Bioresource Technology 97 (2006) 12371242

Bioremediation of toxic heavy metals using acidothermophilic autotrophes

Valentina V. Umrania
*
Department of Microbiology, MVM Sc and HSc College, Saurashtra University, Rajkot 360 002, India Received 12 February 2005; received in revised form 23 April 2005; accepted 30 April 2005 Available online 1 December 2005

Abstract Investigations were carried out to isolate microbial strains from soil, mud and water samples from metallurgically polluted environment for bioremediation of toxic heavy metals. As a result of primary and secondary screening various 72 acidothermophilic autotrophic microbes were isolated and adapted for metal tolerance and biosorption potentiality. The multi-metal tolerance was developed with higher gradient of concentrations of Ag, As, Bi, Cd, Cr, Co, Cu, Hg, Li, Mo, Pb, Sn and Zn. The isolates were checked for their biosolubilization ability with copper containing metal sulde ores. In case of chalcopyrite 85.82% and in covellite as high as 97.5% copper solubilization occurred in presence of 103 M multi-heavy metals on fth day at 55 C and pH 2.5. Chemical analyses were carried out by inductively coupled plasma spectroscopy (ICP) for metal absorption. The selected highly potential isolate (ATh-14) showed maximum adsorption of Ag 73%, followed by Pb 35%, Zn 34%, As 19%, Ni 15% and Cr 9% in chalcopyrite. 2005 Published by Elsevier Ltd.
Keywords: Bioremediation; Toxic heavy metals; Acidothermophilic autotrophes; Chalcopyrite; Covellite

1. Introduction Microbiological processes are of signicance in determining metal mobility and have actual and potential application in bioremediation of metal pollution. These include autotrophic and heterotrophic leaching mechanisms, reductive precipitation, sulfate reduction and metal sulde precipitation (Gadd, 2004). Such microbial processes are gaining increasing interest in the mining industry. Bioleaching of heavy metals, biooxidation of gold ores, desulfurization of coal and oil, tertiary recovery of oil and biosorption of metal ions are examples of the wide variety of potential and actual applications of microorganisms in mining and related elds (Rawlings, 1997; Brierley and Brierley, 1999; Umrania, 2003; Da Silva, 2003).
*

Tel.: +91 281 246 11 04. E-mail address: v_umrania@redimail.com

During the last two decades, extensive attention has been paid on management of environmental pollution and its control due to hazardous materials such heavy metals. Decontamination of heavy metals in the soil and water around industrial plants has been a challenge for a long time. The use of microorganism or its biomass for the recovery of metals from waste streams (Diels et al., 1993; Gadd, 1992) as well as the employment of plants for landll applications (Watanabe, 1997; Scott and Palmer, 1990; Umrania and Agate, 2003) have achieved growing attention. Contamination of soil and ground water due to the use of Cr in various anthropomorphic activities has become a serious source of concern to plant and animal scientists over the past decade (Shanker et al., 2005). Almost all metalmicrobe interactions have been examined as a means for removal, recovery or detoxication of inorganic and organic metal or radionuclide pollutants (Lovley and Coates, 1997; Francis, 1998;

0960-8524/$ - see front matter 2005 Published by Elsevier Ltd. doi:10.1016/j.biortech.2005.04.048

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Stephen and Macnaughton, 1999; Eccles, 1999). The microbial processes for bioremediation of toxic metals and radionucleids from waste streams employ living cells, non-living biomass, or biopolymers as biosorbents. A wide variety of fungi, algae and bacteria are now under study or are already in use as biosorbents for heavy metal remediation (Gadd, 1992; Volesky and Holan, 1995). Recent work (Johnson and Hallberg, 2004; Hall et al., 2005; Hall and Puhlmann, 2004) indicated that most of the transition between metal speciation forms was controlled by microbial behaviour. Whitehead et al. (2005) clearly demonstrated the potential for natural attenuation of acid mine drainage, particularly iron oxidation, by microbial populations, and the subsequent removal of key toxic metals. The cost eective and ecofriendly newer biotechnological processes viz. bioremediation and biobeneciation through microbial metal reabsorption have been widely accepted. Bioleaching/biosolubilization of metal sulde ores is an ideal alternative for the mitigation of pollution even at mining sites. It has been found that maximum rates and yields of metal extraction can be enhanced at elevated temperatures (Norris, 1990). Ultimately there is a need to search such metal tolerant, metal absorbent as well as moderate thermophilic acidophilic organisms for biogeotechnological applications. Bacteria have genes for most elements known to mankind. These genes determine transport systems for uptake of needed nutrients, including for example potassium, phosphorous and iron, for maintaining equilibrium intracellular concentrations balancing needs and toxicity, and for detoxication or elimination of purely toxic elements such as Hg, Pb, As, Cr, Cd, and Ag. Since life arose in an inorganic environment, these genes and the proteins they determine are found in all types of microbes, including prokaryotes, Archaea, and eukaryotes. Some of these genes and proteins show possibilities for their use in environmental measurements, and for clean up or detoxication of waste from human impact (Umrania et al., 1998; Umrania and Joshi, 2002; Umrania and Agate, 2003). In Western India, Rajkot city is metallurgically very active since decades. Many industries such as metal nishing industries, gold and silver rening industries, petroleum rening, iron and steel industries use copper, zinc, chromium, silver, lead and other heavy metals as

important ingredients to develop various types of metal alloys (Umrania et al., 1998). The objective of this work was to examine the potential biotechnological use of acidothermophilic bacteria for bioremediation of toxic heavy metals. Considering natural adaptation theory, industrial drainage samples were collected from such areas to screen and develop metal sulde biooxidizing microbial communities having excellent potency for heavy metal tolerance and bioadsorption activities.

2. Methods 2.1. Sample collection Soil, mud and water samples (Table 1) were collected from the euent disposal site of metal processing industries (higher temperature and presence of toxic heavy metals) from Rajkot city. Samples were categorized as A, B, C, and D as per dierent location of the industry. 2.2. Enrichment and primary screening Primary screening for isolating microbes was done by inoculating selected samples into various autotrophic enrichment broth under dierent incubation conditions (Postgate, 1966). The samples were inoculated into 100 ml of 9 K and MJ medium with 10% (v/v) inoculum and kept at various pH and temperatures ranging from 2.0 to 4.5 and 40 to 65 C, respectively and incubated in steady state for three weeks (Umrania and Joshi, 2002). After this, 10 ml of enriched broth was inoculated into 150 ml of modied 9 K medium containing FeSO4 7H2O and Na2S2O3 as energy source at 55 1 C and pH 2.5 on environmental orbit shaker at 150 rpm for six weeks. 2.3. Secondary screening The cultures were grown in 150 ml of 9 K medium into 250 ml Erlenmeyer ask with FeS, CuS and CuFeS2 on an orbital shaker at 200 rpm, 55 C and pH 2.5. The isolates were monitored by direct count method using Neubers double ruled chamber (Table 2). Titrimatric and colorimetric analysis was carried out for their pyrrohoitite, covellite and chalcopyrite biosolubilizing potency (Vogel, 1962). Control tests were performed

Table 1 Chemical composition of samples Site Gold renery Silver renery Diesel engine alloys foundries Group B and C A and D E and F Nature Mud/soil Water Mud/soil pH 4.5 3.5 3.0 Temperature (C) 55 60 65 Metal elements present (ppm) Cu: 1100; Fe: 580; Au: 78; Ag: 65; SO4 > NO3 > Cl Cu: 860; Fe: 388; Ag: 957; Zn: 640; Ni: 325; Pb: 135; NO3 > Cl Cu: 770; Fe: 6165; Al: 460; Zn: 410; Ni: 250; Cr: 350; Pb: 150; SO4 > Cl > NO3

V.V. Umrania / Bioresource Technology 97 (2006) 12371242 Table 2 Adaptation towards multiple heavy metal gradient by the isolates No. of isolates and group 6 30 18 7 8 3 Total 72 E A B D C F Initial tolerance % FeS 68 75 74 62 67 77 Concentration of metal gradient, M 10
7 6 5 4 3

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10

10

10 4 12 8 1 5 1 31

10 2 7 3 1 2 1 16

Increased tolerance after 12 transfers in 103M (%) 68.5 7678 74 64 6970 79 16 selected

6 30 18 7 8 3 72

6 18 18 5 8 3 58

5 15 18 2 7 3 50

with multi-metal containing articial ore sample and nutrient medium under sterile conditions. 2.4. Adaptation Organisms were exposed to articial multi-metal solution for adaptation and sequential transfers were carried out from survivors. A multi-metal solution was prepared by adding Ag, As, Bi, Cd, Cr, Co, Cu, Hg, Li, Mo, Pb, Sn, and Zn in a concentration gradient from 107 to 103 M (pH 2.5). Gradual adaptation was carried out by incubating at 60 C for ten days in each concentration of metals gradients for metal tolerance. 2.5. Biosorption experiments Control and inoculated samples were analyzed for metal adsorption by inductively coupled plasma optical emission spectroscopy (ICP-OES) (Perkin Elmer, USA).

ing total 72 isolates obtained with autotrophic growth at acidic pH and higher temperatures (Table 2). Gradual addition of multi-heavy metals resulted in the development of metal-tolerant communities, and the level of tolerance increased. During secondary enrichment program the isolates were monitored by direct count method using Neubers double ruled chamber. More than 10 cells per microscopic eld was considered as ++ and more than 25 cells as +++ and counts were recorded (data not shown) (for induction of multi-metal tolerance and bioadsorption). Secondary screening experiments showed an increase in the tolerance to metals other than the metal originally added to soil. Such az-Ravexperimental evidences were also reported by D in a et al. (1994) indicating that there was multiple heavy metal tolerance at the community level. Thus, presence of certain metals as pollutants could induce metal tolerance and absorption of Hg, Mo, Sn, Pb, Ag, which were natively absent in soil. 3.2. Heavy metals tolerance/adaptation

3. Results and discussion It has been shown that bacteria and fungi isolated from polluted environment are frequently tolerant to higher levels of metals than those isolated from unpolluted areas and that tolerant microorganisms are found at higher frequencies in polluted habitats (Arnea th, 1989; Huysman et al., 1994). brant et al., 1987; Ba Table 1 shows the characterization of industrial soil samples of Rajkot city. There was considerable content of Ag, Au, Cr, Cu, Ni, Pb and Zn but As, Bi, Cd, Co, Hg, Mo and Sn were present in negligible quantities. Potentially, any microorganism or cell fraction that exposes negatively charged groups on its surface should have an anity for metal cations (Ehrlich, 1986). 3.1. Primary and secondary screening program The development of acidothermophilic metal tolerant, metal adsorbent and metal sulde solubilizer bacterial strains were achieved as a result of screening program. As a result of enrichment and primary screenThe selected strains exhibited better metal cation anity depending upon soil sample chemical composition and groups (Table 1). In total 72 dierent isolates were obtained at 107 M and tolerance increased with prolonged incubations. Repeated subsequent transfers for twelve times, each for one week into higher concentrations of multi-heavy metals resulted in 58 survivors at 106 M, 50 in 105 M and 31 in 104 M. Gradual decrease in number of isolates was found with increase in concentration gradient and only 16 isolates were recovered at 103 M. The percentage of FeS biooxidation was considered for development of metal tolerance az-Ravin as illustrated in Table 2. D a et al. (1994), by using thymidine incorporation, soil bacterial communities articially exposed to Cu, Cd, Zn, or Ni during a ve to eight months incubation period, observed to have increased metal tolerance compared with those in the a th az-Ravin corresponding unpolluted soil. D a and Ba (1996) conclusively reported that the increase in metal tolerance of the community after adding metals could be attributed to an immediate eect due to the death

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of sensitive species and a later eect due to dierent competitive abilities and adaptation of surviving bacteria. From the nally selected 16 isolates, one designated as ATh-14 was studied for optimization with various physicochemical factors keeping the metal sulde solubilizing ability in view. 3.3. Bioremediation and optimization Fig. 1 shows copper solubilization 85.82% in chalcopyrite after ve days in presence of 103 M multi-metal concentration. Among the heavy metal studied the maximum adsorption was found for Ag 73%, followed by Pb 35.26%, Zn 34%, As 19.03% and Ni 14.85% with chalcopyrite by ICP analysis. In case of covellite (Fig. 2) as high as 97.5% copper solubilized after ve days in presence of 103 M multi-metal concentration. Maximum adsorption was found for Zn (34.35%), followed by Ni (29.4%) and Pb (26.54%) as shown by inductively coupled plasma analysis. The cells were constantly pretreated with iron during development and adaptation processes. Comparative studies with CuFeS2 and CuS revealed that presence of Fe played an important role. Bioadsorption was enhanced in chalcopyrite than in covellite. Similar results were reported by Iwahori et al. (2000), who found that normal bacterial community was sensitive to 0.7 lM of Hg2+ concentration and the most resistant Thiobacillus ferrooxidans SUG 2.2 could grow in autotrophic medium supplemented with Fe2+ at pH 2.5 containing 6 lM Hg2+ concentration, where the amount of mercury volatilized by resistant cells increased to 62% when Fe2+ was added. The optimization with physicochemical parameters such as eect of aeration and agitation (rpm), acidic pH range, size of particles of suldic ore and higher tem-

Fig. 2. Various metals adsorption by ATh-14 during CuS biosolubilization at 55 C, pH 2.5.

perature range is expressed in Figs. 36. Increase in rpm from 100 to 200 resulted increase in rate of FeS biooxidation (Fig. 3). Finely ground particles of pyrrohoitite (100 and 150 lm) exhibited 80% biooxidation on fth day while larger particles of the same required more time period for the same result (Fig. 4). With temperature as a parameter ATh-14 oxidized 80% FeS at 55 C for
ATh-14
80 Biooxidized FeS mg/l 79 78 77 76 75 74 73 100 150 rpm 200 250

Fig. 3. Optimization with various rpm at 55 C by ATh-14 at pH 2.5 on fth day.

ATh-14 81 80 Biooxidized FeS mg/l 79 78 77 76 75 74 100

150 200 mesh size micron

250

Fig. 1. Percent metal adsorbed by ATh-14 during CuFeS2 biosolubilization at 55 C, pH 2.5.

Fig. 4. Eect of various particle size at 55 C by ATh-14 at pH 2.5 on fth day.

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90 80 70 60 50 40 30 20 10 0 0 24 48 72 96 120 144 168 192 216 240 264 288

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40C 45C 50C 55C 60C 65C

metal adsorption in chalcopyrite than in covellite and appeared as a typical extremophile growing at pH 2.5 and temperature of 55 C with 85.82% copper solubilization in chalcopyrite and 97.5% in covellite within ve days in presence of 103 M multi-metal concentration and thus, appeared to be a suitable candidate in bioleaching and bioremediation processes. Acknowledgements The author is thankful to the Principal, and Head, Microbiology Department of MVM Sc and HSc College, Rajkot for their help and providing technical facilities. Thanks are also due to SICART, Vallabh Vidya Nagar to carry out ICP analysis. References
a th, E., Nordgren, A., 1987. Copper tolerance of Arnebrant, K., Ba microfungi isolated from polluted and unpolluted forest soil. Mycologia 79, 890895. a th, E., 1989. Eects of heavy metals in soil on microbial processes Ba and populations: a review. Water Air Soil Pollut. 47, 335379. Brierley, J.A., Brierley, C.L., 1999. Present and future commercial applications of biohydrometallurgy. In: Amils, R., Ballester, A. (Eds.), Biohydrometallurgy and the Environment Toward the Mining of the 21st Century, Part A. Elsevier, Amsterdam, The Netherlands, pp. 8189. Da Silva, E.J., 2003. Biotechnology, development and globalization. ASM News 69, 368369. a th, E., 1996. Development of metal tolerance in az-Ravin D a, M., Ba soil bacterial communities exposed to experimentally increased metal levels. Appl. Environ. Microbiol. 62, 29702977. ., 1994. Multiple heavy a th, E., Frostega rd, A az-Ravin D a, M., Ba metal tolerance of soil bacterial communities and its measurement by a thymidine incorporation technique. Appl. Environ. Microbiol. 60, 22382247. Diels, L., Van Roy, S., Mergeay, M., Doyen, W., Taghavi, S., Leysen, R., 1993. Immobilization of bacteria in composite membranes and developments of tubular membrane reactors for heavy metals recuperation. In: Peterson, R. (Ed.), Eective Membrane Processes: New Perspectives. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 275293. Eccles, H., 1999. Treatment of metal-contaminated wastes: why select a biological process? Trends Biotechnol. 17 462465. Ehrlich, H.L., 1986. What types of microorganisms are eective in bioleaching, bioaccumulation of metals, ore beneciation and desulfurization of fossil fuels? Biotechnol. Symp. 16, 227237. Francis, A.J., 1998. Biotransformation of uranium and other actinides in radioactive wastes. J. Alloys Compd. 271273, 7884. Gadd, G.M., 1992. Microbial control of heavy metal pollution. In: Fry, J.C., Gadd, G.M., Herbert, R.A., Jones, C.W., Watson-Craik, I.A. (Eds.), Microbial Control of Pollution. Cambridge University Press, Cambridge, United Kingdom, pp. 5987. Gadd, G.M., 2004. Microbial inuence on metal mobility and application for bioremediation. Geoderma 122, 109119. Hall, G.H., Puhlmann, T., 2004. Spatial distribution of iron oxidation in the aerobic cells of the Wheal Jane pilot passive treatment plant. Sci. Total Environ. 338, 7380. Hall, G., Swash, P., Kotilainen, S., 2005. The importance of biological oxidation of iron in the aerobic cells of the Wheal Jane pilot passive treatment system. Sci. Total Environ. 338, 4151.

% FeS biooxidation

Time in Hour
Fig. 5. Eect of various incubation temperatures on FeS biosolubilization by ATh-14 at pH 2.5.

100 90

% FeS biooxidation

80 70 60 50 40 30 20 10 0 0 24 48 72 96 120 144 168 192 216 240 264 288

1.5 2 2.5 3 3.5

Time in Hour
Fig. 6. Eect of various medium pH on FeS biosolubilization by ATh-14 at pH 2.5.

120 h and 144 h at 60 C. At lower temperature, activity slowed down as indicated in Fig. 5. At pH 3.0 the rate is highest while at pH 2.5 it slows down may be due to formation of ferric hydroxide jarosite formation and same was seen with pH 2.0 and 1.5 also as in Fig. 6. ATh-14 showed maximum biosolubilizing activity after ve days incubation at 60 C, pH 3.0; with 100 lm mesh size at 200 rpm on an orbital shaker. The potential absorption of silver, zinc, lead, arsenic, cobalt, lithium and nickel by the isolate. ATh-14 indicated the possibilities of exploring the isolate further in the eld of bioremediation of heavy metals from polluted environments. The absorption of silver by isolate, in particular, showed the possibilities of using the isolate for silver recovery from silver containing wastewater.

4. Conclusions From the results, it could be concluded that the bacterial ora isolated from metal processing euent possessed potential in respect of solubilization of copper and bioremediation activity. The isolate ATh-14 developed tolerance for several metals, which could be exploited at mining site where these metals are present either individually or in combination under various physicochemical conditions. The isolate showed highest

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