a thin peptidoglycan layer. These lipid-protein bilayer membranes
contain, proteins, phospholipids, and lipopolysaccharides and separates the outer environment
from the periplasm. In between the outer and the plasma membranes, a gel-like matrix (the
periplasm) is observed in the periplasmic space. Different from Gram-positive bacteria cells
where cell membrane is in close association with the undersurface of the cell wall, Gram-
negative bacteria possess a gap between cell and outer cell membrane. The plasma membrane
and the cell wall (outer membrane, peptidoglycan layer, and periplasm) together form the Gram-
negative envelope.
Biologically produced reagents

Both organic and inorganic reagents are generated through bacterial metabolism which are useful
in mineral beneficiation. As shown in Table 27.1, different types of mineral acids, fatty acids,
polymers and chelating agents are generated by bacteria, fungi and yeasts.
A large variety of fatty acids produced by microorganisms have been tested in the flotation of
various ores. The microbial products can function as flotation collectors, depressants or
dispersants.
Many other inorganic and organic surfactants of utility in mineral processing are also secreted by
microorganisms as illustrated in Tables 27.2 and 27.3. Bacterial polysaccharides and enzymes
would be useful in flotation, flocculation and mineral leaching processes.
3
Table 27.1: Metabolic products of different microorganisms

Microbe Functions Useful products
Acidophilic sulphur and Oxidation of mineral Ferric ions, sulphuric acid

iron bacteria sulphides
Nitrifying bacteria Acidulation Nitric acid
Fermenting bacteria Reduction, Dissolution Formic acid
and chelation
Fungi Complexation and Oxalic, citric, formic and
dissolution gluconic acids
Clostridia Acidulation, Dissolution Acetic acid
Heterotrophic bacteria Polymerization, exopolysaccharides,
dispersion; agglomeration bioproteins and organic
and metal solubilization acids.
Table 27.2: Microbial Exoenzymes
Bacteria Type of enzyme
Bacillus subtilis Amylase (starch)

Paenibacillus polymyxa Pectinase (pectin)
Clostridium thermocellus Cellulose
Bacillus megaterium Proteinase (peptides)
4
Table 27. 3: Some Biological Exopolymers

Bacteria Polymer
Bacillus megaterium Polyglutamic acid

Acetobacter xylinum Cellulose
Agrobacterium tumefaciens Glucan
Paenibacillus polymyxa Polysaccharides
Leuconostoc spp. Dextrans
Streptococcus spp.
Bacterial surface charge, electrical double layer and cell surface

hydrophobicity
Most bacterial cells possess an overall negative charge on the surface of their cell wall at netural
pH, due to the presence of peptidoglycan, which is rich in carboxyl and amino groups. Teichoic
acids containing phosphate rich component also contribute to the negative charge. From a
physico-chemical point, microorganisms can be considered living colloidal particles. The cells
acquire charge through the ionization of amino, carboxylate and phosphate groups, which is pH
dependent. Competition among electrical and chemical forces control surface charge
neutralization. Counter ions can bind to such charged groups. An electrical double layer will be
thus established at the interface. The surface chemical properties of microorganisms can be
characterized by zeta potential and the isoelectric point (IEP). In mineral-bacteria-solution
systems as in beneficiation processes, mineral-solution as well as mineral-bacterial cell interfaces
are formed. Bacterial cells can act as living colloidal particles at the mineral-solution interface.
Increasing ionic strength favours bacterial adhesion to a mineral surface. Bacterial dispersion and
flocculation are governed by electrical forces as in the case of mineral particles.
Different amounts of lipids present on the cell wall are responsible for surface hydrophobicity.
Hydrophobicity is attained by hydrophobic molecules present on cell surfaces. Amino groups
5
confer hydrophobicity. Surface hydrophobicity is important in interfacial interactions among

bacterial cells and mineral particles. Hydrophobic bacteria can readily adhere to surfaces due to
repulsion from the polar water molecule. The effect of electrostatic repulsive force decreases
with increasing cell hydrophobicity.
The role of hydrophobicity in bacterial adhesion to mineral surfaces has been studied. Since
surface charge enhances the chances of polar interactions with water molecules, the more
charged the cell surfaces, the less hydrophobic they would be. Any process that decreases the
cell negative charge can enhance its hydrophobic character. Cells having higher hydrophobicity
and lower electrophoretic mobility are more adherent. Cell surfaces are also covered by a large
number of positively and negatively charged moieties, which lower the hydrophobicity. Cell
appendages such as fimbriae and pili are charged.
Cell surface hydrophobicity depends on the chemical composition and architecture of the cell
wall outer layers. Surface proteins and polysaccharides are involved in bacterial adhesion to
minerals. Relatively hydrophobic portions of protein or hydroxyl-deficient polysaccharide
molecules can be differently oriented. Molecules having both hydrophobic and hydrophilic
portions may confer dual properties. Many organisms possess both hydrophilic and hydrophobic
surface regions. Cell surface hydrophobicity is not a permanent property of the cell envelope and
can be modified depending on environmental conditions. Interfaces offer a better environment
for nutrition and growth of organisms. Bacterial adhesion is dependent on the bio - and surface
chemical properties of the microorganism and also on the interfacial properties of the mineral in
an aqueous system. When grown in a liquid medium, the cells acquire appropriate surface
properties to survive in a liquid medium. If grown on a solid substrate, they will alter their cell
wall and membrane characteristics accordingly.
6
Why bacterial adhesion?

Bacterial adhesion to substrates may be a survival strategy allowing microorganisms in a
nutritionally favourable environment such as the mineral-solution interfaces. Many specialized
structures and complex ligand interactions have evolved in prokaryotes specifically designed for
recognition of surfaces and biofilm formation. Adhesion of microorganisms to mineral substrates
significantly influences microbially-induced mineral beneficiation processes.
7
Lecture 28: Biobeneficiation Of Sulfide Minerals Using Acidithiobacillus Bacteria NPTEL Web Course
Lecture 28
Biobeneficiation Of Sulfide Minerals Using Acidithiobacillus
Bacteria
Keywords: Bioflotation, Bioflocculation, Acidithiobacillus
Bioflotation using Acidithiobacillus ferrooxidans [171 174]

Pyrite-chalcopyrite
Adsorption density of At.ferrooxidans was observed to be higher on pyrite compared to
chalcopyrite. Electrophoretic mobilities of pyrite and chalcopyrite were significantly affected
after interaction with bacterial cells. For example, IEP of pyrite shifted from an initial value at
pH 3.2 to 4.5 after interaction. IEP of chalcopyrite shifted from an initial pH of 2.4 to 4.2 after
similar interaction. Surface adsorption of bacterial cells was the highest from acidic pH to neutral
pH and decreased significantly beyond pH 7. Such a drastic decline in cell adsorption on pyrite
and chalcopyrite is assumed to be due to partial removal of LPS from the cell surfaces.
At.ferrooxidans can be efficiently used for selective removal of pyrite from chalcopyrite (Table
28.1). When the above minerals were individually conditioned with cells before flotation, using
isopropyl xanthate, pyrite was found to be depressed while chalcopyrite exhibited significant
flotability. Conditioning the minerals with a collector prior to bacterial interaction resulted in
better selectivity in separation.
Table 28.1: Bioflotation of Pyrite-chalcopyrite mixture
Pyrite-chalcopyrite mixture conditioned with PIPX

followed by bacterial treatment
Percent weight floation
Pyrite Chalcopyrite
pH 6.5 22 85
pH 8.5 15 82
1
Pyrite-Arsenopyrite
IEP of pure arsenopyrite was at a pH of about 2.5 and there was no significant shifts in its IEP
after interaction with At.ferrooxidans, compared to pyrite. However, after 12 hours of bacterial
interaction, IEP of arsenopyrite shifted to a pH of about 3.2. Kinetics of cell adsorption into
10
pyrite was very fast and significantly higher number of cells (3 x 10 cells / m2) were found to
be adsorbed within about 15 minutes. On the otherhand, bacterial adsorption on arsenopyrite
was slower and 10 times lower than that on pyrite.
SEM studies also revealed higher cell adsorption on pyrite compared to arsenopyrite. Interaction
with bacterial cells (even for 5 min) resulted in depression of pyrite, while the flotability of
arsenopyrite was promoted. The presence of arsenopyrite even inhibited the copper activation of
pyrite, resulting in very good separation between pyrite and arsenopyrite through microbially-
induced flotation (Table 28.2).
Table 28.2 Selective flotation of pyrite-arsenopyrite (conditioned with At.ferrooxidans followed by treatment
with isopropyl xanthate and copper sulfate)
Wt % flotation
pH
Pyrite Arsenopyrite
5 28 92
6 24 96
2
Settling behavior of pyrite and arsenopyrite is the presence and absence of At.ferrooxidans is
illustrated in Table 28.3.
Table 28.3: Settling behavior of pyrite and arsenopyrite is the presence and absence of At.ferrooxidans
Percent Settled
Mineral pH
Cells Control
Pyrite 4.5 96 17
6.5 95 16
Arsenopyrite 4.5 25 22
6.5 24 20
Even at lower cell density, settling rate of pyrite is promoted. There was no significant influence
on the settling rate of arsenopyrite.
Control of cell density is essential to achieve selective flocculation of pyrite from arsenopyrite.
Higher cell densities would result in decrease in selectivity of separation.(Table 28.4).
Table 28.4. Selective flocculation behavior of pyrite-arsenopyrite mixture in the presence and absence of
bacterial cells at neutral pH.
Percent settled
Arsenopyrite pyrite
Inoculated 25 96
Control 20 16
3
Pyrite-chalcopyrite-Arsenopyrite:
Good separation of pyrite-from chalcopyrite and arsenopyrite also could be achieved from 1:1:1
ternary mixture. In the presence of copper sulfate, good pyrite separation from arsenopyrite and
chalcopyrite was achieved by suitably conditioning with bacterial cells and xanthate collector.
For example, 20% pyrite, 85% chalcopyrite and about 80% arsenopyrite could be recovered from
their equal mixtures after bacterial conditioning.
Galena - sphalerite
Use of At.ferrooxidans in the flotation separation of galena from sphalerite was also studied.
Effect of cell population during mineral interaction on the flotation recovery of sphalerite and
galena is illustrated in table 28.5. Control of cell density and interaction time are critical in
achieving selective galena sphalerite separation.
Table 28.5: Effect of inital cell density on the flotability of galena and sphalerite
Percent recovery
Cell population (cells/ml) Sphalerite Galena
102 95 84
104 96 78
108 94 48
109 53 23
Bacteria-mineral interactions result in significant changes in the electrokinetic behaviour of both

the bacterial cells and interacted minerals. Since, mineral flotation and flocculation are
influenced by the changes in their surface chemical properties, bacteria could influence
flocculation and flotation behaviour.
4
At.ferrooxidans selectively adhere to pyrite in coal and has been used to pretreat coal prior to
flotation to achieve significant pyrite depression. The selective depression of pyrite is due to the
specific and selective attachment of bacterial cells on the surface of pyrite. Removal of pyrite
from both synthetic and natural coals can be achieved by microbially-induced flotation.
Utility of Acidithiobacillus sp. for selective removal of galena from sphalerite has been studied.
The depression of galena after interaction with the bacteria was observed. Chandraprabha et al.
reported effective separation of pyrite from both chalcopyrite and arsenopyrite at acidic and
neutral pH through biomodulation using At.ferrooxidans. At.ferrooxidans thus can to play a dual
role, promoting flotation under certain conditions while inducing depression of minerals under
some other conditions. Promotion of flotability of sulphide minerals in the presence of
Acidithiobacillus ferrooxidans could be explained as due to elemental sulphur formation through
biooxidation. Bacterial oxidation over extended periods of time leads to reoxidation of the
sulphur to sulphoxy compounds and to sulphate. Gradual build-up of such oxidized sulfate layers
on mineral surfaces would retard flotation.
Availability of initial cell population as well as the duration of biotreatment are important
variables influencing the flotability of sphalerite and galena. Typical results of flotation recovery
after interaction with bacterial cells are illustrated in Table 28.5. With increase in cell population
and duration of bacterial interaction, flotability of sphalerite and galena was observed to be
deleteriously affected. For sphalerite, 94% recovery achieved after treatment with 10 8 cells/ml
declined to about 53% when the cell population was enhanced ten fold. Flotability of galena was
seriously affected by increasing the initial biomass. Preferential flotation of sphalerite from a
mixed zinc-lead sulphide ore can be achieved through bacterial pretreatment under carefully
controlled conditions.
During bacterial interaction, significant numbers of bacterial cells were observed to be

tenaciously attached to the mineral surfaces. Cell concentrations in the liquid phase as a function
of time of biotreatment for both sphalerite and galena suspensions were monitored. In both cases,
5
about 45% of the cell population were seen to be dispersed in the liquid phase after about one
minute, the rest being attached to the mineral surfaces. The cell population in the liquid phase
increased with time in a sphalerite suspension, while it decreased in case of galena. Higher
numbers of bacterial cells are attached to galena than to sphalerite. Unlike sphalerite, galena
surfaces are seen to be covered with a dense layer of bacterial cells along with their metabolites
and reaction products. Enhanced hydrophobicity of sphalerite compared to galena can be
attributed to the above bacterial attachment behavior.
Bioflotation using Acidithiobacillus thiooxidans [175]
Electrophoretic mobility of pyrite, chalcopyrite and arsenopyrite after interaction with

At.thiooxidans was established. IEP of pyrite shifted from a pH value of about 3.2 to about 4.2
after 12h of bacterial interaction. Similarly IEP of chalcopyrite shifted from an initial value at
pH 2.4 to 3.2 after bacterial interaction. For arsenopyrite, the IEP shifted from an initial value of
2.5 to about 3.3 after similar biotreatment. Surface adsorption of At.thiooxidans can be arranged
in the following order.
Adsorption on pyrite > adsorption on chalcopyrite >> adsorption on arsenopyrite.
The observed difference in the adsorption behavior of bacterial cells on the above minerals plays
a role in their subsequent flotation or flocculation behavior. Bacterial interaction promoted
chalcopyrite flotation, while depressing pyrite.
Differential flotation of pyrite-chalcopyrite mixtures after conditioning with xanthate followed

by cells of At.thiooxidans resulted in 85% flotation recovery for chalcopyrite with only about
18% of pyrite floated.
Similar tests using pyrite-chalcopyrite-arsenopyrite ternary mixtures resulted in good flotation

recovery for chalcopyrite-arsenopyrite along with pyrite depression.
6
In the presence of copper activation, chalcopyrite-arsenopyrite recoveries could be substantially

increased to more than 90% with significant pyrite depression.
Surface chemical changes brought about on sulfide minerals such as galena and sphalerite by
Acidithiobacillus thiooxidans was also studied. After interaction with At. thiooxidans, IEP of
both galena and sphalerite shifted towards higher pH values presumably due to specific
adsorption of bacterial cells. Higher numbers of cells are adsorbed on galena surfaces than on
sphalerite.
Flotation of both galena and sphalerite was carried out before and after interaction with
At.thiooxidans. Flotation recovery of galena was observed to be significantly decreased, while
that for sphalerite was enhanced. After 2 hours of bacterial interaction, galena was totally
depressed while sphalerite completely floated. Significant differences in surface adsorption of
cells onto galena and sphalerite along with the nature of the surface interaction products,
(sulphates and hydroxides), are the contributing factors for the observed flotation behaviour of
galena and sphalerite.
Flotation results for 1:1 galena- sphalerite mixture after interaction with At.thiooxidans are given
in table 28.6.
After interaction with the cells in the absence of collector/activator over 97% galena could be
depressed, while 96% sphalerite was floated.
Addition of collector and activator to the samples initially interacted with the cells
marginally changed the flotation recoveries of the two minerals.
Flotation tests after interaction with cells but in the presence of reduced amounts of collector
and activator, resulted in similar recoveries.
Addition of collector / activator to the mineral mixture which was initially interacted with the
cells, did not significantly improve the flotation recoveries of galena and sphalerite.
Interaction of the cells at pH 2.2-2.5 with the mineral mixture would be sufficient to achieve
7
significant depression of galena and flotation of sphalerite. No separate addition of chemical

regents is necessary.
Results of selective flocculation tests carried out using galena sphalerite mixture under
different conditions are given in Table 28.7.
Selective flocculation tests in the absence of the cells at acidic pH (2.5) resulted in 92%
dispersion of sphalerite and galena and lack of selectivity.
In the presence of cells at pH 2.5, nearly 98% of sphalerite was dispersed together with 70%
of galena.
Selective flocculation tests at pH 9-9.5, in the absence of the cells, resulted in the dispersion
of 70% of sphalerite while 72% galena was flocculated, (partial selectivity).
Interaction with bacterial cells resulted in dispersion of about 96% of sphalerite and
flocculation of over 95% of galena at pH about 9.
Table 28.6: Weight percent Flotation of Galena-sphalerite mixture after interaction with At.thiooxidans
(pH 9)
Percent weight floated

Conditions
Galena Sphalerite
Flotation after bacterial interaction 3 96

(no added collector or activator)
94
Flotation with xanthate and copper 7
sulfate after interaction with
At.thiooxidans
ZnSO4 formed from sphalerite due to bacterial interaction is soluble unlike the case of lead
sulphate formed on galena surfaces. Surface morphology of galena and sphalerite changed after
bacterial interaction. Bacterial cell adsorption was found to be significantly higher on galena
8
along with formation of insoluble lead sulfate as a reaction product. On the otherhand, sphalerite
surfaces exhibited lower cell attachment and indicated surface erosion and corrosion.
Table 28.7 : Selective flocculation of sphalerite-galena mixture in the presence of
Percent settled
Sphalerite Galena
8 8
pH 2-2.5 control
Interaction with
2 30
Acidithiobacillus
thiooxidans at pH 2-2.5
28 72
pH 9-9.5 control
Interaction with
4 95
Acidithiobacillus
thiooxidans at pH 9-9.5
9
Lecture 29: Biobeneficiation Of Sulfide Minerals Using Paenibacillus Polymyxa NPTEL Web Course
Lecture 29
Biobeneficiation Of Sulfide Minerals Using Paenibacillus Polymyxa

Keywords: Paenibacillus Polymyxa, Sphalerite, Galena, Mineral Specific Reagents
Bioseparation of galena-sphalerite in presence of Paenibacillus polymyxa [176-

178]
Utility of a neutrophilic, heterotrophic bacteria, namely Paenibacillus polymyxa in the separation

of galena from sphalerite was also studied. Initially effects of pH, time and cell population on
bacterial adsorption on galena and sphalerite was established. Significantly higher cell
adsorption of galena was observed at neutral pH. There was almost an order of magnitude
difference between the adsorption of cells on galena and sphalerite. Cell adsorption on galena
was found to be independent of pH, while the adsorption density decreased with increase in pH
on sphalerite surfaces.
Electrophoretic mobilities of both galena and sphalerite as a function of pH were measured
before and after bacterial interaction. The IEP of bacterial cells corresponds to a pH of about 2.5
and hence cell surfaces are increasingly negative with increase in pH. After interaction with
galena, bacterial cells became less electronegative. Due to possible adsorption of dissolved zinc
ions on cell surfaces, negative electrophoretic mobilities of cells after interaction with sphalerite
shifted in a positive direction. IEP of sphalerite (pH 2.2) shifted to less acidic pH values after
bacterial interaction. Nonelectrostatic forces such as chemical forces due to secretion of
proteinaceous compounds after bacterial interaction with sphalerite could be responsible for the
observed behavior. After bacterial interaction, electrophoretic mobility of galena became almost
independent of pH and there was no significant shift in its IEP.
The above observed differences in the adsorption and electrokinetic behavior with respect to
interaction of P.polymyxa with sphalerite and galena can be utilized to bring about microbially
induced separation between the above minerals. Flotation behavior of galena and sphalerite was
studied before and after bacterial interaction. In the absence of bacterial interaction, using
copper activator and isopropyl xanthate, upto 95% flotation recovery for sphalerite could be
1
obtained in the pH range of 5-9. After bacterial interaction and in the absence of added collector
and activator, sphalerite could not be floated. However, bio- treated sphalerite could be
efficiently floated if conditioned with xanthate and copper sulfate at a pH in the range 8-9.
However, galena was found to be depressed after interaction with bacterial cells even if collector
addition was made after bacterial interaction. Differential flotation tests were carried out using
1:1 mixtures of galena and sphalerite as illustrated in table 29.1.
Table 29.1: Bioflotation of sphalerite-galena in the presence and absence of Paenibacillus polymyxa
Percent weight flotation

Conditions
Sphalerite Galena
1. In presence of xanthate and copper sulfate 94 99

2. After bacterial interaction (no xanthate and activator) 4 3
3. Bacterial interaction followed by collector and activator 96 5
addition
Bacterial interaction depresses both the minerals when additions of xanthate collector and copper
activator were not made. However, addition of collector and activation after bacterial interaction
promotes flotation of sphalerite and depression of galena.
Flocculation behavior of sphalerite and galena was also established before and after bacterial
interaction at different pH values. In the neutral pH range (6.5 7.2) no significant differences
in their settling rates could be observed. However, in the alkaline pH range of 9.2 9.6,
significant differences in their settling rates were observed after bacterial interaction. At a
settling period of about two minutes, 90% of galena was found to be settled, with only about 5%
of sphalerite flocculated. The role of pH control in selective separation of galena and sphalerite
through microbially induced flocculation-dispersion cannot be underestimated. As indicated
above at alkaline pH of 9 and above, almost complete dispersion of sphalerite and flocculation of
galena could be achieved. Selective flocculation test results for 1:1 mixtures of galena-sphalerite
2
maintained at pH 9-9.5 are illustrated in table 29.2. Bacterial interaction can be efficiently used
to separate galena from sphalerite through selective flocculation.
Table 29.2: Selective flocculation of 1:1 mixtures of galena-sphalerite
Percent settled
Conditions
Sphalerite Galena
Control (pH 9 9.5) 28 72
In presence of cells of P.polymyxa at pH 5 95

9 9.5
The role of bioreagents secreted by P.polymyxa in the separation of galena and sphalerite could
now be examined.
Adsorption behavior of exopolysaccharides and bioproteins was initially examined. Adsorption
density of bacterial polysaccharides on galena was found to be about 50 times higher than that on
sphalerite. Polysaccharide adsorption was pH dependent, especially on sphalerite since it
increased with pH upto 6 8, above which steeply decreased. No such pH dependence was
observed on galena. Adsorption density of bioproteins decreased with increase in pH on both
galena and sphalerite. Higher protein adsorption could be observed on galena.
Effect of interaction of metabolites generated during growth of P.polymyxa on the flotation
behavior of galena and sphalerite was established. Addition of xanthate collector and copper
sulfate to metabolite-interacted sphalerite enhanced its flotability to more than 90% in the pH
range 4.5 to 10.
Such a treatment, on the otherhand, did not improve the flotability of galena. Differential
flotation tests were carried out using 1:1 mineral mixtures under different conditions.
At a pH of about 3 -3.5 (metabolite pH), interaction with bacterial metabolite resulted in
good flotability for sphalerite (over 90%), while over 96% of galena was depressed.
3
Addition of xanthate collector along with copper sulfate to the metabolite-interacted mineral
mixture resulted in efficient depression of galena (95%) and flotation of sphalerite (95%) at a
pH of 9 9.5.
Selective separation of galena from sphalerite could be effectively achieved through
treatment with metabolites of P.polymyxa in the presence of reduced amounts of collector
and activator addition.
Interaction with metabolites at an alkaline pH of 9 9.5, promoted flocculation of galena and

dispersion of sphalerite.
Typical selective flocculation results are presented in table 29.3.
Table 29.3: Selective flocculation with bacterial metabolites

Percent settled
Conditions
Galena Sphalerite
1. Control (pH 3 3.5) 17 7

2. In presence of metabolite at pH 3 -3.5 48 6
3. Control at pH 9 9.5 72 28
4. In the presence of metabolite at pH 9
9.5 93 5
Higher pH in the range of 9 9.5 is favorable for selective separation of galena from sphalerite
through interaction with bacterial metabolites.
Biodesulfurization for environmental protection [171, 183-184, 211]
Microorganisms can be used for removal of sulfide minerals such as pyrite and chalcopyrite from
gangue minerals such as quartz and calcite. It is well established that presence of the above
types of sulfides in tailings containing silicates and calcite is responsible for the formation of
4
acid mine drainage (AMD). Removal of acid-forming sulfides from waste ores and tailings is an
efficient method for prevention of acid mine drainage.
Bioremoval of pyrite and chalcopyrite from quartz and calcite using Paenibacillus polymyxa has
been reported. In the neutral pH range, pyrite and chalcopyrite extribited the highest bacterial
cell adsorption, followed by calcite. Quartz exhibited the lowest cell adsorption. Flocculation
studies indicated that pyrite and chalcopyrite settled very rapidly in the presence of bacterial
cells, unlike calcite and quartz (Tables 29.4, 29.5).
Table 29.4: Selective bioflocculation using P.polymyxa using a mixture of pyrite, chalcopyrite and quartz.
Percent quartz removal from
Desliming stages pyrite-chalcopyrite at pH 7-8
Cells Protein
1 30 29
3 79 78
5 87 82
Table 29.5: Selective bioflocculation of pyrite-chalcopyrite from calcite.

Percent calcite removal at pH 7-8
Desliming stages
Cells Protein
1 15 16
3 62 65
4 72 77
In table 29.6, selective bioflotation results to remove sulfides from quartz are illustrated.
5
Table 29.6: Selective bioflotation for removal of pyrite-chalcopyrite from a mixture containing quartz.

Conditions Pyrite- Quartz
chalcopyrite
1. Control 36 40
2. Interaction with P.polymyxa cells 19 50
at pH 8
3. Interaction with bioproteins 11 93
followed by hexamine addition
4. Interaction with cells followed by 12 94
hexamine addition.
Sulfide minerals such as pyrite-chalcopyrite can be effectively removed from calcite and
quartz through microbially-induced selective flocculation.
Interaction with bacterial cells or bioproteins promoted selective flotation of quartz from
pyrite-chalcopyrite.
Mineral-specific bacterial proteins for beneficiation [206]
Utility of microorganisms and associated extracellular metabolic products in selective flotation

and flocculation of oxide and sulfide minerals has been well established. Paenibacillus
polymyxa, a Gram-positive, neutrophilic, periflagellated heterotrophic bacterium is capable of
producing exo-polysaccharides and proteins, besides organic acids such as oxalic, formic and
acetic acids all of which find use in minerals processing. Adaptation of microorganisms to
different mineral substrates would enable generation of mineral-specific bioreagents.
Role of specific bacterial metabolic products responsible for mineral-specificity need be

understood. Specific affinity of bacterial cells and the role of extra cellular polysaccharides and
6
proteins in influencing bacterial attachment need to be established. Extracellular bacterial

proteins contain numerous kinds of amino groups and could be fractionated using ammonium
sulphate precipitation. Selective separation of pyrite and chalcopyrite from quartz, sphalerite and
galena can be achieved through microbialy-induced flocculation and flotation using various
groups of proteins obtained from protein fractionation. Different protein fractions exhibit
varying surface affinity towards minerals. Growth and adaptation of bacterial cells in the
presence of minerals lead to specific expression of extracellular protein groups which confer
unique surface affinity to a particular mineral. Proteins isolated from such mineral-grown
bacterial cells would then be useful to bring about the desired mineral separation from complex
multisulfides.
Various protein bands observed in the SDS PAGE analysis indicated that extra cellular proteins
consisted of numerous protein groups. Fractionation of such proteins was carried out to isolate
different protein fractions from culture supernatant (metabolite). The separated fractions
(designated as A, B, C and D) were used in adsorption, flocculation and flotation tests.
Adsorption studies with fractionated groups of proteins onto all the minerals were carried out and
the results are shown in Table 29.7. Adsorption density of protein from group A was higher on
pyrite, chalcopyrite and galena (about 3.5 mg/m2). Adsorption density of other group of proteins
(B, C and D) onto pyrite and chalcopyrite were also higher in the range of 4.5-5 mg/m2.
Adsorption density of all groups of proteins was the lowest on sphalerite and quartz. Adsorption
density onto galena was 3.8 mg/m2, 2.5 mg/m2, 2 mg/m2 and 3 mg/m2 for proteins from group A,
B, C and D, respectively Different fractions of bacterial proteins possess different levels of
surface affinity towards various minerals. Also, a single group of protein exhibited varying
adsorption densities towards different minerals. Extracellular bacterial proteins separated from
the metabolic products of P.polymyxa was purified through HPLC and several fractions (upto 30)
were similarly tested. Mineral-adsorbed proteins were also extracted. Numerous proteins are
available in the extracellular bacterial cell matrix. Bacterial cells were also grown in the
presence of various minerals and the protein fractions present mineral-grown cells were
7
characterized through ion exchange chromatography and SDS-PAGE electrophoresis. In the

presence of different minerals, only certain mineral-specific proteins were expressed.
Flocculation behavior of minerals with protein fractions was studied as illustrated in Table 29.8.
All the groups of proteins promoted enhanced flocculation of both pyrite and chalcopyrite. On
the other hand, dispersion of quartz, sphalerite and galena was promoted after similar interaction.
In presence of group C protein, pyrite and chalcopyrite showed the highest settling rate while
other minerals exhibited enhanced dispersion. Selective separation of quartz from pyrite and
chalcopyrite was performed in presence of group C protein. In the presence of 20 mg of group C
proteins 92% of quartz could be removed from pyrite - chalcopyrite at neutral pH.
Selective flocculation tests were also carried out using mixture of sphalerite, pyrite and
chalcopyrite and galena, pyrite and chalcopyrite. At pH 8, 96% and 90% of sphalerite could be
separated using group B and D proteins respectively. Group B and D proteins functioned as a
bioflocculant for pyrite and chalcopyrite, facilitating their rapid settling while dispersing
sphalerite. In the presence of group B and D proteins, 92% and 94% separation of galena could
also be separated from pyrite-chalcopyrite.
Flotation behaviour of various minerals was also established after interaction with various
protein fractions (Table 29.9).
Flotation recovery of quartz was 65%, 80%, 30% and 40% after interaction with groups A,
B, C, D proteins respectively. Group A and B proteins are more effective in the flotation of
quartz.
Both pyrite and chalcopyrite were significantly depressed after similar protein interactions.
70%, 80%, 30% and 65% of sphalerite could be floated after interaction with A, B, C and D
fractions, respectively.
Only group D proteins conferred highest flotability on galena.
8
About 25% of pyrite and chalcopyrite along with 90% of quartz could be floated from a mineral
mixture after interaction with B proteins.
Selective flotation separation of sphalerite from a mixture with pyrite and chalcopyrite was
achieved with group B proteins.
Extracellular bacterial proteins derived from Paenibacillus polymyxa contained various kinds of
amino (protein) groups. Different purified protein fractions were separated and characterized
through controlled ammonium sulfate precipitation and SDS-PAGE. Different protein fractions
exhibited varying surface adsorption density towards minerals such as quartz, pyrite,
chalcopyrite, galena and sphalerite. When bacterial cells were grown in the presence of the
above minerals, mineral-specific proteins were expressed depending on the mineral. Protein
fractions having significant surface affinity towards different minerals can be isolated and used
to bring about selective mineral separation.
Table 29.7: Adsorption of fractionated groups of proteins onto minerals

Adsorption density, mg/m2
Protein fractions
Quartz Sphalerite Galena Pyrite Chalcopyrite
A 1 1.2 3.8 3.6 3.5

B 1 1.2 2.5 5.0 4.8
C 1 1.2 2.0 4.8 4.5
D 1 1.2 3.0 4.6 4.4
9
Table 29.8: Flocculation behavior of minerals with protein fractions

Percent weight settled
Protein fractions
A 38 35 30 90 85
B 30 20 25 85 85
C 30 30 30 94 92
D 38 30 25 85 86
Table 29.9: Flotation behaviour of various minerals with protein fractions
Percent weight floatation

Protein fractions
A 65 70 45 15 20
B 80 80 50 20 16
C 30 30 30 15 10
D 40 65 80 10 15
10
Lecture 2: Biogenesis Of Metals And Minerals NPTEL Web Course
Lecture 2
Biogenesis Of Metals And Minerals
Keywords: Biomineralization, Metal-bacteria Cycles, Biogenesis
Many types of bacteria (autotrophic, heterotrophic, aerobic-anaerobic, mesophile-thermophile,

acidophilic-neutrophilic), fungi, yeasts and algae, inhabit mineral deposits, even under extreme
environmental conditions. Ubiquitous presence of a plethora of microorganisms in mining
environments is pointer towards biogenesis and biomineralization. In figure 2.1 below, bluish
copper sulfate emanating from chalcopyrite containing rocks could be seen. Natural
biooxidation and dissolution of chalcopyrite by native Acidithiobacillus bacteria is a pointer
towards biogenesis.
Fig.2.1: Bluish copper sulfate generated by indigenous Acidithiobacillus in copper-bearing rocks
Geomicrobiology of gold has been well studied. Many microorganisms can solubilise and
precipitate gold. Bacteria and Archaea are involved in the gold biogeochemical cycle. Gold
1
formation, mineralization, reconcentration and precipitation are catalyzed by bacteria. Gold-

bearing sulfide minerals such as arsenopyrite, pyrite and chalcopyrite may be formed in deep
subsurface environments through the activities of sulfate reducing bacteria. Aerobic autotrophs
such as At.ferrooxidans and At.thiooxidans can break-up gold-bearing sulfide minerals.
Microbial metabolism also generates many types of solvents such as amino-acids, cyanides and
thiosulfates which can solubilise gold. Cyanobacteria promote formation of secondary crystals
of gold. Bacteriform gold occurs associated with quartz. Similarly, association of gold with
organic and inorganic carbon can be biomediated. Usage of microorganisms as indicators of gold
occurrences is an interesting outcome. Presence of bacterial spores containing tiny gold particles
on top soils may indicate presence of gold, underneath. Microbially-induced gold cycle in nature
can be assessed at different mine sites. [4]
Microbial activity is an essential constituent in the natural iron cycle. Many iron-involved redox
reactions stimulate bacterial growth which promotes iron dissolution and precipitation. Biogenic
iron oxides generally occur as nano - crystals possessing varying morphology and mineralogy.
Direct bacterial metabolism or biosorption and nucleation processes can result in the formation
of iron minerals. Acidophilic and neutrophilic, aerobic bacteria promotes oxidation of ferrous
iron to ferric oxide. Anaerobic organisms reduce ferric to ferrous and ferrous oxides and sulfides
are formed. Various types of iron oxides such as ferrihydrite, goethite, lepidocrocite, magnetite
and hematite are found in sediments. Iron oxide particulates can occur closely associated with
cell walls containing exopolymers. Extracellular biogenic oxides of iron can be differentiated
from intracellular mineral formations. Iron-oxidizing microbes are isolated from iron rich
seepages. Various types of organic and inorganic intracellular polymeric substances are
generated by microorganisms. Production of intracellular magnetite has been reported in
magnetotactic bacteria, which includes various prokaryotes including anaerobic, sulfate reducing
bacteria. Banded iron formations may represent biogenic iron mineralization. [5-6]
Various microorganisms indigenous to iron ore deposits are isolated to bring about beneficial
iron ore processing to remove impurities such as phosphorus, alkalis, silica and alumina. Fungi
2
and organic and inorganic acid producing heterotrophs and autotrophs isolated from iron ore
mines were found to be useful in dephosphorization.
Microbial formation and degradation of limestone is an important example of biogenesis.

Biological fixation of carbon in carbonates is performed by several bacteria, fungi and algae.
Achromatium oxaliferum is involved in intracellular deposition of calcium carbonate. Calcium
carbonate is a skeletal support structure in many invertebrates, sponges and mollusks. Biogenic
calcium-magnesium carbonates formation in the form of chalk and calcite deposits in nature is
well established. Biodissolution and degradation of limestone can also be brought about by
microorganisms. This will lead to breakdown of cements and concrete structures. Nitrifying and
sulfur-oxidizing bacteria could be detected in limestone samples after degradation. Cyano
bacteria, fungi and algae can cause localised dissolution of limestone, creating pits and fissures.
Such microorganisms bore into limestone and can destroy calcified coatings.
Silicon is taken up and concentrated by organisms. Clays containing silicon influences microbial
growth and attachment. Clays can modify microbial habitat and may influence activity of
bacterial enzymes. Certain bacteria can accumulate silicon, so also some fungi and diatoms.
Microbial silicon mobilizations play a role in weathering of rocks. Silicate minerals can be
biodissolved.
Phosphorous is fundamental to life as a nutritional and structural component of all organisms.

Microbial conversion of organic into inorganic phosphorous and the synthesis of phosphate
compounds is known. Soil organisms like Bacillus subtilis, B.megaterium, Arthrobacter and
fungi such as Aspergillus can participate in natural phosphorous cycle. Microbes can solubilise
many phosphate minerals such as apatite. Microbes involved in phosphate solubilisation include
B.megaterium, Acidithiobacillus, Nitrifying bacteria, Pseudomonads and fungi such as A.niger
and A.flavus. Microorganisms can also cause phosphate immobilization. Phosphorites in nature
are biogenic. Pentavalent phosphorous can be reduced to lower valence states by microbes.
Although arsenic is toxic to life, several microorganisms can grow and metabolize in the
presence of larger concentrations. Many acidophiles inhabit arsenopyrite and orpiment mineral
3
forms in nature. Bacteria may develop resistance to arsenic, both through genetic and mutational
changes. Arsenite-oxidizing bacterial strains have been isolated from soils. Arsenic- bearing
minerals containing iron, copper and sulfur are oxidized by bacteria. For example, arsenopyrite
and enargite are oxidized by Acidithiobacillus to generate As (III) and As (V) under acidic
conditions.
Geomicrobiology of iron is of great interest in metallurgy. Iron is biologically important as a

nutritional source. Acidophiles such as At.ferrooxidans and L.ferrooxidans utilize ferrous ions as
energy source and oxidize them to ferric state. Thermophiles such as Sulfobacillus and
neutrophiles like Gallionella participate in enzymatic iron oxidation. Ferric ion reduction by
bacteria is also known. Examples include Paenibacillus polymyxa, B.circulans and
B.megaterium. The term, iron bacteria is used to represent the organisms that oxidize iron
enzymatically. There are also iron accumulators. Magnetotactic bacteria produce magnetite
through reduction of ferric iron. Magnetite can also be formed extracellularly. Many
sedimentary iron deposits are biogenic. So also banded iron formations. Bacteria and fungi can
mobilize large amounts of iron from minerals. Sulfide forms of iron such as pyrite and
pyrrhotite are biogenic.[5-6]
Many manganese oxide formations in nature are biogenic. Some manganese-oxidising bacteria
include.
Hyphomicrobium
Pseudomonas
Arthrobacter
Leptothrix
Metallogenium
4
Mn (IV) reducing bacteria include
Paenibacillus polymyxa
Bacillus mesentericus
Pseudomonas liquefaciens
Some microorganisms can accumulate manganese oxides. Microbial manganese deposition in

nature is important in biogenesis in soil and water environments. Manganese-oxidizing bacteria
have been detected in marine environments. Bacteria participate in manganese cycle in sea.
Ferromanganese nodules in the ocean floors are formed due to biogenic reactions. The nodules
provide manganese and iron as energy source to Mn (II) oxidising bacteria.
Geomicrobiology of sulfur in nature is important in extractive metallurgy. Bacterial oxidation of

sulfur and its compounds and reduction of sulfate is relevant in bioleaching and bioremediation
of acid mine drainage.
Biogenesis and biodegradation of sulfide minerals in nature lead to formation of many

nonferrous metal sulfides and sulfates. Sedimentary metal sulfides are of biogenic origin.
Hydrogen sulfide is formed from bacterial reduction of sulfates. Many metal sulfides such as
those of iron, zinc, copper and nickel are formed through such biogenic reactions.
5
References (Lectures 1 and 2):
1. R.F. Decker, Biotechnology / Materials: The growing interface, Met. Trans, A, Vol. 17A
(1986), pp 5-30.
2. S.Mann, Biomimetic Materials Chemistry, VCH Publishers Inc. N. Y. (1996).
3. S.Mann, Biomineralization and Biomimetic Materials Chemistry, in Biomimetic Materials
Chemistry, Chapter 1, VCH (New York) (1996).
4. F. Reith, M. F. Lengke, D. Falconer, D. Craw and G. Southam, The Geomicrobiology of
gold, The ISME Journal (2007), 1, 567-584.
5. D. Fortin and S. Langley, Formation and occurrence of biogenic iron-rich minerals, Earth
Science Reviews, 72 (2005), 1-19.
6. H.L. Ehrlich, Geomicrobiology, Marcel Dekker Inc., N. Y. (1990).
6
Lecture 30: Biogenesis And Bio Beneficiation Of Iron Ores Bacillus Subtilis, SRB And Yeasts
NPTEL Web Course
Lecture 30
Biobeneficiation Of Iron Ores Bacillus subtilis, SRB And Yeasts
Keywords: Iron Ores, Biobeneficiation, B.subtilis, Sulfate Reducing Bacteria, Yeasts
Utility of various microorganisms such as Saccharomyces cerevisiae (yeast), Bacillus subtits and
Desulfovibrio desulfuricans (SRB) in the beneficiation of iron ores is discussed below with
respect to silica, alumina and calcite removal.
Use of Bacillus subtilis in iron ore beneficiation. [187]

Bacillus subtilis is a Gram-positive, neutrophilic, aerobic bacterium found associated with iron
ore deposits. Typical morphology and growth curve for B.subtilis are illustrated in Fig.30.1 and
30.2.
Fig. 30.1 : SEM photograph of Bacillus subtilis Fig. 30.2 : Optical density as a function of time during
growth of Bacillus subtilis
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NPTEL Web Course
Scanning electron microscopy (SEM) revealed surface attachment of bacterial cells on hematite,
corundum, calcite and quartz. More profuse and significant bacterial attachment was observed
on hematite. Bacterial adhesion was observed to be lower on quartz than on other minerals.
Adsorption density of cells was also found to be the highest on hematite. Adsorption of cells
varied with pH depending on mineral surface charge variations. For example, adsorption density
of cells on hematite and corundum exhibited the following order:
Adsorption at pH 4 > adsorption at pH 7> adsorption at pH 9.
Bacterial cells extribited an IEP corresponding to a pH 2.2. Bacterial cells are negatively charged
at pH values higher than 2, surface electro-negativity increasing further with increase in pH.
Cell adsorption based on electrostatic forces can then be expected to decrease with increasing pH
on all the above minerals.
When bacterial cells were grown in the presence of various iron ore minerals, different amounts
of extracellular proteins and polysaccharides were secreted. Presence of quartz during bacterial
growth promoted higher secretion of proteins while that of hematite led to enhanced production
of exopolysaccharides.
Mineral-specific proteins were also secreted proteins with molecular weights, 19.9, 33.3, 40.2
and 60.3 kDa when grown in presence of quartz. In the presence of hematite, a new protein of
23.4 kDa was secreted.
Bacterial interaction conferred increased surface hydrophobicity on quartz, followed by

corundum and calcite. Interacted hematite was relatively more hydrophilic. Interaction with
bacterial cells and cell free metabolites significantly increased the flocculation (settling rate) of
hematite at neutral pH. On the otherhand, quartz particles were increasingly dispersed after
similar interaction. Dispersion of corundum and calcite particles was also facilitated by bacterial
interaction, through not to the same extent as observed in the case of quartz.
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NPTEL Web Course
Flotation behavior of various minerals after interaction with B.subtilis is illustrated in table 30.1.
Table 30.1:Flotation behavior of various minerals after interaction with B.subtilis

Interaction
Control Interaction
with cell
with cells
free extract
Hematite 11 5 14
Calcite 13 74 50
Corundum 12 73 30
Quartz 15 92 90
After interaction with bacterial cells and metabolites, hematite was found to be significarfly
depressed. Bacterial interaction promoted the flotation of quartz, corundum and calcite.
Microbially-induced flotation was the highest in case of quartz, followed by calcite and
corundum. Efficient separation of quartz from hematite from their mixtures could be achieved
after interaction with cells and metabolites of B.subtilis.
Hematite-quartz separation in the presence of anaerobic bacteria (SRB),

Desulfovibrio desulfuricans [189]
Morphology of Desulfovibrio desulfuricans and the growth curve are illustrated in fig. 30.3 and
30.4. 8
6x10 1.8
8
5x10 1.6
sulphate concentration(g/L)
Number of cells/ml
8
4x10 cell count 1.4
Sulphate
8 concentration
3x10 1.2
8
2x10 1.0
8
1x10 0.8
0 0.6
0 20 40 60 80 100 120 140 160
Time (min)
Fig. 30.4: Cell number and sulphate
concentration as a function of time during growth
Fig. 30.3 : SEM photograph of Desulfovibrio of Desulfovibrio desulfuricans
desulfuricans
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NPTEL Web Course
For Desulfovibrio desulfuricans, an anaerobe, as the cell count increases, sulfate concentration
decreases. During the log phase and stationary phase, the decrease in sulfate concentration was
steep. During decline phase the decrease in sulfate concentration was nearly parallel to X-axis.
The growth of bacterial cells was monitored in the presence and absence of minerals such as
hematite and quartz. When similar cell growth was attained in the presence of minerals as in
control, adaptation to the minerals was considered achieved. Table 30.2 illustrates extracellular
proteins and polysaccharides generated by Desulfovibrio desulfuricans in the presence and
absence of minerals.
Table 30.2: Extracellular proteins and polysaccharides generated by Desulfovibrio desulfuricans.
Protein Polysaccharide
No minerals 0.25 0.20

In presence of quartz 0.45 0.20
In presence of hematite 0.23 0.70
Extracellular proteins secreted by quartz adapted Desulfovibrio desulfuricans were the highest.
Secretion of extracellular polysaccharides was found to be higher in case of hematite-grown
cells. Bacterial growth in presence of quartz generates higher amounts of bacterial proteins,
while that of hematite produce significant amounts of exopolysaccharides. Negatively charged
quartz surfaces exhibit strong affinity towards amino group containing positively charged
proteinaceous compounds. Hematite exhibit strong affinity towards negatively charged
polysaccharides under neutral to mildly alkaline pH conditions.
Protein profiles of unadapted and mineral-adapted Desulfovibrio desulfuricans were then
established.
Protein bands of molecular weights 105kDa and 25kDa has been attributed to mineral specific
proteins secreted by quartz-grown bacterial cells since they were absent in unadapted and
hematite-adapted cell metabolites. Secretion of higher amounts of mineral specific proteins by
bacterial cells was promoted if grown and adapted in the presence of quartz mineral.
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NPTEL Web Course
Percent adsorption of ruthenium red on Desulfovibrio desulfuricans grown under different

conditions is shown in table 30.3.
Table 30.3: Percent adsorption of ruthenium red on Desulfovibrio desulfuricans grown under different
conditions
Percent ruthenium red
adsorption
In the absence of minerals 42

In the presence of quartz 29
In the presence of hematite 79
Amount of polysaccharides present on the hematite-adapted SRB cells are significantly higher
than that on quartz-adapted cells. SRB cells adapted to hematite become more hydrophilic than
those adapted to quartz which is rendered more hydrophobic due to enhanced secretion of
proteins.
Effects of surface-chemical changes brought about on quartz and hematite due to bacterial
interaction can now be examined with respect to selective separation.
Table 30.4: Flotation separation of hematite and quartz using Desulfovibrio desulfuricans.
Percent weight Floated
Mineral mixture
Quartz Hematite Quartz Hematite
Control 17 5 15 5
Unadapted cells 45 8 48 10
Hematite adapted cells 39 2 37 5
Quartz adapted cells 75 9 76 9
Unadapted metabolite 35 11 39 9
Hematite adapted metabolite 49 16 47 14
Quartz adapted metabolite 84 15 81 13
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NPTEL Web Course
Flotation behavior of hematite and quartz in the absence and presence of bacterial interaction is
illustrated in Table 30.4. In the absence of bacterial interaction, no significant flotation of quartz
and hematite would be possible. Percent weight flotation of quartz was about 45% and 35%
respectively after interaction with unadapted bacterial cells and metabolite, while it increased to
about 75 % and 84% on interaction with quartz-adapted cells and metabolite. Higher surface
hydrophobicity exhibited by quartz is due to adsorption of bacterially produced hydrophobic
proteins. Percent weight flotation of hematite was about 8% and 11% on interaction with
unadapted bacterial cells and metabolite. After interaction with hematite and quartz-adapted
bacterial metabolite about 15% of hematite could be floated. Flotation recovery of hematite
decreased to 2% with hematite-grown cells. Such a hydrophilic surface character of hematite
(unlike quartz) is due to its high affinity towards polysaccharides.
Selective separation of quartz from a binary mixture of quartz and hematite was also studied
after interaction with bacterial cells and metabolite. Interaction with unadapted bacterial cells
and metabolite resulted in only 10% and 9% flotation recovery for hematite. After interaction
with quartz-adapted bacterial cells and metabolite, the percent flotation of quartz from the
mixture was about 76% and 81%, respectively. The above results clearly establish that efficient
separation of silica from hematite could be achieved through selective flotation after interaction
with cells and metabolites of an SRB (Desulfovibrio desulfuricans). However, prior bacterial
adaptation to the respective minerals (especially quartz) is essential to bring about efficient
separation.
Yeast-mediated separation of quartz from hematite and calcite [180-181, 188]

Use of yeast cells instead of bacteria in bio-mineral beneficiation can be expected to be
advantageous since biomass and bioreagents yields will be higher. Utility of Saccharomyces
cerevisiae in the separation of silica from hematite and calcite has been investigated.
Saccharomyces cerevisiae is a unicellular and chemoorganotrophic eukaryote. Typical

morphological features and growth curve are illustrated in Fig. 30.5 and 30.6
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NPTEL Web Course
Fig. 30.5 : SEM of Yeast Saccharomyces Fig. 30.6 : Optical density as a function of time
cerevisiae during growth of Saccharomyces cerevisiae
Adsorption density of yeast cells was found to be significantly higher on hematite and calcite
compared to quartz. Growth of yeast cells in the presence of quartz resulted in the generation of
higher amounts of mineral-specific proteins which were characterized through SDS-PAGE.
Protein bands of molecular weights 8.4 kDa, 14 kDA, 17.7 kDa, 18.8 kDa and 29kDa were
identified as quartz-specific proteins. Exopolysaccharides were increasingly generated when the
yeast cells were adapted or grown in the presence of hematite and calcite. Higher amounts of
such yeast proteins were found to be adsorbed on quartz surfaces, while hematite and calcite
exhibited higher affinity towards yeast polysaccharides.
Yeast-mediated separation of quartz from hematite and calcite was demonstrated through
microbially-induced flotation and selective flocculation as illustrated in tables 30.5, 30.6, 30.7
and 30.8.
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NPTEL Web Course
Table 30.5: Quartz-hematite separation through yeast-induced flotation

Experimental conditions Quartz Hematite 1 : 1 Mixture
alone alone Quartz Hematite
1. Control 12 8 15 14
2. Yeast grown in the absence of 50 10 38 15
minerals
3. Quartz-grown yeast 95 8 93 10
4. Hematite-grown yeast 12 10 14 9
5. Metabolites from quartz-grown yeast 95 8 94 7
Unadapted yeast cells and their metabolites did not bring about significant flotation
separation.
Yeast cells grown in the presence of or adapted to quartz and metabolites from adapted cells
were very efficient in selective separation of quartz from hematite through yeast-mediated
flotation.
Table 30.6: Quartz-hematite separation through yeast-mediated flocculation
Percent weight flocculated
Treatment Quartz Hematite Mixture
alone alone Quartz Hematite
1. Control 30 70 20 75
2. Unadapted yeast 38 76 40 78
3. Quartz-adapted yeast 28 82 23 91
4. Metabolites from quartz-adapted 22 92 20 93
yeast
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NPTEL Web Course
Quartz-grown (adapted) yeast cells and their metabolites promoted dispersion of quartz particles
and significant flocculation of hematite. Efficient separation of quartz from hematite from a
binary mixture could be achieved through interaction with either quartz-grown cells or their cell-
free metabolites.
Table 30.7: Separation of quartz from calcite through yeast-induced selective flocculation.
Percent settled
Process conditions Quartz Calcite 1:1 Mixture
alone alone Quartz Calcite
1. Control 30 60 25 70
2. Unadapted yeast cells 38 40 50 80
3. Calcite-adapted yeast 50 55 50 91
4. Quartz-adapted yeast 28 50 24 90
Calcite-grown yeast cells enhanced settling of quartz particles due to presence of excess
exopolysaccharides.
Quartz-grown yeast cells promoted dispersion of quartz and settling of calcite.
Calcite-grown yeast cells and their metabolites promoted flocculation of calcite.
Efficient selective separation of quartz from calcite could be achieved only by using quartz-
grown yeast cells and their metabolites.
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NPTEL Web Course
Table 30.8: Separation of quartz from calcite through yeast-mediated selective flotation.

Process conditions Quartz Calcite Mineral mixture
alone alone Quartz Calcite
1. Control 18 10 12 8
2. Unadapted yeast 50 28 32 15
3. Quartz-grown yeast 95 12 93 12
4. Calcite grown yeast 50 50 52 40
Quartz-grown yeast cells and their metabolites promoted selective flotation of quartz from
calcite. Interaction with yeast cells and culture conferred surface hydrophobicity on quartz and
hydrophilicity on calcite.
10
Lecture 31: Biobeneficiation Of Iron Ores Using Paenibacillus Polymyxa NPTEL Web Course
Lecture 31
Biobeneficiation Of Iron Ores Using Paenibacillus Polymyxa
Keywords: Paenibacillus Polymyxa, Iron Ore, Silica Removal
Morphological and growth characteristics of Paenibacillus polymyxa are illustrated in fig.31.1

and 31.2.
8
10
10
7
Number of cells/ml
6
9
10
pH
Cell count
pH 5
10
8 4
3
0 10 20 30 40 50 60 70
Time (h)
Fig. 31.1: SEM of Paenibacillus polymyxa Fig. 31.2: Cell number and pH as a function of time
during growth of Paenibacillus polymyxa
Rod shaped
Gram-positive
Periflagellated
Neutrophilic
Heterotroph
Utilize organic compounds as carbon source
Optimum temperature for growth 35 to 40OC
Optimum pH for growth 7
Growth medium Modified Bromfield medium (BFM)
1
Bromfield medium used for growth of P.polymyxa
KH2PO4 0.50 g/L

MgSO47H2O 0.20 g/L
(NH4) 2 SO4 1.00 g/L
Yeast extract 0.15 g/L
Sucrose 5.00 g/L
pH 7.00 g/L
As shown in figs.31.3 and 31.4, cells of P.polymyxa exhibited the highest adsorption on
kaolinite. Percent cell surface coverage was the highest on calcite, followed by hematite and
corundum. Cell coverage was the lowest on quartz. [185-186]
12
10
A. Kaolinite
B. Calcite
C. Corundum
10
11 D. Quartz
E. Hematite
A
10
10
2
Cells / m
B
9
10
C
D
8
10 E
0 2 4 6 8 10 12 14
pH
Fig. 31.3: Adsorption of bacterial cells as a function of pH on different minerals
2
A
100
80
Percent surface coverage

C
60
D
40
A. Calcite
20 B. Hematite
C. Corundum
D. Quartz
0
0 8 16 24 32 40
Period, h
Fig. 31.4: Percent cell coverage on different minerals as a function of time through image analysis
Adsorption behavior of Proteins
Kaolinite > Quartz >> Corundum >> Hematite> Calcite
Adsorption behavior of Polysaccharides
Calcite > Corundum >> Hematite >> Quartz > Kaolinite
4 A. Kaolinite A
B. Quartz
C. Corundum
B
D. Hematite
E. Calcite
2
Adsorption density, mg/m
2 C
1
D
E
0
0 20 40 60
Period, min
Fig. 31.5: Adsorption of bacterial protein on various minerals as a function of time
3
A. Calcite
16
A
B. Corundum
C. Hematite
B
D. Quartz
E. Kaolinite
2
Adsorption density,mg/m
12
E
0
0 20 40 60
Period, min
Fig. 31.6: Adsorption of bacterial polysaccharide on various minerals as a function of time
Quartz and kaolin clay exhibit higher surface affinity to bacterial proteins, while corundum,
hematite and calcite exhibit higher affinity towards exopolysaccharides (Figs. 31.5 and 31.6).
Biomass generation in the presence of different minerals is shown in table 31.1.
Table 31.1: Biomass generation in the presence of different minerals
Substrate Biomass (g /L)
Bromfield medium 1.0

Quartz 0.5
Corundum 1.5
Hematite 1.6
Calcite 10.7
4
Presence of calcite during bacterial growth promoted biomass generation. Proteins and
polysaccharides produced during bacterial growth are shown in table 31.2.
Table 31.2: Proteins and polysaccharides produced during bacterial growth
Polysaccharide, g/L Protein, mg/L
No mineral 0.48 71
Corundum 0.50 53
Hematite 0.55 50
Kaolinite 0.45 74
Quartz 0.44 79
Calcite 0.53 43
Presence of quartz and kaolinite during bacterial growth promoted secretion of bacterial proteins
and that of calcite and hematite promoted production of exopolysaccharides.
As can be seen from results from tables.31.3 and 31.4, bacterial interaction conferred enhanced
surface hydrophobicity on quartz and hydrophility on hematite and corundum. Mineral-
interacted bacterial cells were also affected. For example, interaction with quartz and kaolinite
rendered the bacterial cells more hydrophobic, while interaction with hematite, calcite and
corundum conferred cell surface hydrophilicity. [199-202]
Table 31.3: Variation of contact angle of different minerals before and after interaction with
bacterial cells
Mineral No interaction One hour interaction
Hematite 400 No bubble contact

Corundum 420 No bubble contact
Quartz 520 600
5
Table 31.4: Flotation of bacterial cells grown in presence of different minerals
Substrate Percent floated
No mineral 47
Quartz 90
Kaolinite 96
Hematite 25
Corundum 30
Calcite 20
Bacterial interaction significantly changes the surface chemical properties of the minerals as
shown in table 31.5. IEP of minerals are significantly affected after interaction with bacterial
cells, proteins and exopolysaccharides.
Table 31.5: IEP changes on minerals before and after interaction with cells and metabolic products
Mineral pH corresponding to IEP
Before Cells alone Metabolite Protein Polysaccharide
Interaction
Quartz 1.8 2.0 3.6 3.8 3.3 1.8 2.0
Kaolinite 2.0 2.5 2.6 2.5 2.5 2.0
Hematite 5.8 6.0 2.0 3.2 4.8 2.7
Corundum 7.0-7.2 2.0 4.2 6.6 2.8
Flotability (surface hydrophobicity) of minerals is also influenced by bacterial interaction.

(table.31.6). Quartz and kaolinite are rendered hydrophobic as attested to by increase in their
flotability. On the other hand, flotability of hematite, calcite and corundum is impeded after
bacterial interaction.
Table 31.6: Percent flotability of minerals under different biopretreatment conditions (1

hour interaction period, natural pH)
Control Cells alone Metabolite Protein Polysaccharides
Quartz 4 60 80 98 4
Kaolinite 38 84 80 94 35
Corundum 5 2 6 7 1
Hematite 4 3 4 4 1
Calcite 8 6 7 8 2
6
Interaction with bacterial cells and their metabolic products affected the flocculation-dispersion
of various minerals as shown in table 31.7. Dispersion of quartz and kaolinite was promoted,
while hematite, corundum and calcite increasingly flocculated after interaction with bacterial
cells and metabolic products. Bacterial proteins promoted surface hydrophobicity and flotation
(dispersion) of quartz and kaolinite. Interaction with exopolysaccharides promoted flocculation
and depression of hematite, calcite and corundum.
Table 31.7: Settling of minerals under different biopretreatment conditions
Percent weight settled (one minute) at neutral pH

Mineral Control Cells Metabolite Proteins Polysaccharides
Quartz 58 19 16 19 45
Kaolinite 60 25 17 20 50
Corundum 85 97 92 78 96
Hematite 85 98 96 77 95
Calcite 45 90 93 45 91
As can be seen from the results in table 31.8, efficient separation of quartz from hematite,
corundum and calcite could be achieved through selective flocculation after bacterial interaction.
Separation of kaolinite from hematite was found to be inefficient due to problems associated
with slime coating.
7
Table 31.8: Selective bioflocculation of mineral mixtures

Desliming Separation
stages efficiency,
Percent
Corundum-Quartz 1 89
2 93
3 96
4 98
5 99.6
Hematite-Quartz 1 21
2 52
3 67
4 92
5 95
Hematite-Kaolinite 1 30
2 37
3 42
4 55
Calcite-Quartz 1 25
2 38
3 56
4 71
5 89
6 98
8
Lecture 32: Alumina Separation And Environmental Control In Iron Ore Beneficiation NPTEL Web Course
Lecture 32
Alumina Separation And Environmental Control In Iron Ore
Beneficiation
Keywords: Iron Ore, Alumina Separation, Environmental Control
As illustrated in tables 32.1, 32.2 and 32.3, efficient separation of alumina (corundum) from
hematite requires prior adaptation of bacterial cells in the presence of corundum. Such
preadapted cells were found to secrete alumina-specific bioproteins. Corundum-adapted
bacterial cells and their metabolic products were efficient in the separation of hematite from
corundum through bioflocculation. Proteins separated from adapted cell metabolites could also
be used to separate alumina from hematite. [202]
Table 32.1: selective bioflocculation of corundum-hematite (1:1) mixture using corundum-adapted

cells
Percent
Deslimings hematite
removal
1 50
3 67
5 83
6 99
Table 32.2: separation of corundum-hematite through selective flocculation using corundum-

adapted bacterial metabolite
Deslimings Percent hematite removal

1 52
3 68
5 89
6 99
1
Table 32.3: selective flocculation of corundum-hematite mixture using corundum-adapted bacterial

protein
Deslimings Percent hematite

separation
1 55
3 70
5 98
6 99
Selective separation of hematite and silica from their mixtures containing corundum can also be
brought about using corundum adapted P.polymyxa. (table 32.4).
Table 32.4: Separation of corundum-hematite-quartz mixture through selective

flocculation using corundum-adapted cells
Percent hematite removal Percent silica removal

Deslimings pH pH
7.00 9.00 7.00 9.00
1 58 50 56 50
3 74.8 67 79 76
5 94 83 96 95
6 99 85 99 99
It should be however noted that the adapted cells need be preserved and frequently subcultured
in the presence of alumina to maintain their adapted properties. Subculturing of adapted cells in
the absence of the adapted mineral (corundum) will lead to deadaptation. As shown in table
32.5, deadapted cells lose their aquired mineral specificity.
2
Table 32.5: Separation of corundum-hematite mixture through selective flocculation using cells deadapted
through serial subculturing in mineral-free medium
Percent hematite removal

Metabolite
Number of subculture in
from
mineral-free medium Deadapted cells
deadapted
cells
1 99 98.5
3 66 70
5 33 32
6 17 11
7 0.0 0.0
Applicability of bioflotation and bioflocculation was demonstrated using real iron ore samples.
From an alumina rich iron ore containing upto 15% aluminium oxides and silicates, very
significant reduction in alumina and silica could be brought about through bioflotation or
bioflocculation as illustrated in table.32.6 and 32.7.
Table 32.6: Alumina removal from iron ores through bioflotation and bioflocculation
Conditions Percent concentrate analysis
Bioflotation after interaction with hematite- Fe(T) 69.5

adapted cells. Al2O3 0.3
SiO2 0.95
Recovery ~ 82 Fe
Selective flocculation, 6 deslimings, hematite- Reduction in alumina to 0.5% from the

adapted cells flocculated iron concentrate
Similarly, significant removal of silica could be obtained through bioflotation and

bioflocculation from another silica rich iron ore, containing upto 32% silica and 45% iron.
Typical results are illustrated in table 32.7.
3
Table 32.7: Silica removal from iron ores through bioflotation and bioflocculation
Concentrate
Experimental conditions Percent Fe Percent SiO2 Percent
Fe-Recovery
1. Flotation without
biotreatment using 0.15
No significant silica removal
kg/ton of amine collector
at pH 9.0
2. After 5min bacterial 50 10 90

interaction followed by
collector-less floatation
3. After bacterial treatment 67 0.9 90

as above followed by
flotation in the presence
of 0.15 kg/t of amine
collector
4. Selective bioflocculation 61 3 75
(in presence of cells) at
pH 7.0 with 6 desliming
stages
Bio-environmental control in mineral beneficiation [200]
Microbially-induced iron ore beneficiation would be more cost effective, energy-efficient and
environment-friendly compared to other chemical alternatives. Native microorganisms
inhabiting iron ore deposits are preferred since such organisms are already acclimatized to the
mining environment and are known to be involved in biomineralization. For example, FeOOH
sheaths formed by Leptothrix incorporates exopolysaccharides which protect the organisms
against metal toxicity. Magnetotactic bacteria synthesize magnetic minerals such as magnetite.
Since phosphorous is an essential micronutrient promoting microbial growth, phosphorous-
specific organisms colonize phosphorous-enriched zones of iron ore deposits. An anaerobe,
Shewanella oneidensis can recognize goethite which it prefers to colonise on, and not alumina or
gibbsite! Such organisms may prove to be critical in the separation of a desired impurity mineral
such as alumina or apatite from iron oxides.
4
Many mining organisms are also found to be capable of bioremediation of toxic effluents. For
example, Bacillus spp, Pseudomonas spp, yeasts and algae are capable of metal accumulation,
biosorption and biodegradation. The use of Paenibacillus polymyxa in the biodegradation of
several toxic flotation collectors commonly used in iron ore and sulfide mineral flotation has
been reported. For example, active cultures of P.polymyxa could efficiently degrade and remove
dodecylamine within about 8 hours. Similar biodegradation and efficient removal of oleate and
xanthate collectors could also be achieved. Typical results are presented in Table 32.8.
Table 32.8: Biodegradation of different collector reagents under various conditions of treatment
Percent degradation
Reagents
Cells alone Cells in fully grown
culture Cell-free metabolite
DAA (Dodecylamine) 100% (6h) 100% (2 h) 45%
DA-16 (Silica collector) 100% (8h) 100% (2 h) 45%
Sodium oleate 75% (5h) 80% (8h) 30%
Isopropyl xanthate 40%(5h) 100% (5h) 100% (10 h)
Cationic collectors such as amines exhibited pronounced biosorption onto bacterial cell surfaces
and the surface-adsorbed amines were also utilized and degraded by bacterial metabolism.
Adsorbed amine and oleate collectors from the floated iron ore surfaces could also be effectively
stripped through biological conditioning using P.polymyxa. Such biological surface stripping of
reagents from floated concentrates is of practical significance in subsequent pelletization process.
Improved pellet quality requires moisture control and mineral surface hydrophobicity plays a
determinative role.
Biotreatment of ore tailings would enhance settling rates of fines and would enable iron recovery
from wastes as well as water harvesting.
5
Restoration of ore mined sites requires functional microbial community for soil remediation and
fertility. Many mining microorganisms serve as biological indicators for ore mineralization and
also serve as pointers to disturbed mine sites.
Abandoned mine rehabilitation

Biorestoration of ore mined areas can be efficiently used. Native functional microbial
community will be useful in soil development, biogeochemical activity and for successful plant
community establishment.
Abandoned mine rehabilitation can be achieved through beneficial microorganisms such as
Rhizobia and Mycorrhizal fungi(AMF) Biological indicators of disturbed mine sites will serve
as monitors to identify polluted soils and waters..
Soil phosphate solubilizing microorganisms and cellulolytic population are used as biological
indicators of iron mined lands Brazil.
Restoration of tailing dams

Processed ore tailings containing moderate amounts of heavy metals such as iron, copper,
manganese, lead and zinc can be readily bioremediated. Microorganisms colonise such man-
made habitat. Soil bioremediation and phytoremediation are practiced. Biological restoration of
tailing contaminated soils will be cost effective and environment - friendly.
Metabolic activity of iron reducing bacteria will enhance natural or

engineered bioremediation of contaminated sites
Increasing surface hydrophobicity of iron ore fines through microbial treatment.
Dewatering of concentrates and tailings through microbes.
Microbially-induced flocculation/sedimentation of iron ore fines in tailing ponds.
Water harvesting and recirculation.
Microbial dephosphatisation of iron ores, slags and furnace dusts.
6
Lecture 33: Biobeneficiation Of Industrial Minerals NPTEL Web Course
Lecture 33
Biobeneficiation Of Industrial Minerals
Keywords: Industrial Minerals, Clays, Bauxite, Silica Sands
Biobeneficiation using Sulfate Reducing Bacteria (SRB)

Unlike acidophilic, aerobic autotrophs, sulphate reducing bacteria (SRB) such as Desulfovibrio
are anaerobic heterotrophs reducing sulphate to sulphide at an optimum pH of 6 7 with the
involvement of hydrogenase.
SO4- - + 4H2 = S- - + 4H2O
H2S and other metal sulphides are produced during their growth. From a flotation view point,
these bacteria could function as biological sulphidising agents. Many oxidized ores could be
sulphidised in the presence of SRB. Flotation of cerusstie was improved after pretreatment with
SRB. Besides, they can also be used as effective desorbents for xanthate coatings from mineral
surfaces. Selective separation of lead and zinc from sulphidic ores can be achieved after
pretreatment with SRB. Lead recovery in concentrate after bacterial treatment was about 95%,
with the sphalerite recovery only about 5% after SRB interaction. Selective separation of
chalcopyrite and molybdenite after biotreatment with SRB has been attempted.
The capability of SRB to produce large volumes of H2S and to convert metals and their salts to
sulphides could be effectively used in a number of mineral processing applications. Bacterial
reaction can be so controlled as to generate either a sulphide product (promotion of flotation) or
H2S gas (depressant). Bioconversion of iron oxides to ferrous sulphide, malachite to covellite,
silver carbonate/chloride to argentite and smithsonite to sphalerite could be achieved. SRB has
been increasingly used in environmental control and generation of biomaterials. Dissolved metal
ions in acidic and neutral effluents can be precipitated as sulfides after treatment with SRB.
1
Biogenic metal sulfides can be produced through interaction with SRB from different industrial
minerals.
Biobeneficiation of industrial minerals [214-226]

Silicon containing compounds in the form of clays stimulate microbial activity. Clays modify
microbial habitat. Some fungi are known to accumulate silicon. Biological reactions in the
silicon cycle is of practical significance. Many autotrophic and heterotrophic organisms can
break the siloxane bond, Si-O-Si. Biological destruction of quartz increases at alkaline pH.
Bacterial breaking of Si-O-Si or Si-O-Al bonds or removal of cations from the lattice of silicate,
lead to disintegration of the silicate lattice. Microbially produced ligands, organic or inorganic
acids, ammonia or amines or extracellular polysaccharides can solubilise silicates. Pseudomonas
generates 2-keto gluconic acid from glucose dissolving silicates of calcium, zinc and magnesium.
Fungi such as Aspergillus niger degrade spodumene. Sarcina ureae releases silicon from
plagioclase and quartz. Bacillus mucilaginosus produces polysaccharides that can react with
silica and silicates. Biological processes are of significance in mineral beneficiation with respect
to removal of silica, silicates, and oxides of iron, calcium and aluminium.
Biologically mediated silica removal from magnesite ores has been achieved using Bacillus
circulans, Bacillus mucilaginosus and Bacillus licheniformis. Desiliconisation of bauxite, iron
ores, manganese and phosphate ores could be achieved through appropriate microorganisms.
The removal of silica from bauxite using silicate bacteria Bacillus circulans was reported.
Biologically produced concentrates yielded higher values of alumina using growing cells. Low-
grade bauxites can be bioleached by Aspergillus niger, Bacillus circulans and Pseudomonas
aeruginosa. Bacillus mucilaginosus and Aspergillus niger were found useful for the removal of
Al2O3, SiO2 and Fe2O3 from bauxite.
Bacterial removal of iron from clays is of industrial importance in the production of commercial
grade raw materials. Oxalic and citric acids produced by Aspergillus niger could remove iron
from clays. The iron content of different kaolins could be brought down through bacterial
action.
2
Removal of calcium and iron impurities from low-grade bauxite using Paenibacillus polymyxa
for abrasive and refractory applications has been reported.
A microbial survey of some Indian bauxite mines revealed the indigenous presence of various
autotrophic and heterotrophic bacteria and fungi associated with the ore body and water bodies in
the vicinity. Bacteria of the type Acidithiobacillus, Bacillus and Pseudomonas are implicated in
the weathering of alumino-silicates, precipitation of iron-oxyhydroxides, dissolution and
conversion of alkaline metal species, and formation of alumina, silica and calcite minerals.
Fungi such as Cladosporium can reduce ferric iron and dissolve alumina silicates. Biogenesis
plays a significant role in bauxite mineralization.
Paenibacillus polymyxa can selectively remove calcium and iron impurities from low-grade
bauxite. An industrial scale formulation of Bromfield medium (called ISF-2) based on cane
sugar and tap/mine water, was developed to culture Paenibacillus polymyxa under asceptic
conditions. The bioleached residue was found to be enriched in alumina with only trace amounts
of iron and calcium. A column bioreactor, which can be operated either in fluidized bed mode
for coarser particles or in total recycle slurry mode for the finer particles was designed, and
tested for biobeneficiation.
Biological removal of phosphorous and silicon from manganese ores is yet another example.
Hypomicrobium sp, and Aspergillus niger are efficient phosphorous removers. Rock phosphates
can be solubilized by Rhizobium and Bradyrhizobium. Bacillus mucilaginous also aids in
phosphorous removal from ores.
Biogenesis of clay minerals

Clay minerals are biologically converted under environmental conditions and iron-containing
diagenetic minerals as iron hydroxide nanocrystals and biogenic clay particles are deposited on
fungal hyphae and bacterial cell envelopes.
3
Natural clay formations present a unique subsurface microbial habitat. Many types of
microorganisms are trapped during deposition of clay layers.
Iron is present in brown and yellow - tanned kaolins as ferric oxides and hydroxides (hematite
and goethite) as a structural replacement in kaolinite, smectite and mica. In grey kaolins iron is
present as pyrite or lower sulfides. Sulfur contents in clays are of biogenic origin.
Clay minerals are ubiquitous in soils and sediments. Together with microorganisms, clays serve
as catalytic surfaces in sedimentary deposits. Biogeochemical cycles are involved in clay
mineral formation and conversion. Similarly, iron and sulfur cycles in clay minerals are mediated
by microorganisms.
Microbial clay reduction has been proved at temperatures and pH encountered in soils. Organic
complexing agents enhance the bioavailability of ferric ions for reduction. Ferric ions associated
with clay minerals can be biologically reduced.
Aerobic microorganisms generating ferric ion specific chelating agents called siderophores
influence iron cycle in clays. Iron is an essential micro nutrient for many microbes. In
oxygenated neutral environments, availability of iron is controlled by the solubility of iron
hydroxides. Aerobic microorganisms produce siderophores to mobilize iron. For example,
siderophore-producing Pseudomonas mendocina, acquire smaller concentrations of iron from
kaolinite. Bacterial siderophore, Desferrioxamine B inhances the dissolution of iron, silicon and
aluminium in kaolinite.
Changes in the composition of iron minerals can occur due to indigenous microorganisms.
There exists a bacteria iron cycle in clay mineral formations.
Increase in magnetite content.
Synthesis of unstable hydroxides such as:
ferrihydrite. 5Fe2O3 . 9H2O
akaganeite ( FeOOH) and
lepidocrocite FeOOH
Formation of ferrous sulfides and pyrites.
Alterations in morphology, structure and composition
4
Simplified iron bacteria cycle in clays is illustrated below:
Iron containing Dissimilatory reduction FeS2

minerals in clay (Iron-reducing bacteria)
Hematite Reductive dissolution
(Bacillus, Nitrobacteria, SRB
Magnetite
Clostridium,
Goethite Desulfovibrio) Fe++ Fe+++ Fe (OH)3
Complexation due to
Lepidocrocite IOB
microbial metabolites
Silicates
Magnetite
Fe+++
Goethite
Ferrihydrites
Lepidocrocite FeOOH
Hematite Goethite
Both oxidation and reduction of iron and precipitation occur mediated by various
microorganisms.
Potential for industrial biotechnology to remove iron from kaolin
A biological process for iron removal from clays would be cost-effective, flexible and
environment-friendly.
Depletion of high grade kaolin deposits necessity to mine low grade kaolin which are colored
due to presence of iron impurities. Development of flexible biotechnology for kaolin
beneficiation, where either microbial ferric reduction or ferrous oxidation would be stimulated,
depending on the iron mineralogy in the kaolin. For commercial quality kaolin, ferric reduction
of hematitic and goethitic iron would enhance the quality of coloured and hard kaolins.
Use of indigenuous ferric reducing bacteria for kaolin whitening would be beneficial.
Stimulation of microbial ferric oxidation has the potential for efficient clay whitening. Ferric iron
reducing microorganisms can remove ferric oxides from kaolin. For example, Bacillus sp. and
Shewanella have been used for iron removal from clays.
5
Selective flocculation is a preferred method for removal of titania. In some selective flocculation
methods bacterial cells can be used as flocculants. Bio reagents such as proteins and
polysaccharides can selectively flocculate the titania particles to settle to the bottom of a
thickener leaving the product kaolin to be recovered from the supernatant in dispersed form.
Microbiological reducing agents will also be useful. Biotechnological methods for leaching
kaolin using Aspergillus niger, Enterobacter aerogens and Leuconostocmesenteroides have been
proposed. Bioreducing agents such as hydrosulfides, oxalic acid etc, can solubilise ferric oxides
and leach them selectively.
Microbial flocculation of clay fines

Finely divided clays, or clay-slimes, are generated during the beneficiation of phosphate and
potash ores. The use of microorganisms for dewatering clay-slimes and enhancing settling rates
would be beneficial. Microorganisms such as Xanthomonas and Azotobacter (all polymer-
producing microbes) grow actively in presence of phosphatic clay-slimes. These organisms
induce flocculation when grown in the presence of the phosphatic clay-slimes. Phosphatic clay-
slimes can be effectively flocculated using live organisms. Biopolymers isolated from
L.mesenteroides, Xanthomonas and Beijerinckia indica, flocculated phosphatic clay-slimes at a
rate comparable to the synthetic polymer, polyethylene oxide (PEO).
Microorganisms such as Bacillus sp play an important role in the dissolution of silicate structure
and in the genesis of clay minerals. Interaction with these bacteria can remove upto 50% of free
iron occurring in kaolin and about 20% of bound iron in mica after a month of bioleaching.
Prolonged bioleaching led to the removal of more than 50-60% of bound iron. Biodegradation of
iron oxyhydroxides and partial destruction of mineral structure lead to transformation of one
mineral form to another, leading to improvement of qualitative properties of kaolins and quartz
used in ceramic and glass industries.
Some examples of bio-beneficiation of clays:

Iron leaching from oxide minerals by microorganisms known since 1954.
Eg: Iron removal from quartz sands, kaolins and clays.
Use of bacterial and fungal metabolites at high temperature will enhanced iron removal rates.
6
Bacillus cerius isolated from kaolin deposits could be used to remove more than 50% of free
and 20% of bound iron.
Fungal strains of the type Aspergillus, Penicillium, Paecilomyces can be used for iron
removal from clays.
High amounts of oxalic acid produced by A.niger will selectively dissolved ferric oxides.
Stimulation of activity of natural microflora inhabiting clay deposits through nutrient
additions in situ.
Bacterial growth and production of organic acids and their metabolites, decrease pH, and
alter Eh leading to transformation of iron minerals.
Combination techniques such as initial iron removal by magnetic separation prior to and after
microbial treatment would enhance efficiency.
For example, iron content in kaolin could be reduced from 0.8 to 0.2%.
In kaolin samples, development of microorganisms resulted in appearance of small amounts of
dissolved iron in the supernatant (7.0 mg/L) and pronounced increase in iron extracted during
magnetic separation (10 to 58%)
De-ironing of commercial porcelain mass
Bacterial development in the inner layers of porcelain mass supplement nutrient medium
First stage: Growth of aerobic and facultatively anaerobic bacteria
Second stage: Anaerobic (SRB, iron reducing)
Microbial removal of pyrite can be achieved from low-grade clay using a sulphur and iron-
oxidizing bacterium, such as Acidithiobacillus ferrooxidans. For example, about 90% of pyrite
could be removed in about 10 days at pulp densities upto 70% (w/v). With the refined clay no
red colour due to the presence of pyrite was developed after firing, and its whiteness was similar
to that of a high-grade clay. Biotreatment with Acidithiobacillus thiooxidans in combination
with At.ferrooxidans at higher acidity (pH 0.5 1.0) would be more efficient for iron sulfide
removal from clays.
Ferric oxide impurities, which reduce refractoriness and whiteness from porcelain and pottery,
could be biologically removed from even low-quality clays by indigenous dissimilatory iron
reducing microorganisms. Insoluble ferric oxides in clay fines and slimes can be leached out as
7
soluble ferrous iron through biological ferric reduction. By such microbial treatment, the
whiteness of clay increased from 60% to 80% and the redness decreased from 14 to 3.5.
Microbial polysaccharides can aid such ferric reduction.
8
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10
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13
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14
Lecture 38: Acid Mine Drainage Mechanisms And Control NPTEL Web Course
Lecture 38
Acid Mine Drainage Mechanisms And Control
Keywords: Acid Mine Drainage, Chemolithotrophs, Mechanisms, Control
Acidophilic chemolithotrophs such as Acidithiobacillus are responsible for the generation of

acid mine drainage from abandoned and closed mines, waste piles and processed tailings.
Microbiological aspects and mechanisms of acid mine water generation from sulfide mines
are illustrated. Methods and industrial applications of bioremediation technologies
applicable to mining industries are critically analyzed in lectures 38-40 [245-250].
Acid mine drainage

Acidic sulfate and metal ion-rich waste waters and effluents from mining operations are a major
problem. The origin of acidic metal rich drainage, called, acid mine drainage (AMD) is many,
such as abandoned and closed sulfide ore mines, waste tailing dumps and mined over - burden.
Acid mine drainage can be defined as metal-rich acidic water formed by chemical and
biochemical reactions between water and waste ores, rocks and over - burden. The run off
formed is usually contaminated with dissolved metal ions and acidic constituents originating
from existing or abandoned metaliferrous and coal mines (fig.38.1). Such acidic drainages can
seep into ground water table and contaminate surface and ground waters, disrupt growth of
aquatic animals and plants and corrode infrastructure installations. In this respect, closed and
abandoned mines are more prone to acid mine drainage. [245]
1
Fig. 38.1:Brownish yellow colored acid mine drainage from a sulfidic mine.
Chemical composition of AMD vary widely from mine to mine. Generally reported composition
corresponds to
pH 16
CaCO3 0 50.000 mg/L
Fe 1 10,000 mg/L
Mn 1 50 mg/L
SO4-- 1 25,000 mg/L
A variety of metals could be present such as Cu, Zn, As, Cr etc.
Primary factors that influence AMD include,
Type of sulfide mineral

Oxygen availability
pH
Temperature
Presence of alkaline constituents
Exposed surface area and particle size
Presence and type of bacteria
2
Photographs of a tailing dump site and an abandoned mine cavity containing acidic water are
illustrated in fig. 38.2.
Fig. 38.2: A tailing dump site and an abandoned mine cavity containing acidic water
Bacterially-mediated pyrite and other mineral sulfide oxidation generates sulfuric acid containing
ferric and other dissolved metal ions.
2FeS2 + 7O2 + 2H2O = 2Fe++ + 4SO4-- + 4H+
2Fe++ + O2 + 2H+ = 2Fe+++ + H2O
2Fe+++ + 6H2O = 2Fe (OH)3 + 6H+
FeS2 + 14Fe+++ + 8H2O 2SO4- - + 15Fe++ + 16H+
Acidophilic bacteria such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans

play a significant role in accelerating the above acid-producing reactions. These bacteria
catalyze the oxidation of ferrous ions and sulfides. [246]
3
Other than pyrite, a number of sulfide minerals such as sphalerite, chalcopyrite, covellite, galena
and millerite can be biooxidized causing acid mine drainage.
ZnS + 2O2 = Zn++ + SO4
PbS + 2O2 = Pb++ + SO4 - -
NiS + 2O2 = Ni++ + SO4- -
CuFeS2 + 4O2 = Cu++ + Fe++ + SO4- -
CuS + 2O2 = Cu++ + SO4- -
The simultaneous presence of reduced iron and oxygen promotes the growth of acidophiles such
as Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans and At.thiooxidans. Thermophilic
organisms such as Sulfolobus and other sulfur-oxidizers can also participate in AMD generation.
Biooxidation of sulfide to sulfur and to sulfate is significant and could determine rate of acid
generation.
2S + 3O2 + 2H2O = 2H2SO4 (Acidithiobacillus)
12FeSO4 + 3O2 + 6H2O = 4Fe2 (SO4)3 + 4Fe (OH)3 (At.ferrooxidans)
Biooxidation of sulfur can take place from a pH of 8 to 1.5, in the presence of neutrophilic and
acidophilic organisms. A succession of bacterial species is involved, resulting in the decrease of
pH from neutral to acidic levels. Similar bacterial mutualism may be involved with respect to
temperatures and other environmental parameters such as Eh, metal ion concentrations and
oxygen levels.
Bioacid generation will enhance dissolution of other metals and minerals.
There are a number of laboratory procedures to determine acid generation capacity of mine
wastes and the screening tests include determination of
Conductivity
Chemical composition
4
Acid-Base Accounting (ABA)

pH
Mineralogy
Balance between acid generation and acid neutralization can be evaluated by acid-base
accounting. Maximum potential acidity and acid neutralizing capacity can be thus ascertained.
The above are static tests, based on single measurements. There are kinetic tests to simulate field
conditions, such as column tests, which can be used to establish sulfide activity, weathering
behavior, role of bacteria in acid generation and to evaluate sulfide mineral oxidation rates and
metal dissolution. Larger sample volumes can be used in column tests which can be carried out
over extended periods of time (even years). Typical sampling sites are illustrated in fig. 38.3.
Fig. 38.3 Abandoned mine and tailing sites for sample collection
Based on the acid-generation-capacity, mine wastes can be classified as potentially acid forming
and non-acid forming materials.
Interactions between members of microbial consortia are critical in AMD generation. Mutualism
exists between heterotrophs and autotrophs, mesophiles and thermophiles and aerobes and
anaerobes. Archaea are abundant in AMD environments. Iron oxidising Ferroplasma have been
isolated from acidic streams. Thermoplasma, Sulfolobus spp, and F.acidarmanus could be
isolated from different mines. Acidiphilum can adapt to temperatures upto 450C.
5
Acid generating capacity can be characterized through knowledge of
Amount of acid producing sulfides present.

Amount of acid neutralizing minerals
Other contaminants.
Rate of acid generation is influenced by type of sulfide mineral, available surface area and
particle size, type and amount of acid neutralizing minerals, water and oxygen as well as
presence of bacteria.
Prevention or minimization of AMD can be based on different approaches such as [245-246].
Sealing / flooding of underground mines

Storage of tailings underwater.
Mineral wastes blending
Sealed waste heaps for land storage
Use of proper surfactants
Coating as microencapsulation
Control of dissolved oxygen in waters that are in contact with ore burden and wastes would
minimize growth and activity of sulfide and iron oxidising acidophiles. Soil cover and water
cover to prevent oxygen diffusion into waste tailings and exposed rocks would be beneficial.
Shallow water covers can prevent contact between sulfide minerals and oxygen. Tailings
covering with a layer of organic or sediment may be useful to limit oxygen diffusion and to
prevent erosion due to high winds and tides of water. Blending as a preventive strategy involves
appropriate mix of acid consuming and acid neutralizing minerals so as to maintain a higher
neutral pH of the composite mixture. Surface coatings using phosphate (ferric phosphate) or iron
oxide/silica coatings are also recommended. Biocides such as anionic surfactants (SDS) can
inhibit bacterial activity and growth.
However, all the above AMD elimination strategies may not be worthwhile as a long term
measure.
6
Remediation of existing acid drainages and polluted water and soils may be a more suitable
engineering strategy. In this respect, both active and passive remediation strategies need be
considered.
Active process: Continuous application of neutralization and precipitation of metals.
Passive process: Use of natural and constructed wet land technology-low cost and maintenance.
Various biological and abiotic remediation approaches can be classified as follows:
Abiotic processes:
Lime neutralization along with aeration (active) - Use of lime, limestone, sodium
hydroxide.
Anoxic limestone drains (passive) Alkali addition maintaining ferrous iron not
precipitating on limestone.
Bio-processes:
Use of hybrid bioreactor systems (active)

Aerated wetlands (passive)
Reactive (permeable) barriers (passive)
Bioremediation depends on the ability of microorganisms to immobilize metals and produce

alkaline conditions. Phytoremediation is yet another approach. In aerobic wetlands, Typha and
Phragmites are used. Microbial alkalinity generation processes include sulfate reduction, iron-
manganese reduction and denitrification. Photosynthetic organisms can generate alkalinity.
Dissimilatory sulfate reduction by anaerobes can also generate alkalinity.
SO4- - + 2CH2O + 2H+ = H2S + 2H2CO3
Dissolved metal ions can be biologically precipitated and removed through SRB.
M++ + H2S = MS + 2H+
7
Many of the bioprocesses for AMD control depend on passive systems such as constructed
wetlands and compost bioreactors.
Aerobic wetlands are used to treat alkaline mine waters. If necessary, pH of the mine water can
be increased through lime additions. Relatively shallow operating on surface flow will maintain
aerated conditions. Macrophytes can be planted to regulate water flow and to stabilize ferric
precipitates. There may be iron oxidation in the wetlands due to both abiotic and biotic
processes.
Bioremoval of arsenic will be facilitated in aerobic wetlands. Solubilisation of arsenopyrite

(FeAsS) into As(III) and As(V) occurs due to microbiological reactions in the presence of
acidophiles. The solubilized arsenic species may be removed by adsorption into ferric iron and
formation of FeAsO4 (scorodite). Arsenic-tolerant Thiomonas can oxidize As(III) to As(V),
besides oxidising ferrous ions and detoxify arsenic polluted waters.
In compost bioreactors, anaerobic environment prevails. Biogenic sulfides and alkalinity will be
generated. Organic matter in the compost (manure, vegetable matter, saw dust etc) provides
reducing conditions and promotes growth of anaerobic bacteria.
Composite aerobic-anaerobic wetland systems have also been introduced.
8
Lecture 3: History And Methods In Biohydrometallurgy NPTEL Web Course
Lecture 3
History And Methods In Biohydrometallurgy
Keywords: Historical Perspectives, Methods, Biohydrometallurgy
In lectures 3-5, historical perspectives in bioleaching, methods in biohydrometallurgy,

microorganisms implicated in bioleaching and types of bioleaching of ores and
concentrates are discussed [7-17].
Biohydrometallurgy as part of mineral or mining biotechnology incorporates several

microbially-mediated processes, such as
Bioleaching
Biooxidation
Bio-induced surface chemical changes and
Bioremediation
Bioleaching involves leaching (dissolution) of a metal from a solid mineral facilitated by activity
of microorganisms (bacteria, archaea). On the otherhand, in biooxidation brought about by
bacterial action, the valuable metal is just liberated from the mineral matrix and remains enriched
in the leached solid residue. (Bioliberation of gold entrapped in pyrite and arsenopyrite matrix).
Bioleaching is commercially used to process copper, uranium, zinc, copper and nickel-containing
ores. Bio-induced surface chemical changes confer surface hydrophobicity or hydrophilicity on
interacted minerals promoting their flotation, flocculation, dispersion or depression. Such
microbially-induced beneficiation processes are essentially interfacial reactions without any
mineral dissolution. Use of microbes in detoxification and decontamination of polluted soils and
water is referred to as bioremediation. For example, acid mine drainage is caused by microbial
activity and its remediation can be brought about using native organisms.
1
Historical perspectives
Role of microbial processes in mineral dissolution was not recognized till late 1900s. However,
natural leaching of metals from rocks was reported even as early as 20-70 AD. Georgius
Agricola (1494-1555) referred to copper leaching from ores and leachates from mines. The
history of bioleaching begins much earlier than one might ponder. In china and India, natural
recovery of copper and other base metals such as zinc from solutions emanating from rocks was
known almost 2000 years ago. The Rio Tinto in Spain owes its nomenclature to the presence of
reddish brown water having higher concentrations of ferric ions. Natural iron and copper
dissolution from minerals through activity of native microorganisms was later recognized at
RioTinto. The contribution of sulfur and iron-oxidising Acidithiobacillus ferrooxidans in
generating ferric-ion containing sulfuric acid which can dissolve minerals such as copper sulfides
was scientifically established in 1947. Since the 1950s research activities on the use of
Acidithiobacillus picked up and commercial applications of bioleaching particularly in copper
dump and heap leaching began to emerge. The run-of-mine lean grade copper ores were stacked
in waste dumps over 100m in height and leached using acidic solutions for economic copper
extraction at the Bingham mines of Kennecott Copper Company during the late 1950s and early
1960s. Heap and in situ mining using native microorganisms were developed later.
Heap bioleaching of uranium was reported in the 1960s, mainly in Canada.
Commercial applications of biohydrometallurgy on a larger scale began in the 1980s for heap
bioleaching of secondary copper sulfides and oxidized ores. Numerous copper heap bioleach
systems have been commissioned since the 1980s.
In 1986, bioreactor processing for refractory sulfidic gold-bearing concentrates was

commercialized at Fairview mines in South Africa. Since then, several gold-bioreactor industrial
operations have been initiated. Piloting and prototype stirred tank reactor systems for
thermophilic bioleaching of base metal (copper, zinc, nickel) were then established. In 1999,
commercial cobalt bioleaching from pyritic concentrates was established.
2
Some year-wise developments in metals biotechnology since 1947 are listed below:
(Adapted from presentation at Mintek 75 Technical conference, June 2009 by P.J. van Staden)
1947: Acidithiobacillus ferrooxidans was identified (Colmer A.R., Hinkle M.E. (1947).
Science 106:253-256)
1950s: Leach-dumps for copper. Kennecott
1960s: Dump / heap leaching for copper, uranium lean ores - insitu leaching.
1965: Iron and sulfur oxidizing archaea
1977: First international biohydrometallurgy meeting, Braunschweig, Germany.
1980: Lo Aguirre, first heap bioleaching plant.
1985: In-situ uranium heap leaching using intermittent flooding and forced aeration.
1986: Fairview S.Africa: first commercial refractory gold biooxidation plant
(many gold bioreactors to follow).
1987: Paques anaerobic systems for effluent treatment.
1993: Forced aeration on heap bioleach systems.
1995: Bioleaching of chalcopyrite concentrate developed and evaluated on commercial
scale.
1997 2000: BioNic and BioZinc for nickel and zinc bioleaching.
1999: Cobalt bioreactor plant, Uganda.
2000: Commercial scale applications of archaea.
2002: Penoles, Mintek, BacTech Chalcopyrite concentrate bioleach pilot plant, Mexico,
2002: BHP Billiton / Alliance Copper commercial demonstration plant for
copper- enargite concentrate
2002: GEOCOAT Thermophilic Bioleachng of Chalcopyrite Concentrates, Field Trials
2003: Demonstration. BioCopTM, BHP. Billiton (chalcopyrite)
2006: High temperature heap bioleaching, transitional primary / secondary copper ore
(Mintek)
3
Major companies that developed biooxidation processes
Newmont BIOPROTM Heap bioleaching of sulfidic refractory gold ores (0.09 2.0
g/t) 2000.
BHP Billiton BIOCOPTM agitated tank bioleaching for copper sulfides.
BIONICTM agitated tank bioleaching for nickel sulfides.
BIOZINCTM agitated tank bioleaching for zinc sulfides.
High Temp. Heap bioleaching of chalcopyrite ore.
Gold Fields BIOXTM Agitated tank biooxidation / leach for copper sulfides and
refractory sulfide gold concentrates.
Bac Tech Bac Tech / Mintek-agitated tank oxidation / leaching of copper sulfides
Environment and refractory sulfide gold concentrates.
Geo Biotics GEOCOATTM Bioheap leaching of sulfide concentrates.
Methods in biohydrometallurgy
Bioleaching is critically dependent on factors such as bacterial concentration, activity and its
growth, rate of biooxidation reactions, and iron oxidation ability of bacteria.
Development of adapted cells play a vital role in the efficient and faster dissolution of minerals.
Bacterial adaptation to toxic ions and ore minerals significantly enhances dissolution rates of
sulphides, reduce the lag phase and decrease the solution pH. Bacterial tolerance limit can be
increased by serial subculturing and adaptation to higher metal concentrations. Adapted strain of
Acidithiobacillus ferrooxidans exhibited high copper and nickel extraction from a copper and
nickel concentrate. Enhanced sphalerite dissolution has been observed by using a pyrite -
adapted Acidithiobacillus ferrooxidans strain. In the simultaneous presence of zinc, copper is
less toxic to Acidithiobacillus ferrooxidans and copper toxicity can be minimized.
Major current commercial applications of bioleaching are extraction of copper and uranium from
low grade ores and waste ore burden through heap bioleaching and insitu mining and
4
enhancement of gold recovery from sulphidic, refractory ores and concentrates, through stirred
bioreactor processing. Bioreactor processing of pyritic cobalt concentrates has been
commercialized in 1999. The relevance of bioleaching for the extraction of other base metals
such as zinc, lead, cobalt, nickel and molybdenum need also to be stressed. Another significant
application of biooxidation is in the processing of complex multisulphide ores and concentrates.
Bioleaching of metal concentrates such as the ones containing copper and zinc in bioreactors is
of great commercial interest. All these applications have direct relevance to India.
Three methods have been commercially employed for extraction of metals from lean grade ores
and waste overburden.
In situ leaching or solution mining

Dump leaching
Heap leaching
All the above methods have been practiced over the last four decades with advantages such as
low cost, suitability for different mines and lean grade ores and environmental acceptability.
In situ leaching
Microbial in situ leaching (or solution mining) is a unique method of metal extraction from run
of mine ores and uneconomical underground ore bodies. Underground mined stopes can be
leached. Leach solutions are injected and percolated into rock cavities and the pregnant solution
recovered by pumping. Selective blasting of rock can be done to gain access to regions of rich
mineralization.
Application of in situ leaching depends on the geological and topographical features of the ore
body, and also on the hydrological and rock-mechanical properties of the host rock. Feasibility of
in situ leaching need be established with respect to rock permeability and accessibility.
5
Dump bioleaching
Mining operations over several years produce millions of tonnes of waste ore. Waste ore dumps
remain at the mine site and cannot be economically processed for extraction of metals by any
conventional methods. Bioleaching offers an economical alternative to process such overburden.
Most of the dumps are generated without any planning and contain different sizes of ore
particles. Dump geometry conducive for efficient leaching need be analyzed. Dumps positioned
parallel to each other and greater in length than width and height are called finger dumps which
have dimensions of the order of 800 m x 35 m x 200 m. Permeability of the fragmented rock
mass in the dump is important. Too much of fines affect percolation of leaching solution.
Dump leaching involves percolation of acid solution through the mineralised particles across the
mass. Native microorganisms are allowed to proliferate by providing optimum growth
conditions. Metals are biologically solubilized and the effluent collected at the bottom is
enriched with the metal content. The leach solutions are treated to recover the dissolved metal.
Several methods have been practised for efficient irrigation of the waste dumps.
Formation of shallow square or rectangular ponds covering the top dump. The lixiviant
(sulphuric acid solution at a pH of about 2) is pumped to the ponds through a network of
distribution lines. Sections of such ponds can be flooded to permit continuous availability
of percolating solution. Intermittent leaching with intervals of wetting and rest cycles is
preferable.
The leach solutions can be distributed on the upper portions and sides of the dump
through sprinklers. Compressed air can be used and leach solutions are allowed to flow
through the cross sections in the dump.
Since the biooxidation of sulphides is exothermic, temperatures of the order of 60-80oC can be
attained inside the dump. Microbial activity under different temperature conditions need be
6
understood for efficient leaching. Acidithiobacillus ferrooxidans and Acidithiobacillus

thiooxidans which are the most important acidophilic bacteria present in the dump have optimal
activity at temperatures of the order of 30-35oC. With rise in temperature, mesophiles undergo
thermal death. Presence of several thermophilic autotrophs which can tolerate higher
temperatures has been reported in the dumps. Bioleaching with better reaction kinetics can thus
be maintained even at higher temperatures. Synergistic activity between mesophiles and
thermophiles bring about biooxidation of the sulphides in dump leaching operations.
Heap bioleaching
As different from dumps, heaps of low grade ores or waste materials could be prepared on
impervious bottoms with control exercised with regard to amount of the ore materials, particle
size, height and irrigation methodology. The heap bioleaching otherwise is similar to dump
leaching. The following aspects need be considered.
Topography, geological and mineralogical aspects

Geography, environmental and ecological features
Operational procedures including particle size distribution, dimensions and irrigation
techniques.
Heaps are arranged in the immediate vicinity of the mines. Ground impermeability is important.
Nonporous pads are created on which heaps are arranged. Impermeable bottoms can be made of
concrete, asphalt, pitch, coal-far or synthetic polymeric sheets. Slopes need be maintained to
permit collection of percolated leach solution. Particle size distribution and porosity of the ore
particles in the heap should be controlled to enable percolation of leach solution and oxygen
diffusion. In many commercial heaps, run-of-mine ores containing about 4% - 100 mm and 25%
- 20 mm particles are used. Excessive fines result in slime formation, impeding percolation. In
case of excessive fines, prior agglomeration may become essential. The height of the heap need
be so chosen to attain adequate oxygen diffusion through natural draft. Heap sizes of 6-10 m
7
height, 137 m length and 107 m width containing more than 50,000 tonnes of ore are common.
Typical heap design is shown in fig.3.1.
Acid solution
Heap bioleach pad

Leach solution
recovery and
metal extraction
Oxidation and bacterial growth pond.

Metal free acid solution
Addition of energy source + nutrients
Fig 3.1: Heap leaching set-up
Commercial scale bioleaching started in the form of dump leaching during the 1950s. Heap
bioleaching of secondary copper sulfides has since taken over very effectively, especially in
Chile. Bioleaching of refractory sulfidic gold ores in heaps was also tested on a demonstration
scale and evaluated (Carlin, Nevada).
In copper heap bioleaching crushed ores are blended and the fines agglomerated. Agglomerated
and acid-preconditioned ore is then stacked (6-10 m high) on lined pads or top of a prior-leached
ore. Air supply is provided through piping and irrigated with raffinate. Pregnant solutions are
collected at the base of the heap. Copper is recovered through solvent extraction-electrowinning
route and the solution recycled to the heap. Leach period could be 200-300 days with upto 85%
recovery.
Unlike copper, heap bioleaching for enhanced gold extraction has not yet been widely
commercialized. Data based on pilot trials are available. Even though, there are several
8
similarities between both copper and gold ore bioheap leaching, some essential differences do
exist. The precious metal gets enriched in the bioleached residue and has to be lime-treated and
cyanided to extract gold.
Strict and frequent monitoring of bioheaps is essential. Pregnant leach liquors are analyzed for
redox value, pH and metal concentrations. Similarly, ore samples from different depths of the
heaps need be so assayed. Temperature and oxygen levels at different heap locations need be
assessed. Bacterial counts are generally made in liquid samples. But role of solid-attached
organisms also need be understood.
Heap bioleaching microbiology has not been well understood. Many parameters influence
microbiology, such as acid addition, nature and amount of sulfide minerals, solution chemistry,
availability of oxygen and nutrients as well as temperature. Dominance of Acidithiobacillus
group of organisms will be determined by pH, iron and sulfur content in the heap materials.
Temperatures in different regions of the heap will be higher due to exothermic reactions and
moderate and extreme thermophiles can play a role in mineral dissolution.[7 - 15]
9
Lecture 43: Laboratory Experiments In Metals Biotechnology I NPTEL Web Course
Lecture 43
Laboratory Experiments In Metals Biotechnology - I
Keywords: Microscope, Petroff Hausser Counter, Bacterial Isolation
The subject matter of METALS BIOTECHNOLOGY is interdisciplinary dealing with

microbiological, chemical and metallurgical aspects. It thus becomes essential to familiarize
with microbiological aspects such as identification, growth and culturing of relevant
microorganisms, enumeration and growth protocols, bioleaching techniques,
bioremediation strategies as well as other laboratory procedures for culturing mining
bacteria and evaluation of their metabolites. In lectures 43-45, laboratory techniques and
research strategies in metals biotechnology are illustrated [259-266].
Microbiological techniques
Microscopy
Microscope is an optical instrument consisting of a combination of lenses to view magnified
images.
Phase - contrast microscope
Unpigmented living cells are not visible clearly in bright field due to little difference in contrast
between cells and water. A phase contrast microscope adjusts small differences in refractive
indices and cell density into easily detectable changes in light intensity. Living cells can be very
clearly viewed at different magnifications. A photograph of phase contrast microscope is given
in fig.43.1.
Phase contrast microscope has two additional plates - annular diaphragm and phase shifting
plate. Such a modification permits only a ring of light to pass through the condenser and
subsequently to the objective. When incident light falls on a specimen, two rays emerge. When
1
direct and defracted beams join, phase differences are apparent and contrast in the image is
obtained.
Phase contrast microscopy is useful to assess microbial motility, endospores, shape, and
microbial encrustations or inclusions. For bacterial cultures generally used in
biohydrometallurgy, such as Acidithiobacillus sp. phase contrast microscopic examination is
very useful.
2
Fig. 43.1: Phase Contrast Microscope
Cell Count using Petroff Hausser counter
Bacterial cell counts can to be made using Petroff Hausser counter which is depicted in fig. 43.2.
Cell count of bacteria has to be taken by directly putting a drop of culture on to the counter slide
and cell number measured under a phase contrast microscope. The counter consists of ruling
covering a square millimeter. The center square millimeter is ruled into 25 groups, each
consisting of 16 squares. All the 25 groups are separated with triple ruling where as each of the
single squares of 16 square are singly ruled. The height of the ruling wires is 0.02 mm in height.
The area of each squares are 1/400 mm2. The bacterial cells have to be counted in this center
square.
Typical calculations are given below:
Depth of small square = 1/50 mm
Area of small square = 1/400 mm2
3
Volume of small square = 1/50 x 1/400 mm3 = 1/50 x 1/400 x 10-3 cm3.
No of cells per ml = Average no. of cells counted per small square/volume in cm3
No of cells per ml = Average no. of cells counted per small square x 20.000 x 1000
No of cells per ml = Number of cells x 20 x 106 / 16
No of cells per ml = Number of cells x 1.25 x 106
Fig 43.2: (a) A photograph and (b) Schematic diagram of a Petroff Hausser counting chamber
4
Isolation, evaluation and characterisation of mining microorganisms

Sources for isolation of Acidithiobacillus, Leptospirillum and Sulfate Reducing Bacteria are the
following:
Mine water collected at sulfide ore sites
Acidic mine water drainage
Mine sediments with moisture
Ore samples from open cast and underground mines of coal and sulfide mineral ore deposits
Solid and liquid samples from tailing dams.
The above samples are inoculated in enrichment media; to obtain enrichment cultures. One can
also use desired prescribed media for various acidophiles, neutrophilic and anaerobic SRB.
Isolation of acidophiles
Enrichment in 9K medium Appearance of brownish colour and ferric-salt precipitation on

incubation. For adaptation, desired metal ions or metal ores and concentrates can be present
during bacterial subculturing and growth.
Isolation of pure culture using solid agar 9K medium - silica gel plates can also be used
impregnated with the medium. From the colonies grown on solid medium, inoculations are
made into liquid nutrient media. Purity of cultures is verified.
Method of end-point dilution can be used to isolate pure cultures of At.ferrooxidans.

Purity check for possible contamination by other acidophiles.
The isolated culture are to be maintained in 9K medium (in the presence of metal ions or ore
concentrates, if necessary) in a refrigerator at 4 0C. Transfers should be made atleast once in two
weeks.
Various isolated strains of At.ferrooxidans can be classified with respect to their respective iron
oxidation rates (or iron oxidation index).
For At.thiooxidans, rate of sulfate production from sulfur can be used for similar classification.
For Sulfate Reducing Bacteria, sulfate reduction rates can be used for classification.
5
Media composition
The media composition for At.ferrooxidans, At.thiooxidans and Leptospirillum ferrooxidans are
given in table 43.1, 43.2 and 43.3 respectively.
Table 43.1: Silverman and Lundgren medium (9K medium) for Acidithiobacillus ferrooxidans
Components g/L
(NH4)2SO4 3.0
KCl 0.1
K2HPO4 0.5
MgSO4 0.5
Ca(NO3)2 0.01
Distilled water 1000 ml
The above constituents have to be dissolved in 1000 ml distilled water and the pH has to be
adjusted to 1.9-2.0 with 10M H2SO4 and sterilize by autoclaving (Solution A). 44.8 g of
FeSO4.7H2O should be dissolved in 100 ml of medium and filter sterilize (Solution B). The
media has to be prepared by mixing the two solutions.
Table 43.2: Basal media for Acidithiobacillus thiooxidans
Components g/L
(NH4)2SO4 2.0
MgSO4 0.5
K2HPO4 0.25
Sulphur powder 10.00
H2O 1000 ml
pH 2.4
The medium should be sterilized without Sulphur powder by autoclaving. Later Sulphur powder
should be added and sterilize by tyndallization.
6
Table 43.3: Silverman and Lundgren medium (9K medium) for Leptospirillum ferrooxidans
Components g/L
(NH4)2SO4 3.0
KCl 0.1
K2HPO4 0.5
MgSO4 0.5
Ca(NO3)2 0.01
Distilled water 1000 ml
pH 1.2
The above constituents have to be dissolved in 1000 ml distilled water and the pH has to be
adjusted with 10M H2SO4 and sterilize by autoclaving (Solution A). 44.8 g of FeSO 4.7H2O
should be dissolved in 100 ml of medium and filter sterilize (Solution B). The media has to be
prepared by mixing the two solutions.
Growth of chemolithotrophs on solid media

Special techniques are used to grow autotrophic bacteria on solid media. Isolated colonies grown
on plates have to be transferred to liquid medium and cultures should be maintained only in
liquid media for preservation.
Solid medium for the growth of Acidithiobacillus ferrooxidans
The composition of solid or agar medium for Acidithiobacillus ferrooxidans is shown in table
43.4.
Solution A: FeSO4.7H2O (33.4g/L)
Table 43.4: Solution B: Basal salts
Components g/ L
(NH4)2SO4 6.0
KCl 0.2
MgSO4.7H2O 1.0
Ca(NO3)2 0.02
pH has to be adjusted to 2.5
7
Solution C
Purified agar has to be added at a concentration of 0.7%. Agar should be added to distilled water
and stirred for 20 min and the supernatant discarded. The same process has to be repeated thrice
to remove all the organic matter.
Solution A has to be filter sterilized. Solutions B and C are autoclaved separately, cooled and all
the three solutions have to be mixed. Later the medium should be poured into petriplates.
Identification Studies
The characteristic features of genus Acidithiobacillus (At.ferrooxidans, At.thiooxidans)
Strictly aerobic
Gram-negative
Motile ,Mono flagellated
Rod shaped
Cells are 0.3 x1 to 3 m in size
Non-spore forming; best growth at 25-35
Able to oxidise iron, sulfide, elemental sulfur, thiosulphate.
T.thioparus , grows at neutral pH
Responsible for acid mine drainage, due to production of sulfuric acid
The characteristic features Leptospirillum ferrooxidans
Vibrious, spiral-shaped, pseudococci
0.1m in diameter and 6m to 12m in length
Gram negative, obligate chemolithoautotrophs
8
Oxidises ferrous ions to ferric
Motile (mono flagellated), obligately aerobic, with a generation time of 6-16h
Oxidase positive, catalase or peroxidase positive
Optimum temperature is 28 to 30C
On solid media, colonies appear in small red-brown colour
Some phenotypic and biochemical characteristics of At.ferrooxidans, At.thiooxidans and

Leptospirillum ferrooxidans are given in table 43.5.
Table 43.5: Phenotypic and biochemical characteristics
Phenotypic
At. Ferrooxidans L.ferrooxidans At.thiooxidans
characteristics
Vibrios 1 m
Cell morphology Rods 0.5-1.5 m Spirilla Rods 0.5-1 m
(2-5 turns)
Endospores - - -
Motility (+) ++ (+)
Reaction to Grams
- - -
stain
2+
Fe oxidation + + -
S oxidation + - +
Biochemical characteristics
Oxidase test + + -
Catalase test + + -
Symbols: +, positive; -, negative; ++, strongly motile; (+), limited motility
Typical scanning electron micrographs and digital photographs of At.ferrooxidans,

At.thiooxidans and Leptospirillum ferrooxidans are illustrated in fig.43.3.
9
At. ferrooxidans L. ferrooxidans
At. thiooxidans Growth of At. ferrooxidans and

L. ferrooxidans on agar plates
Growth of At. ferrooxidans and Growth of At. thiooxidans in liquid

L. ferrooxidans in liquid media medium
Fig. 43.3: Scanning electron micrographs and digital photographs of At.ferrooxidans, At.thiooxidans and
10
A typical growth curve of bacteria is depicted in fig. 43.4 which shows the initial lag,
logarithmic, stationary and decline or death phase of bacteria. Bacteria are cultured in
Erlenmeyer flasks and placed in a shaking incubator to attain full growth. (fig. 43.5).
Fig. 43.5: Shaking incubator

Fig. 43.4: Bacterial growth in batch mode
Growth kinetics
Growth kinetics of Acidithiobacillus ferrooxidans
Figure 43.6 shows the typical growth curve of Acidithiobacillus ferrooxidans. As can be
observed, the lag phase for this strain extends up to 12h. This is followed by the exponential
growth phase up to 38h. The maximum cell number corresponds to 2x108 cells /mL. The redox
potential values continuously increase from 280 to 540mV with increase in time. This is in
agreement with the increase in the ferric concentration and decrease in the ferrous concentration.
The pH increases initially from 1.9 to 2.5 and then drops to about 2.2. The decrease in pH may
be attributed to the formation of acidic ferric sulphate. There was also a simultaneous decrease in
11
dissolved ferric iron concentration observed, presumably due to its precipitation either as ferric
hydroxide or jarosite or both.
The change in the ferrous and ferric concentrations as a function of time during growth of
At.ferrooxidas shown in Figure 43.6. The time taken for the complete decrease in ferrous iron
concentration is about 40h. A steep increase in ferric concentration is observed from 10 to 40h.
10
8 2.5 550
2.0x10 B
Fe and Fe conc (g / L)
A 2.4
8
8
500
1.6x10
No.of cells / mL
2.3 450 6
ESCE in mV
8
1.2x10 2.2 pH Fe3+
3+
400 4 Fe2+
7
8.0x10 2.1
Cell count 350 2
pH 2.0
2+
7
4.0x10 ESCE 300
1.9 0
250
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Time (hours) Time (hours)
Fig43.6: (A) Cell number, pH, ESCE as a function of

time during growth of At. ferrooxidans (B) Ferrous and ferric concentration as a function of
time during growth of At.ferrooxidans
The trends with respect to increase in the ferric concentration complement the decrease in the
ferrous concentration as a function of time. This further testifies to enhanced bacterial activity.
Growth kinetics of Leptospirillum ferrooxidans
Growth curve of Leptospirillum ferrooxidans is similar to that of Acidithiobacillus ferrooxidans

except for the pH. For Leptospirillum ferrooxidans growth pH is 1.2.
Growth kinetics of Acidithiobacillus thiooxidans
From Figure 43.7 it is evident that the lag period of growth extends up to 24h, beyond which the
exponential growth phase can be observed up to 130h. The maximum growth attained was 8 x
108 cells/ml.
12
The variation in the pH values for the sample as a function of time is shown in Figure 43.7. As
the cell concentration increases, the pH of the solution decreases from 2.0 to 0.5 by 220h. Such a
significant pH decrease during bacterial growth is due to production of sulphurous and sulphuric
acid, by oxidation of the sulphur present in the medium.
From Fig. 43.7 it is also evident that the sulphate concentration continuously increases as a
function of time. The sulphate concentration increases from 1.6g to 28g/L in 240h.
A B
Fig. 43.7: (A) Cell number as a function of time during

growth of Acidithiobacillus thiooxidans (B) pH & SO4 conc. as a function of time during
growth of Acidithiobacillus thiooxidans
13
Estimation of Iron
Iron can be estimated spectrophotometrically by 1, 10-phenanthroline method. Iron (II) reacts
with 1, 10-phenanthroline to form an orange-red complex, [(C12H8N2)3 Fe]2+ . Iron (III) should
be reduced with hydroxylamine chloride and then reacted with 1, 10-phenanthroline. The color
intensity of iron-1, 10-phenanthroline complex is independent of acidity in the pH range 2-9 and
is stable for long periods. 1mL of known concentrations of iron (II) has to be taken and buffered
with 2 ml of 0.2M potassium hydrogen phthalate solution at pH 3.9. To this, 10 ml of 0.15%
solution of 1, 10-phenanthroline should be added and made up to 25 ml using pH 2 solution. The
absorbance is measured using UV- visible spectrophotometer. The absorbance need be measured
against a reagent blank at 512 nm wavelength. A similar procedure should be adopted for total
iron (as ferrous) after reduction of the sample with 4mL of 10% hydroxylamine hydrochloride
solution for half an hour. The calibration graph is shown in Fig. 43.8 using which, the
concentration of iron in the samples can be determined.
Fig. 43.8: Calibration curve for Iron
Concentrations of total iron, iron as ferric and iron as ferrous can be determined.
14
Estimation of Sulphate
Sulphate has to be estimated spectrophotometrically after precipitation as barium sulphate. 1ml

of the sulphate sample should be added to 5 ml of conditioning reagent and left undisturbed for
30 min. For the preparation of conditioning reagent, dissolve 75g of NaCl in 30 ml concentrated
HCl, 50 ml glycerol, 100 ml isopropanol, and the solution should be made up to 1000 ml using
Milli-Q water. Then the solution has to be made up to 100 ml using Milli-Q water. To this 0.3g
of barium chloride should be added with continuous stirring. The barium sulphate precipitate so
formed is allowed to stand for 5min and then the concentration of sulphate has to be determined
in the spectrophotometer at 420 nm wavelength using against a reagent blank. A typical
calibration graph is shown in fig.43.9.
Calibration curve for sulphate
Fig. 43.9: Calibration curve for Sulphate
15
Estimation of Copper
Copper can be estimated using atomic absorption spectrophotometer in air acetylene oxidizing
flame at 327.4 nm wavelength. Stock solution to be prepared by dissolving 1g of copper metal in
1:1 HNO3 and making it up to 1000 ml using Milli-Q water. Various solutions for calibration
ranging from 1-4 mg/l of copper have to be prepared from the stock solution. The calibration
curve is shown in fig. 43.10.
Fig. 43.10: Calibration curve for Copper
16
Lecture 44: Laboratory Experiments In Metals Biotechnology II NPTEL Web Course
Lecture 44
Laboratory Experiments In Metals Biotechnology - II
Keywords: Column Leaching, Shake Flak Leaching, Sulphate Reducing Bacteria
Leaching strategies in the laboratory
Bioleaching can be defined as a hydrometallurgical dissolution process assisted by

microorganisms for the recovery of metals from their ores/concentrates.
The bacterial oxidation of sulphide minerals permits biodissolution of metal ions of interest such
as zinc from sphalerite and, copper from chalcopyrite. In case of precious metals, such as gold
and silver, bacterial oxidation liberates the precious metal embedded in pyrite or arsenopyrite
matrix by solubilising the sulphide mineral and leaving behind exposed gold or silver in the ore
which can be subsequently solubilized by cyanide solution. Acidithiobacillus ferrooxidans and
Leptospirillum ferrooxidans are the most widely used organisms in bioleaching of sulphide
minerals. There are two important mechanisms involved in bacterial oxidation, namely, direct
and indirect mechanisms:
1. Direct mechanism, in which the bacteria Acidithiobacillus ferrooxidans/ Leptospirillum

ferrooxidans attaches to the sulphide mineral and oxidizes the mineral, which provides energy
for bacterial growth.
2. Indirect mechanism in which ferrous iron is oxidized to ferric (Fe2+) by the bacteria (energy
source) in the liquid phase. Fe3+ in turn can chemically leaches sulphide mineral.
For example, biodissolution of arsenopyrite can be stated as
FeAsS + 3.5O2 + H2O = Fe3+ + SO42- + H2AsO4-

FeAsS + 7H2O = Fe2+ + H3AsO3 + 11H+ + 11e- + SO42-
1
Presence of Acidithiobacillus ferrooxidans / Leptospirillum ferrooxidans increases the rate of

arsenopyrite oxidation catalyzing the iron oxidation.
Bacteria
2+ +
2Fe + 0.5O2 + 2H 2Fe3+ + H2O
The presence of iron (III) in solution further favours arsenopyrite oxidation.
FeAsS + 13Fe3+ + 8H2O 14Fe2+ + SO42- + 13H+ + H3AsO4 (aq)
Shake flask leaching (Agitation leaching for ore fines / concentrates)
Microbial Leaching of Copper
The most commonly investigated minerals of copper are chalcopyrite, chalcocite and covellite.
These minerals can be leached by direct bacterial attack (At.ferrooxidans) and / or indirectly by
ferric iron as the oxidant. Bacterial oxidation of chalcopyrite can be seen by the following
overall reaction.
4CuFeS2 + 11O2 + 6H2O 4CuSO4 + 4Fe(OH)3 + 4S
2S + 3O2 + 2H2O 2H2SO4
In two 500 ml Erlenmeyer flasks containing 180 ml 9K medium (i.e. without ferrous sulfate),
add 10 g of the given chalcopyrite ore or arsenopyrite concentrate. Add a few drops of thymol
(1%) which is a bactericide, into one of the flasks. This flask serves as a control. Incubate the
experimental flask with 10 ml of actively growing culture of Acidithiobacillus ferrooxidans.
Incubate both the flasks at room temperature on a rotary shaker at 200 rpm. Estimate dissolved
copper, arsenic and iron at regular intervals of 48 hours.
2
Column leaching (Percolation leaching for coarser ores)
Airlift percolators can be designed and fabricated easily. A typical arrangement is illustrated in
fig. 44.1.
The columns are designed with 400mm height and 50mm diameter. The columns are packed
with the ore sample on glass wool, which is used as the filtering medium. The liquid level has to
be maintained at a height of 1.5 cm above the bed of the ore sample. These types of columns are
set up in order to simulate the typical conditions prevailing in the actual heap leach sites. The test
conditions are similar to those used in the agitation leaching tests. Air compressor is used to
ensure continuous recirculation of the leached liquor.
Leachant
Sample
To Air
compressor
Glass-wool
Fig. 44.1: Typical set up for Column leaching with a schematic representation
3
Scanning electron micrographs of arsenopyrite concentrate prior to leaching and after leaching in
presence of Acidithiobacillus ferrooxidans is depicted in fig. 44.2. The attachment of
Acidithiobacillus ferrooxidans onto the arsenopyrite concentrate is illustrated in fig. 44.3.
Fig. 44.2: Arsenopyrite concentrate (a) before leaching and (b and c) after leaching
Fig. 44.3: Acidithiobacillus ferrooxidans attached onto arsenopyrite concentrate

(b
)
Perform bioleaching tests as a function of time and monitor Eh, pH and dissolved metal ion
concentrations frequently. Compare metal leaching rates in the presence and absence of bacteria.
Bioremediation using Sulphate Reducing Bacteria

The media composition for sulphate reducing bacteria like the Barrs medium and Postgates
medium are given in table 44.1 and 44.2.
4
Table 44.1: Barrs medium for Sulphate reducing bacteria
Components g/L
K2HPO4 0.5
NH4Cl 1.0
CaSO4 2.0
MgSO4 2.0
Sodium lactate 7.0
Ferrous ammonium sulphate 0.5
SodiumThioglycollate 0.2
Yeast extract 1
Distilled water 1000ml
pH 7.6
The medium should be sterilized by adding all the components except ferrous ammonium
sulphate and sodium thioglycollate consequently for three days till the pH remains constant.
Ferrous ammonium sulphate and sodium thioglycollate should be filter sterilized and added to
the medium.
Table 44.2: Postgates medium
Components g/L
Tryptone 10.0
Sodium sulphite 1.0
Sodium sulphate 1.0
Ferric citrate 0.5

Distilled water 1000ml
pH 7-7.5
The above components are sterilized by autoclaving.
5
Characteristic features of Sulphate Reducing Bacteria
Sulphate reducing bacteria, next major group of bacteria found in the mine ecosystem.
The genera Desulfotomaculum and Desulfovibrio are the most predominant members.
Desulfotomaculum nigrificans and Desulfovibrio desulfuricans are the most abundant

members of the genera Desulfotomaculum and Desulfovibrio respectively in mine
ecosystem.
Use sulphate or other reducible sulphur compounds as terminal electron acceptor and
being reduced to H2S.
Desulfotomaculum are straight rods, 3-9m in size, with rounded or pointed ends.
Stain Gram negative, Obligate anaerobes
Spore forming
oval to round, causing slight swelling of the cells
Motile with peritrichous or polar flagella
Desulfovibrio, curved or occasionally straight rods, some time sigmoid or spirilloid,

2.5-1m in size.
Obligate anaerobes, motile
Non spore forming
6
Digital photographs and scanning electron micrograph of Sulphate reducing bacteria are
illustrated in fig.44.4.
(a) (b) (c)
Fig. 44.4: Growth of SRB in (a) broth medium (b) agar medium and (c) SEM of Desulfovibrio spp
Some phenotypic characteristics of Desulfotomaculum spp and Desulfovibrio spp are given in the
table 44.3.
Table 44.3: Phenotypic characteristics Desulfotomaculum spp and Desulfovibrio spp
Phenotypic Desulfotomaculum Desulfovibrio

characteristics
Cell morphology Rods 0.5-1.5 m Curved 2.5-1m
Endospores + -
Motility (+) ++
Reaction to Grams - -
stain
2+
Fe oxidation - -
3+
Fe reduction - -
S oxidation - -
Growth at 50 C + -
Symbols: +, positive; -, negative; ++, strongly motile; (+), limited motility
7
Growth Kinetics of Sulphate Reducing bacteria (SRB)
The growth curve in Fig 44.5 shows the cell number and change in sulphate concentration as a
function of time during growth of Desulfotomaculum. Sulphate concentration steepily decreases
with time due to bacterial reduction.
1.0
8
3.0x10
Sulphate Conc ( g / L)
8 0.8
2.5x10
No. of Cells / mL
Cell count
8
2.0x10 Sulphate conc 0.6
8
1.5x10 0.4
8
1.0x10
0.2
7
5.0x10
0 20 40 60 80 100
Time h
Fig 44.5: Cell number as a function of time during growth of SRB
Prepare solutions containing of copper, iron and zinc ions at neutral pH and establish metal
removal by additions of SRB. Monitor metal removal as a function of time.
Study single metal, bimetal and tri-metal combinations using SRB.
8
Lecture 4: Microorganisms In Biohydrometallurgy NPTEL Web Course
Lecture 4
Microorganisms In Biohydrometallurgy
Keywords: Mining Microorganisms, Acidithiobacillus, Thermophiles
Some chemolithotrophic bacteria useful in biohydrometallurgy are listed below:

T.caldus
L.ferrooxidans
Arthrobacter sp.
Acidianus brierleyi
Sulfobacillus thermosulfidooxidans
Sulfolobus rivotincti
Sulfolobus metallicus
Metallosphaera sedula
For microbiological and experimental discussions on Acidithiobacillus bacteria, refer to

lectures 43 and 44.
Acidithiobacillus
Acidithiobacillus is known for its ability to oxidize elemental sulphur and sulphur containing
compounds, but the conditions (such as pH, temperature) may vary depending on the physiology.
Bacteria of the genera Acidithiobacillus are aerobes and are obligate or facultative
chemolithotrophs. They grow at pH values between 0.5 9 as acidophiles and neutrophiles.
They are mesophiles having optimum temperatures for growth at 25- 300C. Some are
0
moderately thermophilic such as At. caldus, oxidising sulphur above 40 C and have been used in
bioliberation of gold from pyrite and arsenopyrite.
1
Chemolithotrophic bacteria including the genus Acidithiobacillus, can oxidise various sulphur
compounds (i.e. S- -, So, S2O4, S2O3- -, SO4- -).
2S + 3O2 + 2H2O = 2H2SO4

Na2S2O3 + 2O2 + H2O = Na2SO4 + H2SO4
2H2S + O2 = 2So + 2H2O
H2S + 2O2 = H2SO4
Moreover, some species derive energy from oxidation of ferrous ions. (At.ferrooxidans and L.
ferrooxidans)
2FeS2 + 7O2 + 2H2O = 2FeSO4 + 2H2SO4

4FeSO4 + O2 + 2H2SO4 = 2Fe2(SO4)3 + 2H2O
Main species of Acidithiobacillus include At. thioparus, At.dentrificans, At.thiooxidans, At.

intermedius and At.ferrooxidans.
Leptospirillum
Leptospirillum ferrooxidans (L.ferrooxidans) is a moderately thermophilic iron oxidizer which
can oxidize only ferrous ions, and can grow at higher temperatures and also at stronger acidity
levels. L.ferrooxidans has a higher affinity for Fe++ than At.ferrooxidans and a lower affinity for
Fe+++, a competitive inhibitor.
L.ferrooxidans can tolerate higher concentrations of toxic metals such as uranium, molybdenum
and silver than At.ferrooxidans.
Thermophiles
Thermophilic iron-oxidising bacteria can be classified into moderate and extreme thermophiles.
Optimum temperature for growth and leaching are in the range between 65 0 and 850C for
2
extreme thermophiles and about 400 to 600C for moderate thermophiles. A number of
thermophilic microorganisms, such as Sulfolobus have been enriched and isolated from
bioleaching environment. The bacteria grow chemolithotrophically at the expense of iron. Some
are facultative autotrophs.
A moderately thermophilic bacterium, Sulfobacillus thermosulfidooxidans, is a Gram-positive,

nonmotile, sporeforming, rod-shaped eubacterium, with optimum growth at 28-600C. They are
facultative autotrophs. They can also grow autotrophically on Fe (II), S, or mineral sulphide as
energy source.
Examples of the extreme thermophiles are Sulfolobus acidocaldarius and Acidianus brierleyi,
both in the genera of Archaebacteria. There are four of these, namely, Sulfolobus, Acidanus,
Metallosphaera, and Sulfurococcus. All are aerobic, extremely thermophilic and acidophilic
bacteria oxidising ferrous ions, elemental sulphur and sulphide minerals between 55 and 90 0C
and pH 1 5.
Acidithiobacillus ferrooxidans is the most studied bioleaching bacterium.
They are Gram-negative rods (0.5 x 2 m) and occur singly or in pairs. Cell mobility is achieved
by means of a single polar flagellum. They are generally characterized by the following
properties.
Chemolithotrophic growth and maintenance energy is derived from the oxidation of ferrous
ions or reduced valence sulphur compounds.
Autotrophic carbon dioxide is used as a cellular carbon source and nitrogen and phosphorus
needed as nutrients for growth and synthesis along with the trace minerals, K, Mg, Na, Ca
and Co.
Aerobic-oxygen is the electron acceptor and in contact with air derive oxygen and carbon
dioxide.
Mesophilic-bacterial growth and iron oxidation occurs at temperatures 20 0C and 300C.
3
Acidophilic bacterial growth at a pH 1.5 to 2.5.

They are regarded as obligate chemolithotrophs which derive energy from the oxidation of
ferrous ions and reduced sulphur compounds. Carbon in the form of carbon dioxide is
assimilated through the Calvin-Benson cycle. They can also grow on elemental sulphur or
mineral sulphides under anaerobic conditions using ferric ions as the electron acceptor.
Mesophilic and thermophilic bioleaching bacteria
T.ferrooxidans first isolated from mine drainage in 1947 and in 1951 Temple and Colmer
isolated and described the acidophile.
Microorganisms cultured from leach systems in waste rock dumps at the Bingham mine.
Numerous investigations on At.ferrooxidans followed. Leptospirillum ferrooxidans
described in 1972.
At.thiooxidans and At.Caldus were used in leaching.
Importance of L.ferrooxidans and use of mixed cultures containing At.ferrooxidans,
At.thiooxidans and L.ferrooxidans was later established.
Molecular biology techniques indicated dominant presence of L.ferrooxidans in stirred tank
reactors for gold at pH 1.6 and temperature of 400C. In many columns, also L.ferrooxidans
was found to dominate.
New species such as Leptospirillum ferriphilum isolated which were moderate thermophiles
(2002).
Another organism Ferroplasma, an iron oxidizing archaeon identified (2000).
At.Caldus and L.ferrooxidans were dominant iron oxidizers functioning at 450C, acidic
conditions.
Use of thermophiles in bioreactor and bioheap leaching was found to be useful since
exothermic biooxidation of sulfides generates heat.
There is considerable interest to use thermophiles in the leaching of chalcopyrite.
Moderate and extreme thermophiles were identified and isolated.
In actual situations, bioleaching processes involve consortia of microorganisms.
4
Typical microorganisms inhabiting waste copper ore dumps and heaps are given in table 4.1.
Table 4.1:Microorganisms present in waste ore dumps
Acidithiobacilli Heterotrophic bacteria

Acidithiobacillus ferrooxidans Bacillus
Acidithiobacillus thiooxidans Pseudomonas
T. denitrificans
Iron-oxidisers Fungi and yeasts

Gallionella Aspergillus
Lepotothrix Penicillium
Cladosporium
Saccharomycae
Sulphate reducing Thermophiles such as

Desulfovibrio spp. sulfolobus
5
Microbiological advancements with potential benefits to heap bioleaching are given in table 4.2.
Table 4.2: Microbiological aspects in heaps with their potential benefits

(adapted from Brierley, C.L, Hydrometallurgy, 59 (2001), 249-255).
Microbiological developments Potential benefits
Use of bacteria that can initiate oxidation at Effective bioheap leaching of high acid
higher pH values and condition the ore for consuming ores.
conventional bioleaching bacteria
Comprehensive recognition of adaptation or Use of low quality water for make-up of
succession process of the bacteria to the leach solutions and operation of the bioheaps
changing character of the leach solutions. lower capital and operating costs.
Realization of succession of thermophiles in Bioheap leaching of more refractory ores-
bioheaps, interpreting their function and effects increase metal recoveries with shorter leach
on minerals and precipitates. time, at reduced costs.
Understanding of heterotrophic and Better understanding of conditions that could

Chemolithotrophic microorganisms in bioheaps optimize bioheap performance.
related to function and nutrients.
6
Lecture 5: Reactor Bioleaching And Developments In Bioleaching Of Concentrates NPTEL Web Course
Lecture 5
Reactor Bioleaching And Developments In Bioleaching Of
Concentrates
Keywords: , Reactor Leaching, Bioleaching Of Metal Concentrates, Recent Developments
Metal sulfide concentrates are generally bioleached in stirred tank reactors (agitation leaching).
Several designs of bioreactor configurations are available (Fig.5.1)
Feed
Air Input
Air Agitated Tank Reactor
Pachuca Reactor
Fig. 5.1: Typical bioreactor designs
1
Bioleaching of base metal concentrates
A number of companies have developed biooxidation processes for use in metal extraction.
Some of these have found commercial application while others are still in the experimental or
pilot plant stages:
Newmont Mining BIOPRO Process heap leaching of refractory gold ores
Gold Fields, Ltd BIOX Process agitated tank oxidation of refractory

gold ores
BHP Billiton, Ltd.

BioCOP Process agitated tank oxidation and leaching
of copper sulfides
BioNIC Process agitated tank oxidation and leaching
of nickel sulfides
BioZINC Process agitated tank oxidation and leaching
of zinc sulfides
BacTech Enviromet BacTech/Mintek Process agitated tank oxidation and

leaching of copper sulfides
GeoBiotics, Inc.
GEOCOAT Process heap leaching sulfide mineral
concentrates
BacTech Enviromet Corp., in conjunction with Mintek, has developed proprietary technologies
for the high temperature leaching of copper concentrates. Working in conjunction with Industrias
2
Penoles SA de CV, they operated a 2.2-metric tons per day (mt/d) stirred-tank copper-
concentrate bioleach demonstration plant in Monterrey, Mexico during 2001.
BHP Billiton and Codelco, in a joint venture, Alliance Copper Ltd., constructed a demonstration
plant at Chuquicamata, Chile, to produce 20,000 tons of cathodes a year starting in 2003 using
Billitons patented BioCOP process, to treat dirty concentrates containing high levels of
arsenic (enargite, Cu3AsS4).
The following process developments are note-worthy:

The GEOCOAT Process A heap leaching process capable of bioleaching base
metal and refractory sulfidic gold concentrates. The concentrate slurry is coated onto a
support rock, or substrate material, and stacked in a bioxidation heap. The ratio of
support rock to concentrate is in the range of 5:1 to 10:1 by weight. The heap can be
inoculated with thermophilic bacteria and a process leach solution consisting of sulfuric
acid, ferric iron and nutrients applied to the heap. Low-pressure blowers provide air
supply through a system of perforated pipes placed under the heap. The exothermic
nature of the oxidation reaction increases the heap temperature to at least 50C.
The BioCOP Process Carried out in a stirred reactor containing sulfuric acid into
which air is blown. Thermophilic microorganisms are used at a temperature between
60C and 90C. Limestone is used to maintain the pH of the solution and to provide
carbon dioxide for bacterial growth. Copper concentrate is fed to the reactor. Pregnant
solution containing 30-40 grams per liter is sent to solvent extraction electrowinning.
Retention time is about 10 days.
The BacTech/Mintek Process Countercurrent reactors used. Two proprietary

bioreactors were tested in Mexico; - (a) the Circox bioreactor developed for the
biotreatment of municipal sewage and industrial waste water and licensed from Paques
Bio Systems B.V. of the Netherlands. The Circox bioreactor used an airlift to circulate
3
the solids within the reactor, and (b) the BAR (BacTech Aerated Reactor).
Thermophilic microorganisms used at temperatures between 25C and 55C. A pH of 0.5
to 2.5 is maintained. Nutrients are added to the leach liquor. Retention time is about of
30 days.
Bioleaching parameters and developments:

Parameters influencing bioleaching are illustrated in table 5.1.
Table 5.1: Factors and parameters influencing bacterial mineral oxidation (Adapted from
H.Brandl; Microbial leaching of metals (www.wiley-vch.de/books/biotech/pdf/v10_branpdf))
Physicochemical Temperature Mass transfer, nutrients,

parameters pH Ferrous concentration
redox potential
oxygen, carbon dioxide
Microbiological factors Microbial diversity Metal tolerance and toxicity

Population diversity Whether adapted or not
and density
Spatial distribution
Mineral properties Mineral type and composition Surface area, galvanic

Mineral dissemination, interactions
porosity
Processing parameters Leaching mode (in situ, heap, Stirring rate (in case of tank
dump, or tank) leaching operations)
Pulp density Heap geometry (in case of heap
leaching)
4
Status of bioleaching for base metal and refractory gold-bearing concentrates.

Commercial viability of bioleaching proven for refractory gold concentrates
Mesophile bioleaching applied commercially for treatment of cobaltiferrous pyrite (Kasese,
Uganda)
Continuous high temperature thermophile bioleaching demonstrated at pilot scale for
chalcopyrite and pentlandite.
Construction of demonstration plants for treatment of copper concentrates.
Bioleaching is technically feasible and economically viable as a process option for treatment
of base metal and multimetal complex concentrates.
Biological extraction of cobalt from pyritic concentrates was commercialized in 1999. (BRGM,
France) at Kasese Project at the Kilembe mine in Uganda. An inoculum of mesophilic iron-
oxidizing bacteria used for bioleaching cobalt in a stirred-tank reactor system. The plant uses
solvent extraction-electrowinning for recovery of the cobalt. The promising developments in
bioleaching of cobalt and nickel may lead to immediate commercialization of
Biohydrometallurgical processing of base metals other than copper. Details of cobalt bioreactor
operations are given below:
Recovery of cobalt from cobalti-ferrous pyrite
Tonnage (t/d) : 241
Primary Reactor Size (m3) : 1350
Start Date : 1998
Cobalt Content (%) : 1.4
Sulphide Content (%) : 41
Challenges for commercial development
Slow leach kinetics and need for large reactors;
High power consumption for oxygen supply;
Confidence that high plant availability will be achieved;
Economic recovery of precious metals.
5
New developments in Biohydrometallurgical reactor designs

Aerated trough bioreactor
Low energy bioreactor
Falling laminar liquid film principle
Inclined plate bioreactor
Biorotor
Future biotechnological initiatives
Modelling, design and development of bioreactor assemblies-low energy-high efficiency-

stirred tank and Pachuca.
o New type of reactor of systems
Comprehensive study of microbial composition of both bioheap systems and stirred-tank
reactors-interactions among various microflora
Techniques to enhance biomass growth
o Electrochemical, Bacfox, inert substrates,
o Growth reactors-genetic engineering
Development of metal-tolerant and substrate-adapted special strains
o Genetically modulated super bugs!
Rapid, accurate and simple techniques to monitor bacterial activity in bioleach systems.
High temperature heap bioleaching (Inoculation of heaps)
High temperature tank bioleaching using thermophiles
Bioprocessing of dirty and complex ores and concentrates
Leaching of silicate-matrix minerals and alkaline ores
Agitated tank bioleaching for uranium concentrates
Nano particles produced by microbes Biomaterials processing, Biomimetics.
Surfactants (flotation reagents, flocculants) through microbes in mineral beneficiation
Organic acid leaching using heterotrophic bacteria and fungi
Sulfate reduction in metals recovery and effluent disposal - Biogenic sulfides
Waste treatment (slags, ash, scrap) Bioleaching to recover values from wastes.
6
7. Brierley C.L, Bacterial succession in bioheap leaching, Hydrometallurgy, 59 (2001), 249-

255.
8. Natarajan, K.A., 1998, Microbes, Minerals and Environment, Geological Survey of India,
Bangalore.
9. Rossi, G., 1990, Biohydrometallurgy. McGraw Hill, New York.
10. Brierley, C.L., 1978, Bacterial leaching, CRC Critical Reviews in Microbiology, 4, pp.
207-262.
11. Brierley, C.L., August 1982, Microbiological mining, Scientific American, pp. 44-54.
12. Acevedo, F., Gentina, J.C. and Bustos, S., 1993, Bioleaching of minerals a valid
alternative for developing countries, Journal of Biotechnology, 31, pp. 115-123.
13. Brierley, C,L., and Brierley, J.A., 2001, Present and future applications of
biohydrometallurgy, Hydrometallurgy, 59, pp. 233-239.
14. Anon, May 31, 2002, Bioleaching Moves forward, Mining J., pp. 392.
15. Miller, P.C., Rhodes, M.K., Winby, R., Pinches, A. and Van Stadan, P.J., 1999, Minerals and
Metallurgical Processing, 16, pp. 42-50.
16. Olson G.J, Brierley J.A and Brierley C.L, Bioleachng Review Part B: Progress in
bioleaching: applications of microbial processes by the minerals industries, App. Microbiol.
Biotechnol 63, (2003), 249-257.
17. Gericke M, Neale J.W and van P.J Staden-A.Mintek perspective of the past 25 years
in minerals bioleaching, J.South African Inst. Min. Met., 109 (2009), 567 583.
7
Lecture 6: Bioleaching Mechanisms NPTEL Web Course
Lecture 6
Bioleaching Mechanisms
Keywords: Direct Mechanism, Indirect Mechanism, Contact Mechanism
In lectures 6 and 7, mechanisms involved in interaction of At.ferrooxidans with sulfide

minerals are discussed with respect to direct and indirect attack as well as role of microbial
attachment [18-35].
Classical understanding of biooxidation reactions involving Acidithiobacillus ferrooxidans

involve two possible mechanisms; namely,
Direct and Indirect mechanisms.
In the direct mechanism, bacteria directly oxidize the minerals and solubilise the metal.
MS + H2SO4 + O2 = MSO4 + S + H2O
S+1 O2 + H2O = H2SO4
Bacterial attachment to the mineral surfaces is a prerequisite to the direct attack.
In indirect attack, the bacteria oxidises ferrous to ferric form which is the oxidizing agent for the
minerals.
MS + 2Fe+++ = M++ + 2Fe++ + S
2Fe++ + O2 + 2H+ = 2Fe+++ + H2O
Both direct and indirect mechanisms may occur simultaneously along with other
physicochemical reactions in real bioleaching systems.
1
There are several reports on direct cell attachment on several sulfides and the role of ferric ions
bound to the cell surface
Bioleaching of sphalerite by At.ferrooxidans was observed to be through an indirect mechanism

with the bacterial contribution only to oxidize ferrous ion to ferric.
Fe++ = Fe+++ + e (Bacterial oxidation)
ZnS + 2Fe+++ = Zn++ + 2Fe++ + S
The above tests were carried out by maintaining the desired redox potentials through control of
ferric to ferrous ion ratio.
However, later studies revealed that when ferrous concentrations were higher, zinc leaching by
the bacteria increased, presumably due to removal surface sulfur layers through chemical
oxidation by ferric ions.
Sand et al (1995) proposed indirect mechanism promoted by ferric ions. The role of ferric ions
bound by extracellular polymeric substances (EPS) for binding of bacterial cells onto pyrite was
proposed. [22]
Two indirect mechanisms for the sulfur part in the mineral after initial reaction with Fe +++ were
also proposed. [22-28]
FeS2 + 6 Fe+++ + 3H2O = S2O3- - + 7Fe++ + 6H+
S2O3- - + 8 Fe+++ + 5H2O = 2SO4- - + 8Fe++ + 10H+
The thiosulfate mechanism differs from the polysulfide mechanism relevant for acid soluble
sulfides such as ZnS.
For example, ZnS can interact with acid as
ZnS + 2H+ = Zn + H2S
H2S + 2Fe+++ = S + 2Fe++ + 2H+
2
On the other hand, pyrite sulfur is mainly oxidized to sulfate (82%), S (16%) and the rest S 4O6
and S5O6- -, unlike sulfur in ZnS (mainly elemental S and about 5% as sulfate).
In case of pyrite, bacterial oxidation of ferrous ions and also of sulfur and polythionates to
sulfate can occur. All the above contributions are considered indirect.
Tributsch (1999) found the rate of biodissolution of sulfides with At.ferrooxidans to be

approximately proportional to the solubility products.
ZnS, FeS, PbS are easily dissolved, while MoS 2, FeS2 are difficult to dissolve.
Biology of leaching bacteria is more complex and recent developments in molecular biology,
surface analysis and surface chemistry have enabled better understanding of mechanisms
involved in biooxidation processes.
Direct enzymatic oxidation of the sulfur moiety of sulfide minerals as suggested in the direct
attack mechanism is questioned. Alternately, nonenzymatic sulfide oxidation by ferric ions in
association with reoxidation of ferrous iron enzymatically is a newly proposed mechanism
having sub-mechanisms such as contact and noncontact mechanisms. [22-28]
Non-contact mechanism involves ferrous oxidation in solution by planktonic bacteria. However,

the oxidized ferric ions come in contact with mineral surfaces where they get reduced (by
oxidation of the sulfide mineral).
Recent review by Rohwerder et al. (2003) outlines the current- understanding as follows [23]:
Two different mechanisms thiosulfate and polysulfide pathways.

Reaction pathway determined by mineral species (acid dissolution)
Reactivity of sulfides with protons is a criterion (acid dissolution)
Electronic configuration is important in determining solubility criterion.
Acid insoluble sulfides-Thiosulfate pathway Eg:- Pyrite, Molybdenite
Free thiosulfate is oxidized to sulfate (via) tetrathionate and other polythionates.
3
Elemental sulfur also is produced in absence of S-oxidizers. Based on the necessity of an

electron transfer reaction, iron oxidising bacteria can leach acid-insoluble sulfide in acid
solutions.
Acid soluble sulfides-polysulfide pathway.
Eg:- sphalerite, galena, chalcopyrite, arsenopyrite etc
Combination of acid attack and ferric oxidation.
In both the above mechanisms, the role of bacteria would be generation of ferric ions and
ratio between ferric to ferrous ions determines the redox potential. Control of redox potential
is needed to promote biooxidation of the mineral.
Acidophilic sulfur oxidizers can contribute to transformation of intermediate sulfur
compounds ultimately to sulfuric acid.
In the absence of sulfur oxidation (as in abiotic conditions), surface accumulation of sulfur
layers would impede mineral dissolution. Thus, sulfur oxidizers can influence dissolution
kinetics.
Electrochemical properties of sulfides can control leaching kinetics-Redox potentials play an
important role.
4
Lecture 7: Bacterial Attachment To Minerals NPTEL Web Course
Lecture 7
Bacterial Attachment To Minerals
Keywords: Bacterial Attachment, Surface Morphology, Mineral Specificity
Role of bacterial attachment
It is now well established that leaching bacterial cells attach to sulfide mineral surfaces. Such
attachment is thought to be predominantly influenced by extracellular polymeric substances
(EPS). EPS may consist of various sugars, saturated fatty acids, and also ferric ions.
Several forces may be involved in attachment of At.ferrooxidans onto different sulfide minerals.
Electrostatic forces play a role in certain cases. IEP of bacterial cells is observed to be at a pH of
about 2-2.5 before interaction with sulfur and other sulfide minerals.
Bacterial interactions with sulfur and sulfide minerals can change the IEP of cells. Electrostatic
attraction between positively charged cell surfaces and negatively changed sulfide surfaces
become possible. Depending on which sulfide mineral the bacteria are interacting, the
composition of EPS may change. For example, cells grown on sulfur do not adhere to pyrite due
to modified EPS. Hydrophobic interactions may play a role in attachment of At.ferrooxidans to
sulfur particles. Similarly, cells grown in the presence of ferric, cupric, zinc and arsenic ions
could exhibit different surface attachment and hydrophobic properties. IEP changes of the cell
surfaces are also observed after bacterial interaction (growth) in the presence of various metal
ions or metal sulfide concentrates.[29-35]
Bacterial attachment depends on several factors such as surface morphology, surface defects,
crystallinity etc. Attachment to specific sites may be promoted. Chemotaxis may play a
prominent role guiding bacterial mobility to substrates containing nutrients. Chemotactic
migration of cells of At.ferrooxidans towards energy yielding regions containing ferrous ions or
reduced valence sulfur compounds has been established.
1
Electrochemical mechanisms involving anode and cathode areas on the substrate need be also
considered. Dissolution occurring in the EPS layer is reported. Sulfide mineral surfaces can
develop micro anodic and cathodic areas due to several reasons such as metal concentration and
oxygen gradients, surface defects, and heterogeneous surface morphology. Local anodic
dissolution on mineral surfaces can occur under the conditions with oxygen reduction at the
cathodic areas, with the attached bacterial cells catalyzing the electrochemical reactions and
promoting electron transfer reactions.
A galvanic model for selective mineral dissolution in the presence of At.ferrooxidans is

illustrated in fig. 7.1.
Fig.7.1: Galvanic biodissolution mechanism.
As could be seen in the figure, chalcopyrite, being anodic to pyrite, undergoes selective
dissolution. Oxygen reduction to water occurs on cathodic pyrite surfaces. Ferrous ions are
oxidized by the bacterial cells. Elemental sulfur formed on chalcopyrite surfaces due to its
oxidation is also effectively removed by its biooxidation to sulfate. Chalcopyrite dissolution is
thus catalytically promoted by bacterial activity.
2
In pyrite dissolution, possibility of a thiol group of cysteine participation in At.ferrooxidans is

considered. Role of thiol groups in sulfide mineral oxidation or enhanced ferric concentration in
the extracellular polymer layer is an essential requisite for direct bacterial contact and pyrite
dissolution. Corrosion pits and direct contact of bacterial cells could be observed
microscopically. Both contact and indirect leaching may be operational. Bacterial mineral
attachment can result in changes in surface pH. Sulfur oxidation by ferrous-grown
At.ferrooxidans was also purposed.
Sulfur or sulfide ore-grown At.ferrooxidans exhibited higher sulfur oxidation ability.

Biooxidation of both ferrous ions and sulfide / sulfur is important and bacterial growth in the
presence of substrates may influence the rate of oxidation of either ferrous ions or sulfide
species. Iron-binding component has been identified as aporusticyanin, located on the outer
surface of the cell. Bacterial attachment is shown to be influenced by aporusticyanin, which is
the principal protein responsible for selective adhesion of At.ferrooxidans on pyrite and other
mineral sulfides. Mineral adhered and unbound aporusticyanin were quantified by SDS-PAGE
and the quantity of adhered rusticyanin to a mineral was assessed as a function of the mineral.
[29-31]
Cells grown on soluble iron adhered specifically and efficiently to pyrite. 92% of ferrous grown
cells were found to be so attached unlike sulfur-grown cell which exhibited only lower surface
affinity to pyrite. Cell surface-located aporusticyanin functions as a mineral-specific receptor for
pyrite adhesion.
The discussions on bioleaching mechanisms in the presence of At.ferrooxidans cite several

scenerios for biooxidation of mineral sulfides. The following possibilities generally emerge:
Indirect mechanism in which bacterial cells oxidize ferrous ions in solution to ferric ions
which solubilizes the mineral.
Indirect contact mechanism in which attached bacterial cells oxidize ferrous ions within the
layer of EPS and bacteria and the ferric ions within this layer solubilise the mineral.
3
Direct contact mechanism which assumes direct bacterial mineral oxidation by biological
means. Such a biooxidation of the mineral could occur even in the absence of ferric ions.
Direct biodissolution of iron-free sulfides can be explained through such a mechanism.
Cooperative bioleaching involves cooperation between mineral attached organisms and free
cells in solution. Attached cells releases chemicals to solution that serve as energy to those
in solution.
Is bacterial attachment essential to initiate bioleaching?
What is the role bacterial mineral adhesion?
How does bacterial adaptation (and growth) to mineral particles (concentrates) and
toxic metal ions influence surface attachment and bioleaching efficiency?
Studies have shown correlation between electrokinetic properties and hydrophobicity with
respect to mineral adhesion of At.ferrooxidans Generation of a newer surface component with
proteinaceous properties by cells grown in presence of substrates such as sulfur, sulfide mineral
particles as well as some metal ions such as copper. At least partial removal of lipolysaccharides
along with exposure of surface proteins may facilitate cell adhesion on solid substrates. Higher
protein exposure confers increased cell surface hydrophobicity which facilitates enhanced
adhesion to hydrophobic substrates such as sulfur.
Typical scanning electron micrograph depicting cell attachment to a mineral substrate is shown
in Fig.7.2.
Fig. 7.2: Scanning electron micrographs illustrating attachment of At.ferrooxidans on sulfide minerals
4
Studies have shown a down regulation of exposed polysaccharides in mineral-grown cells. Also,
changes in surface morphology of the cells of At.ferrooxidans were observed when grown on
sulfur and sulfide mineral substrates compared to ferrous grown cells.
When At.ferrooxidans are grown in the presence of minerals, newer proteinaceous compounds
are seen to be synthesized leading to significant surface chemical changes conferring increased
hydrophobicity
Iron oxidation rates by At.ferrooxidans grown on different substrates were also studied. Cells
grown on ferrous ions in a 9K medium oxidized all the iron within about 45 hours. On the
otherhand, those grown on sulfur and thiosulfate exhibited a decreased rate for ferrous oxidation
(It took about 120 hours for complete iron oxidation). Ferrous oxidation machinery is inducible
unlike the sulfur pathway which is constitutive. Growth on solid sulfur needs prior surface
adhesion unlike in the presence of thiosulfate and ferrous ions (which are soluble substrates). It
has been observed that for At.ferrooxidans grown in the presence of solid substrates such as
sulfur, pyrite and chalcopyrite, prior surface adhesion is essential for its enzymatic machinery to
come into close contact for dissolution of the mineral. Such an adhesion is not required in case
of solution substrates such as ferrous iron and thiosulfate. Proteinaceous substances were seen to
be secreted when cells were grown on such solid substrates. Induction of more hydrophobic
surface proteins and down regulation of exposed polysaccharides lead to significant changes in
cell surface properties, including surface hydrophobicity. Sulfur pyrite and chalcopyrite
grown At.ferrooxidans were observed to be more efficient in the bioleaching of chalcopyrite
compared to ferrous grown and thiosulfate grown cells. The lag phase for iron oxidation was
seen to be significantly extended for cells grown on the above solid substrates. [33 35]
5
18. H.L.Ehrlich, Past, present and future of biohydrometallurgy in Biohydrometallurgy and

the environment-toward-mining of the 21st century, IBS, (1999).
19. T.A Fowler and F.K. Crundwell, Leaching of zinc sulfide by Thiobacillus ferrooxidans:
experiments with a controlled redox potential indicate no direct bacterial mechanism;
App. Env. Microbiol. 64, (1998) pp 3570-3575.
20. T.A Fowler and F.K. Crundwell, Leaching of zinc sulfide by Thiobacillus ferrooxidans:
bacterial oxidation of the sulfur product layer increases the rate of zinc sulfide dissolution
at high concentrations of ferrous ions; App, Env. Microbiol 65, (1999), 5285-92.
21. D.E. Rawlings, Heavy metal mining using microbes, Ann. Rev. Microbiol, 56 (2002), 65-
91.
22. W. Sand, T. Gehrke, P.G.Jozsa and A.Schippers, Biochemistry of bacterial leaching-
direct vs indirect bioleaching, Hydrometallurgy, 59, (2001), pp 159-175.
23. T. Rohwerder, T.Gehrke, K.Kinzler and W.Sand, Bioleaching review. Part A: Progress in
bioleaching: fundamentals and mechanisms of bacterial metal sulfide oxidation, App.
Microbiol. Biotechnol; 63, (2003), pp 239-248.
24. T.Gehrke, R.Hallmann, K.Kinzler and W. Sand, The EPS of At.ferrooxidans - a model
for structure-function relationships of attached bacteria and their physiology; Water Scc.
Technol. 43, (2001), pp 159-167.
25. W.Sand, T.Gehrke, R. Hallmann, and A.Schippers, Sulfur chemistry, biofilm, and the (in)
direct attack mechanism a critical evaluation of bacterial leaching. Appl. Microbiol
Biotechnol 43, (1995), 961-6.
26. W. Sand, T. Gehrke, P.G.Jozsa and A.Schippers, Direct versus indirect bioleaching.
Biohydrometallurgy-and-the-environment-towards the mining-of- the 21st century-PT-
A, In: R. Amils, A. Ballester, editors, New York - Wiley, (1999), pp. 27-49.
27. A.Schippers and W.Sand, Bacterial leaching of metal sulfides proceeds by two indirect
mechanisms via thiosulfate or via polysulfides and sulfur. Appl Environ Microbiol 65,
(1999), pp. 319-21.
6
28. H.Tributsch, Direct versus indirect bioleaching, Biohydrometallurgy and the environment
toward the mining of the 21st century PT A, In: R.Amils, A. Ballester, editors, New York:
Wiley, 9, (1999), pp.51-60.
29. I.Suzuki, Microbial leaching of metals from sulfide minerals, Biotech. Adv. 19, (2001),
pp 119-132.
30. T.Sugio et al. Appl. Environ. Microbiol, 60, (1994), pp 722-25.
31. R.C. Blake II, K. Sasaki and N. Ohmura, Does aporusticyanin mediate the adhesion of
Thiobacillus ferrooxidans to pyrite, Hydrometallurgy 59, (2001), pp 357-372.
32. Y.Rodriguez et al., Hydrometallurgy 71, (2003), pp 47-66.
33. Preston Devasia, K.A. Natarajan, D.N. Sathyanarayana and G. Ramanda Rao Surface
chemistry of Thiobacillus ferrooxidans relevant to adhesion on minerals App. Environ.
Microbiology, 59 (1993), pp. 4051-4055.
34. P. Devasia, K.A. Natarajan and G. Ramananda Rao, Role of bacterial growth conditions
and adhesion in the bioleaching of chalcopyrite by Thiobacillus ferrooxidans, Minerals
and Metallurgical Processing, May 1996, p82.
35. Preston Devasia and K.A. Natarajan, Adhesion of Acidithiobacillus ferrooxidans to
mineral surfaces, International Journal of Mineral Processing, Vol. 94 (2010), pp 135-
139.
7
Lecture 8: Metal Toxicity In Leaching Bacteria NPTEL Web Course
Lecture 8
Metal Toxicity In Leaching Bacteria
Keywords: Metal Toxicity, At.ferrooxidans, Adaptation
Bioleaching in the presence of At.ferrooxidans generates toxic metal ions which could act as
poisons for bacterial growth. The role of metal toxicity in leaching, possible mechanisms as
well as development of metal-tolerant At.ferrooxidans are discussed in lectures 8 9 [36
41].
Why metal tolerant strains?
Bioleaching of
Sulfide mineral concentrates such as chalcopyrite, Arsenopyrite, sphalerite etc.

Gold-containing pyrite-arsenopyrite concentrates.
Accumulation of toxic metal ions, such as
Cu++
Fe++, Fe+++
As+++, As+++++ accumulate in the bioleached leach liquor, introducing heavy metal ion toxicity.
Inhibitory concentrations of some metal ions towards At.ferrooxidans:
Fe++ 0.5M
Cu++ 5 x 10-2 M
Zn++ 5 x 10-2 M
Ag+ 10-3 M
As+++ 10-3 M
1
At.ferrooxidans is more tolerant to heavy metals than most of the other heterotrophic
bacteria.[36-37]
It has been suggested that plasmids are also involved in their metal resistance
Some reported inhibitory levels of metals during bacterial ferrous oxidation are:
Cobalt, Nickel and Zinc > 10g/L
Copper > 1g/L
Uranium < 0.7 g/L
Silver < 0.05g/L
Molybdenum < 0.005g/L
The most toxic metals are uranium, silver and molybdenum. Adaptation of bacterial cells to
various metal ions will enhance their tolerance (Table 8.1)
Table 8.1: Heavy metal tolerance of At.ferrooxidans
Natural limits Highest tolerance achieved

(mol/L) through adaptation (g/L)
Cu 0.87 55
Zn 1.83 120
Ni 0.85 150
U 0.004 1 (without adaptation)
0.05 12 (after adaptation)
Mo 0.0008 0.08
Metal tolerance depends on relative metal ion toxicity.

Uranium and molybdenum exert highest toxicity.
2
Necessity for adaptation?
To enhance bacterial efficiency in the leaching of sulfide concentrates such as chalcopyrite,

arsenopyrite, sphalerite, millerite, pentlandite. etc.,
Dissolved metal ions
Two types of adaptation
Solid substrate (ores and concentrates)
a) Repeated subculturing in presence of increased levels of added toxic metal ions in 9K

medium.
- Increase dosage of toxic metal in instalments
- In one instalment of higher dosage and wait till lag period is overcome.
b) Subculturing at increasing pulp densities of sulphide mineral concentrates.
Some important factors concerning metal toxicity and metal tolerance. [38-41]
Metal ion / substrate tolerance reversible.

Need to preserve adapted strains under induced stress
Cells in a population develop metal tolerance after some structural rearrangement of the cell
material associated with cytoplasmic membrane.
Protecting mechanism is metal-specific
Cell surface membrance changes to detoxify
Metal-specific proteins? Chelation?
Cell surface changes as well as proteinaceous compounds secreted in the metabolite?
Adaptation to mineral sulphide switching from Fe++ oxidation machinery to S-oxidation-
Prolonged lag periods for ferrous ion oxidation exhibited by cells grown on S and mineral
sulfides.
Surface changes make cells more hydrophobic
Better/more efficient mineral adhesion by adapted bacteria
3
Copper adapted cells more hydrophobic

Difference FTIR-spectra indicated significant presence of NH3, NH2, NH CONH3, COCH3,
CH2, CH and COOH groups (proteinaceous).
Presence of more surface proteins increased cell surface Hydrophobicity.
Increased adhesion of Cu-adapted cells on pyrite and chalcopyrite.
Cu-adapted cells exhibit IEP shift to higher pH values (2 to 4.7) on treatment with
proteinase K, reverts to initial value.
Cu-adapted cells adsorb more Cu++ on surface.
Copper toxicity [38 39]
Ferrous ion oxidation in 9K medium by an unadapted strain of At.ferrooxidans in the presence

and absence of cupric ions is illustrated in Fig. 8.1.
In the absence of toxic cupric ions, it took about 42 hours to completely, biooxidize all
ferrous ions.
It took about 80 hours and 280 hours respectively to oxidize all the ferrous ions in the added
presence of 10 and 20 g/L of cupric ions, indicating the toxic and inhibitory role of copper
(toxicity increasing with higher copper concentrations).
A prolonged lag phase for ferrous oxidation corresponds to the period during which some
sort of rearrangement of cell material associated with its membrane takes place.
Once this lag phase is crossed, ferrous ion oxidation proceeds at the same rate as in control.
Completion of one such cycle of iron oxidation is an indication that the entire cell
populations have achieved the ability to overcome metal toxicity.
4
Control
++
1st subculture (10 g/L Cu )
2nd subculture
++
8 1st subculture (20 g/L Cu )
2nd subculture
6
Fe , g/L
4
++
0
0 100 200 300 400 500
t (h)
Fig. 8.1: Ferrous ion oxidation in 9K media by unadapted Acidithiobacillus ferrooxidans in absence and
presence of 10 and 20 g/L Cu++.
Repeated sub culturing enables the cells to gain tolerance and get adapted to the toxic metal
ion content and adaptation is considered achieved if the iron oxidation rate in the presence of
toxic metal ions reaches the same level as that of the control (in absence of toxic ions).
Repeated subculturing in progressively increasing concentrations of the toxic metal ion will
lead to bacterial adaptation. However, adaptation may be possible in a single subculturing, if
the cells are allowed to oxidize all the iron, however prolonged the lag phase may be.
Exposing cells to higher toxic metal ion levels till they are capable of oxidizing all the iron could
be yet another single step procedure for adaptation of At.ferrooxidans.
Cell surfaces preadapted to cupric ions were also found to be more hydrophobic than those
which were unadapted. Bacterial growth in the presence of toxic ions such as Cu ++ results in the
generation of stress-induced proteins. Even growth of At.ferrooxidans in the presence of
chalcopyrite and pyrite minerals induced such protein secretion which renders the cell surfaces
more hydrophobic. Such enhancement of cell surface hydrophobicity will enable higher
5
adsorption (attachment) of At.ferrooxidans onto sulfides such as pyrite and chalcopyrite. Copper
and mineral-adapted bacterial cells are more efficient in the leaching of chalcopyrite.
Electrophoretic mobilities of unadapted and copper-adapted cells of At.ferrooxidans as a function

of pH are shown in Fig. 8.2. Compared to unadapted cells, those preadapted to copper extribited
significant shifts in isoelctric point (IEP) towards higher pH values. Increased positive surface
charges on adapted cells are attributed to enhanced secretion of amino-group containing proteins.
FTIR spectra taken on cell surfaces which were grown in the presence of copper-containing
medium revealed the prominent presence of NH2-groups.
Surface-chemical changes due to the presence of newly secreted proteins are brought about due
to preadaptation to toxic metal ions such as copper.
0.8
0.0
Electrophoretic mobility
-0.8 C
B
-1.6
-2.4
A
0 4 8
pH
Fig. 8.2: Electrophoretic mobility as a function of pH for cells of At.ferrooxidans grown in the (A) absence and
presence of (B) 10g/L and (C) 20 g/L of copper sulfate.
Another important aspect regarding development of metal-tolerant strains of At.ferrooxidans is

that the acquired toxic metal tolerance is found to be a temporary trait brought about by
membrane-related changes due to exposure to toxic stress. For example, when copper-tolerant
strains were back-cultured repeatedly in a 9K medium in the absence of cupric ions, the initially
6
acquired metal tolerance was found to be lost. In order to preserve such metal-tolerant strains, it
becomes necessary to store and resubculture them in the presence of the same concentration
levels of the toxic metal ions (such as copper). Same observation was found to be true in case of
bacterial strains preadapted to sulfide mineral concentrates as well.
Arsenic toxicity [40]
Biooxidation using At.ferrooxidans is used in the liberation of encapsulated gold particles from
arsenopyrite. Interaction of At.ferrooxidans with arsenic and iron sulfides and the role of arsenic
toxicity are therefore practically significant.
Arsenic is toxic to At.ferrooxidans, resulting in significant decrease in the growth rate and
eventual death. The levels at which arsenic becomes toxic to At.ferrooxidans are not clearly
known. Bacterial cells can be adapted to tolerate much higher concentrations of arsenic species
than it naturally would allow. Tolerance levels for arsenic ranging anywhere from 1mg/L upto
40g/L have been reported. Arsenic present in the trivalent form is found to be three to eight
times more toxic to At.ferrooxidans than As (V).
Toxicity of As (III) and As (V) on cells is dependent on the availability of an energy source.
It has been documented that At.ferrooxidans does not oxidize arsenite to arsenate, but they can
oxidize arsenic-containing minerals such as arsenic sulfide, arsenopyrite and enargite.
Arsenate formed in the cultures was either a result of oxidation from ferric iron or autoxidation
in conjunction with the metabolite, but not by the bacteria itself.
Exposure of cells of Acidithiobacillus ferrooxidans to arsenic (as As+++ and As+++++) resulted in
increased cell surface hydrophobicity and decreased electrophoretic mobility. Strains grown in
presence of arsenic exhibited stronger surface adsorption on arsenopyrite indicating that
hydrophobic interactions alone are not principally responsible for the initial adsorption of
adapted strains of At ferrooxidans.
7
Bacterial resistance to arsenic was found to be mainly due to active efflux system and not
because of decreased membrane permeability. Neither the bacterial cells nor the ferric ions were
capable of oxidizing or precipitating arsenite ions directly. Presence of ferric ions in the EPS
was necessary for binding or entrapment of arsenic ions in the EPS during the growth of
At.ferrooxidans. Bacterial EPS of ferrous-grown unadapted cells were able to uptake arsenate
ions due to the strong affinity of ferric ions towards arsenate ions.
Both arsenate and arsenite ions were co-precipitated in the presence of ferric ions, formed during
bacterial growth, resulting in the formation of crystalline jarosite, scorodite and amorphous ferric
arsenate or jarosite, tooeliete and amorphous ferric arsenite phases respectively. Co-precipitation
of arsenic with the ferric hydroxide precipitate formed during growth of At. ferrooxidans can be
made use of for remediation of arsenic in arsenic contaminated soils and waters.
8
Lecture 9: Development Of Metal-tolerant Acidithiobacillus Ferrooxidans NPTEL Web Course
Lecture 9
Development Of Metal-tolerant Acidithiobacillus ferrooxidans
Keywords: Metal Tolerance, Multimetal Tolerance, Serial Subculturing
Multi-metal toxicity
How to quantify metal tolerance in relation to unadapted and adapted strains of

At.ferrooxidans?
A comparison index termed Toxicity Index (TI) can be used which is the ratio of the
period required for complete iron oxidation by a strain in presence of a given
concentration of toxic metal ion to that required by an unadapted strain in the absence of
the toxic metal.
For a normally 9K grown strain of At.ferrooxidans, it takes upto 42-45 hours for
complete ferrous ion oxidation. Considering a sampling period of 8 hours, a strain can be
considered fully metal tolerant if the TI is in the range of 1-1.2.
For TI higher than 1-1.2, the added metal ion is considered toxic to the organism. [41]
Ferrous ion oxidation ability of different strains of At.ferrooxidans in the presence and absence
of varying concentrations of toxic metal ions such as ferric, cupric and zinc is illustrated in table
9.1.
1
Table 9.1: Iron oxidation rates for At.ferrooxidans in the presence and absence of toxic metal ions.
Time for complete

Type of strain Toxic metal ion ferrous oxidation in TI
9K medium
Normal strain grown ---- 42h 1

in 9K medium
Unadapted strain in Fe+++ 5g/L 74h 1.8

the presence of 10g/L 140h 3.3
different toxic metals
Cu++ 5g/L 45h 1.1
10g/L 80h 1.9
Zn++ 10g/L 45 1.1

20g/L 50 1.2
40g/L 95 2.3
Relatively, among the three types of toxic metal ions, ferric ions confer higher toxicity, followed
by cupric ions for the growth of At.ferrooxidans with respect to ferrous ion oxidation. Zinc ions
are relatively less toxic compared to ferric and cupric ions for a given metal ion concentrations.
For a strain preadapted to 10 g/L of ferric ion, the toxicity index decreases from 3.3 to 1.1
if exposed to a 9K medium containing upto 10g/L of cupric ions.
Such a Fe+++ ion preadapted strain aquires tolerance towards higher concentrations of
copper and zinc also.
Iron oxidation behavior of metal-tolerant At.ferrooxidans are illustrated in table 9.2.
2
Table 9.2: Iron oxidation behavior of metal-tolerant At.ferrooxidans
Time for complete

Type of strain Toxic metal ion TI
Fe++ oxidation(h)
At.ferrooxidans 5g/L of Fe+++ 50 1.2

preadapted to 25g/L 10g/L of Fe+++ 85 2.0
of cupric ion
10g/L of Cu++ 45 1.1
25g/L of Cu++ 45 1.1
20g/L of Zn++ 45 1.1

++
40g/L of Zn 60 1.4
At.ferrooxidans 5g/L of Fe+++ 50 1.2

preadapted to 40g/L 10g/L of Fe+++ 85 2.0
of ZnSO4
10g/L of Cu++ 45 1.1
25g/L of Cu++ No significant iron ----
oxidation
20g/L of Zn++ 42 1.0

++
40g/L of Zn 42 1.0
Preadaptation to 25g/L of cupric ions, reduces toxicity index towards ferric ion toxicity
and enhances also both copper and zinc tolerance.
3
Preadaptation to 40g/L of Zinc ions, on the other hand did not significantly enhanced cupric ion
tolerance (eg: 25g/L of Cu++)
Multimetal tolerance in At.ferrooxidans is illustrated in table 9.3
Table 9.3: Multimetal tolerance in At.ferrooxidans
Metal ion concentration, g/L Time for complete

Type of strain TI
Fe +++
Cu ++
Zn ++ Fe++Oxidation
Normal unadapted 5 20 120 2.9

5 40 Insignificant iron ---
oxidation
5 10 --- 125 3.0
5 25 --- Insignificant iron ----

oxidation
10 20 115 2.7
10 40 Insignificant iron ---
oxidation
5 10 20 130 3.1
5 10 40 Insignificant iron ---
oxidation
Preadapted to 10g/L of Fe+++ --- 25 40 Insignificant oxidation ---
5 25 40 Insignificant oxidation ---
Preadapted to 25g/L of Cu++ --- 25 40 55 1.3

5 25 40 58 1.4
Preadapted to 40g/L of Zn++ --- 25 40 No significant oxidation ---
5 25 40 No significant oxidation ---
4
Metal ion toxicity in the presence of multiple toxic metal ions presents a different picture.
If the concentration of any metal ion in the mixture attains the inhibitory concentration level
when present alone, the biological iron oxidation would be impeded even of the other metals (as
binary or ternary mixture) are present in lower non-inhibitory levels.
Effect of ternary metal ion combinations in a solution is more detrimental (inhibitory) to

bacterial growth than similar levels when present as a binary combination.
Metal toxicity exhibited by a single metal ion (when present alone) is retained even when more
than one (binary or ternary) metal is present simultaneously.
At.ferrooxidans preadapted to 25g/L of copper sulfate was found to exhibit reasonable tolerance
to all metals such as copper, ferric and zinc, even when present in binary or ternary
combinations. The observation implies that copper adapted strains of At.ferrooxidans may be the
best suited for bioleaching of multimetal concentrates containing copper, zinc and iron sulfides.
To demonstrate the advantages of preadapted metal-tolerant At.ferrooxidans in bioleaching,

copper dissolution and zinc dissolution from their respective concentrates were monitored using
different types of bacterial strains as illustrated in fig.9.1 and 9.2. As expected, use of copper
adapted strains resulted in higher copper extraction from a chalcopyrite concentrate and that of
zinc-adapted strains produced highest zinc recovery from a sphalerite concentrate. Unadapted
cells were relatively inefficient in the bioleaching of both concentrates. Zinc-adapted cells were
unable to tolerate higher dissolved copper in solution. [38, 39, 41]
On the otherhand, copper-adapted cells were capable of tolerating higher zinc concentrations.
Besides the adaptation of the bacterial cells to dissolved toxic metal ions, preadaptation through
continuous growth in the presence of the metal concentrates themselves would be beneficial. It
has been experimentally established that preadaptation of bacterial cells to the concentrate
substrates is essential to ensure more efficient bioleaching. (see Figs 9.1 to 9.3).
5
50
A Copper adapted bacteria)
B Unadapted cells)
C Control)
40 A
30
% Cu
B
20
C
10
0
0 20 40 60
Period, d
Fig. 9.1:Bioleaching of chalcopyrite-pyrite multiconcentrate using adapted and unadapted strain of

At. ferrooxidans.
++
20 (A) At.ferrooxidans preadapted to 40 g/L Zn
++
(B) At.ferrooxidans preadapted to 25 g/L Cu
(C) Unadapted strain
(D) Control
15 A
B
Zinc (g/L)
10
C
5 D
0
0 5 10 15
(days)
Fig. 9.2: Bioleaching of sphalerite concentrate with different strains of At.ferrooxidans.
6
++
3 (A) At.ferrooxidans preadapted to 25 g/L Cu
++
(B) At.ferrooxidans preadapted to 40g/L Zn
(C) Unadapted strain
(D) Control
A
Copper (g/L) 2
1
B
C
D
0
0 5 10 15
Days
Fig. 9.3: Bioleaching of chalcopyrite concentrate using different strains of At.ferrooxidans.
7
References(Lectures 8-9):
36. I.A. Chisholm, L.G.Leduc and G.D.Ferroni, Metal resistance and plasmid DNA in
Thiobacillus ferrooxidans, Antonie Van Leeuwenhoek, 73, (1998), pp 245-254.
37. O.H Tuovinen, S.I.Niemela and H.G.Gyllenberg, Tolerance of Thiobacillus ferrooxidans
to some metals, Antonie Van Leeuwenhoek, 37, (1971), pp 489-496.
38. A.Das, J.M. Modak and K.A. Natarajan, Surface Chemical Studies of Thiobacillus
ferrooxidans with Reference to Copper Tolerance, Antonie van Leeuwenhoek, Journal
of Microbiology, 73 (1998), pp 215-222.
39. K.A. Natarajan, K. Sudeesha and G. Ramananda Rao, Retainability of copper tolerance
in Thiobacillus ferrooxidans Antonie van Leeuwenhoek, 66 (1994) pp. 303- 306.
40. M.N. Chandraprabha and K. A. Natarajan, Mechanism of arsenic tolerance and
bioremoval of arsenic by Acidithiobacillus ferrooxidans, J. Biochem Tech. 3(2), (2011),
pp 257-265.
41. A.Das, J.M. Modak and K. A. Natarajan, Studies on multi-metal ion tolerance of
Thiobacillus ferrooxidans, Minerals Engineering, 10, (1997), pp 743-749.
8

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Lecture 11: Biohydrometallurgy Of Copper Dump And Heap Leaching NPTEL Web Course

Keywords: Heap Leaching, Dump Leaching

Dump (heap) bioleaching

Table 11.1: Some industrial heap bioleaching operations for copper

Heap bioleach plant Copper, t / year

Lince II, Chile, 1991 - Oxides / Sulfides 27 000

Cerro Colorado, Chile, 1993 - Chalcocite, covellite 1.0 lakh

Quebrada Blanca, Chile, 1994 - Chalcocite 75 000

Ivan-Zar, Chile, 1994 - Oxides / Sulfides 10 000 12 000

Punta del Cobre, SA/Chile, 1994 Oxides / Sulfides 7 000-8 000

Escondida, Chile, 1995 - Oxides / Sulfides 200 000

Hellenic Copper Mines/Cyprus, 1996 - Oxides / Sulfides 8 000

Dos Amigos, Chile, 1996 - Chalcocite 21 000

Cerro Verde, Peru, 1977 - Oxides / Sulfides 54 200

Lomas Bayas, Chile, 1998 - Oxides / Sulfides 60 000

Zaldivar, Chile, 1998 - Chalcocite 150 000

Nifty Copper, Australia, 1998 - Chalcocite, Oxides 16 000

S & K Copper, Monywa, Myannar, 1999 - Chalcocite 40 000

Morenci, Arizona, 2001 - Chalcocite, Pyrite 380 000

Jinchuan Copper, China, 2006 - Chalcocite, Enargite, Covellite 10 000

Constellation Copper Corporation/Lisbon Valley, Utah, 2006 - Chalcocite, Oxides 27 000

BHP Billiton/Chile, Spence, 2007 Sulfides, Oxides 200 000

Factors affecting leaching rates

Keywords: Developments In Heap, Thermophile Leaching, Chalcopyrite Leaching.

Heap bioleaching of chalcopyrite ores need several controls such as

Control heat transfer Humidity and aeration control.

Heap (bio) leaching of copper ores is a practical technology.

The StickBugsTM process, currently being developed, is capable of generation of sufficient

Thermophile bioleach status

Successful batch bioleaching of chalcopyrite (98%), enargite (98%), and sphalerite

Bac Tech/Mintek process (using moderate thermophiles)

Successful bioleaching of Mount Lyell copper concentrate in 1997.

Fig.12.1: Schematic representation of chalcopyrite coating on a support rock in a heap configuration.

Fig. 12.3: Pilot Plant for High Temperature Heap Bioleaching

Heap bioleaching tests on Iranian low grade chalcopyrite ores

About 0.6% copper as average could be achieved through a 70:30 blend.

References (Lectures 10-12):

Bioleaching of nickel ores and concentrates

Typical bioleaching reactions involving nickel sulfides are illustrated below:

(Ni Fe)9 S8 = 9Ni++ + 9Fe++ + 8S + 18e

2S + 2H2O + 3O2 = 2H2SO4

NiS + H2SO4 = NiSO4 + H2S

NiS + 2Fe+++ = Ni++ + 2Fe++ + S

In a pyrite-chalcopyrite-pentlandite-pyrrhotite combination, galvanic dissolution of pentlandite

In laboratory bioleaching tests using a copper-nickel-iron sulfide ore in the presence of

Nickel-copper-iron sulfide concentrate grown adapted At.ferrooxidans, could extract about

Bioleaching of nickel from laterites

reduction or through use of reducing agents secreted by microorganisms. The following

Use of biologically-derived reducing and metal-complexing organic acids.

Reductive dissolution of goethite coupled to sulfur oxidation is given below

6 - Fe O.OH + S + 10H+ = 6 Fe++ + SO4 - - + 8H2O

Acidophiles such as At.ferrooxidans and Acd.organivorans were found to catalyse reductive

Keywords: Polymetallic Schist, Heap Bioleaching, Talvivaara Experience

Heap bioleaching of a polymetallic nickel ore at Talvivaara, Finland.

Heap bioleaching conditions are summarized below:

Ore supply 25 Mtpa

Heap dimensions 8m high (dynamic) for primary.

The following organisms were detected in column tests at acidic pH.

Metals recovery flowsheet is illustrated in fig. 14.2.

Fig. 14.2: Metals recovery flowsheet.

Table 14.1: Heap leaching data for various minerals

Pentlandite 0.57 % 0.07 %

References (Lectures 13-14):