Review

Biological approaches for treatment of distillery wastewater: A review

Deepak Pant
a
, Alok Adholeya
a,b,
*
a
Centre of Bioresources and Biotechnology, TERI University, DS Block, India Habitat Centre, Lodhi Road, New Delhi 110 003, India
b
Biotechnology and Management of Bioresources Division, The Energy and Resources Institute, DS Block, India Habitat Centre,
Lodhi Road, New Delhi 110 003, India
Received 12 June 2006; received in revised form 13 September 2006; accepted 25 September 2006
Available online 7 November 2006
Abstract
Euent originating from distilleries known as spent wash leads to extensive soil and water pollution. Elimination of pollutants and
colour from distillery euent is becoming increasingly important from environmental and aesthetic point of view. Stillage, fermenter and
condenser cooling water and fermenter wastewater are the primary polluting streams of a typical distillery. Due to the large volumes of
euent and presence of certain recalcitrant compounds, the treatment of this stream is rather challenging by conventional methods.
Therefore, to supplement the existing treatments, a number of studies encompassing physico-chemical and biological treatments have
been conducted. This review presents an account of the problem and the description of colour causing components in distillery waste-
water and a detailed review of existing biological approaches. Further, the studies dealing with pure cultures such as bacterial, fungal,
algal and plant based systems have also been incorporated. Also, the roles of microbial enzymes in the decolourization process have been
discussed to develop a better understanding of the phenomenon.
2006 Elsevier Ltd. All rights reserved.
Keywords: Colour removal; Distillery euent; Enzymes; Melanoidin; Microorganisms; Wastewater treatment
1. Introduction
Production of ethanol from agricultural materials for
use as an alternative fuel has been attracting worldwide
interest because of the increasing demand for limited
non-renewable energy resources and variability of oil and
natural gas prices. In India this demand is projected to
go up because of a law for mixing 5% ethanol with petrol
and further raising this amount to 10% (The gazette of
India, 2002). Besides this, the other common usages of eth-
anol are in the form of industrial solvent and beverages. In
the year 1999, there were 285 distilleries in India producing
2.7 10
9
L of alcohol and generating 4 10
10
L of waste-
water each year (Joshi, 1999). This number has gone up
to 319, producing 3.25 10
9
L of alcohol and generating
40.4 10
10
L of wastewater annually (Uppal, 2004). Over
the years as the sizes and number of distilleries have grown,
bigger conventional aerobic-treatment plants have been
built to deal with the constantly increasing euent vol-
umes. Space and money to construct these installations
are the biggest hindrances for such investments (Fumi
et al., 1995).
In India, there are a number of large-scale distilleries
integrated with sugar mills. The waste products from
sugar mill comprise bagasse (residue from the sugarcane
crushing), pressmud (mud and dirt residue from juice
clarication) and molasses (nal residue from sugar
crystallization section). Bagasse is used in paper manufac-
turing and as fuel in boilers; molasses as raw material in
distillery for alcohol production while pressmud has no
direct industrial application (Nandy et al., 2002). The eu-
ents from molasses based distilleries contain large amounts
of dark brown coloured molasses spent wash (MSW). In
the distillation process, ethanol ranges from 5% to 12%
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.09.027
*
Corresponding author. Address: Biotechnology and Management of
Bioresources Division, The Energy and Resources Institute, DS Block,
India Habitat Centre, Lodhi Road, New Delhi 110 003, India. Tel.: +91 11
24682100/24682111; fax: +91 11 24682144/24682145.
E-mail address: aloka@teri.res.in (A. Adholeya).
Bioresource Technology 98 (2007) 23212334
by volume, hence it follows that the amount of waste varies
from 88% to 95% by volume of the alcohol distilled. An
average molasses based distillery generates 15 L of spent
wash L
1
of alcohol produced (Beltran et al., 2001).
MSW is one of the most dicult waste products to dispose
because of low pH, high temperature, dark brown colour,
high ash content and high percentage of dissolved organic
and inorganic matter (Beltran et al., 1999). The biochemi-
cal oxygen demand (BOD) and chemical oxygen demand
(COD), the index of its polluting character, typically range
between 35,00050,000 and 100,000150,000 mg L
1
,
respectively (Nandy et al., 2002).
Worldwide environment regulatory authorities are set-
ting strict norms for discharge of wastewaters from indus-
tries. In India for instance, distillery industry had been told
to achieve zero discharge of spentwash by December 2005
according to the charter of Central Pollution Control
Board, the apex pollution control authority (CPCB,
2003). It further says that till 100% utilization of spentwash
is achieved, controlled and restricted discharge of treated
euent from lined lagoons during rainy season will be
allowed by SPCBs/CPCB in such a way that the perceptible
colouring of river water bodies does not occur. Physical,
chemical and biological treatment approaches have been
employed for the treatment of distillery wastewater. This
review focuses mainly on lab and eld scale biological
approaches. The overall objective of the work is to present
a literature review on the state of the art in this eld and
address the issues requiring further research.
2. Pollution and toxicity prole of distillery euent
The production and characteristics of spentwash are
highly variable and dependent on feedstocks and various
aspects of the ethanol production process. Wash water
used to clean the fermenters, cooling water blow down,
and boiler water blow down further contributes to its
variability (Duarte et al., 1997). In a distillery, sources of
wastewater are stillage, fermenter and condenser cooling
water and fermenter wastewater. The liquid residues during
the industrial phase of the production of alcohol are:
liquor, sugar cane washing water, water from the condens-
ers and from the cleaning of the equipment, apart from
other residual water. This extract is extremely polluting
as it contains approximately 5% organic material and fertil-
izers such as potassium, phosphorus and nitrogen. The
amount of water used in this process is large, generating
a high level of liquid residues (Borrero et al., 2003).
The MSW is a potential water pollutant in two ways.
First, the highly coloured nature of MSW can block out
sunlight from rivers and streams, thus reducing oxygena-
tion of the water by photosynthesis and hence becomes det-
rimental to aquatic life. Secondly, it has a high pollution
load which would result in eutrophication of contaminated
water courses (FitzGibbon et al., 1998). Due to the pres-
ence of putriciable organics like skatole, indole and other
sulphur compounds, the MSW that is disposed in canals
or rivers produces obnoxious smell (Mahimaraja and
Bolan, 2004). Undiluted euent has toxic eect on shes
and other aquatic organisms. The estimated LC
50
for dis-
tillery spent wash was found to be 0.5% using a bio-toxicity
study on fresh water sh Cyprinus carpio var. communis
(Mahimaraja and Bolan, 2004). Impacts of distillery eu-
ent on carbohydrate metabolism of freshwater sh, C. car-
pio were studied recently by Ramakritinan et al. (2005).
The respiratory process in C. carpio under distillery euent
stress was aected resulting in a shift towards anaerobiosis
at organ level during sublethal intoxication.
Spent wash also leads to signicant levels of soil pollu-
tion and acidication in the cases of inappropriate land dis-
charge. It is reported to inhibit seed germination, reduce
soil alkalinity, cause soil manganese deciency and damage
agricultural crops (Kannabiran and Pragasam, 1993; Agra-
wal and Pandey, 1994). However, eect of distillery euent
on seed germination is governed by its concentration and is
crop-specic. In a study by Ramana et al. (2002) the germi-
nation percent in ve crops decreased with increase in con-
centration of the euent. The germination was inhibited in
all the ve crops studied with concentration exceeding 50%.
At the same time, organic wastes contained in distillery
euent are valuable source of plant nutrients especially
N, P, K and organic substrates if properly utilized (Pathak
et al., 1999). For instance, distillery euent in combination
with bioamendments such as farm yard manure, rice husk
and Brassica residues was used to improve the properties
of sodic soil (Kaushik et al., 2005). The use of fungi for
bioconversion of distillery waste into microbial biomass
or some useful metabolites has been recently reviewed by
Friedrich (2004). The end products of bioconversion are
fungal biomass, ethanol, enzymes etc. and substantially
puried and decolourized euents. Recently enhanced pro-
duction of oyster mushrooms (Pleurotus sp.) using distill-
ery euent as a substrate amendment have been reported
(Pant et al., 2006).
3. Colorants in distillery wastewaters
The molasses wastewater from alcoholic fermentation
has a large amount of a brown pigment. The colour is
hardly degraded by the conventional treatments and can
even be increased during anaerobic treatments, due to
repolymerization of compounds. Phenolics (tannic and
humic acids) from the feedstock, melanoidins from Mail-
lard reaction of sugars (carbohydrates) with proteins
(amino groups), caramels from overheated sugars, and
furfurals from acid hydrolysis mainly contribute to the col-
our of the euent (Kort, 1979). During heat treatment, the
Maillard reaction (non enzymatic reaction) takes place
accompanied by formation of a class of compounds known
as Maillard products. The reaction proceeds eectively at
>50 C and it is favored at pH 47 (Morales and Jimnez-
Perez, 2001). Melanoidins are one of the nal products of
the Maillard reaction. They are complex compounds with
their structures not fully understood.
2322 D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334
Melanoidin is one of the biopolymers that is hardly
decomposed by microorganisms and is widely distributed
in nature. Melanoidins have antioxidant properties, which
render them toxic to aquatic micro and macroorganisms
(Kitts et al., 1993). However, melanoidins present in Span-
ish sweet wines were studied by Rivero-Perez et al. (2002)
and they reported that high molecular weight brown pig-
ments (mostly hypothesized to be melanoidins) isolated
by dialysis were correlated with the colour of the wines
but not to their antioxidant activity. Melanoidins, or
related formation products can occur in dierent processes
of beverage manufacture, such as heat concentrated juices
and musts, beers or wines (Kroh, 1994). From studies using
13
C and
15
N CP-NMR spectrometry, Hayase et al. (1986)
conrmed the presence of olenic linkages and conjugated
enamines which were suggested to be important for the
structure of the chromophores in melanoidin. The reduc-
tion in intensity of absorption for the ozonated sample
indicates a cleavage of the C@C on ozonation.
For melanoidins formed from carbohydrates and amino
acids, a new model of a basic melanoidin skeleton mainly
built up from amino-branched sugar degradation products
was suggested by Cammerer et al. (2002). They indicated
that oligo- and polysaccharides reacted in the Maillard
reaction preferentially as complete molecules at the reduc-
ing end under water-free reaction conditions. Another
approach to estimate the chemical structure of melanoidins
was suggested by Kato and Hayase (2002). A blue pigment
(Blue-M1, C
27
H
31
N
4
O
13
) was isolated from the reaction
mixture of D-xylose and glycine in 60% ethanol stored at
26.5 C for 48 h (or 2 C for 96 h) under nitrogen, whose
chemical character was comparable to that of a nondialyz-
able melanoidin preparation obtained from the reaction
mixture of D-xylose and butylamine neutralized with acetic
acid in methanol incubated at 50 C for 7 days. Recently,
the empirical formula of melanoidin has been suggested
as C
1718
H
2627
O
10
N. The molecular weight distribution
is between 5000 and 40,000. It consists of acidic, polymeric
and highly dispersed colloids, which are negatively charged
due to the dissociation of carboxylic acids and phenolic
groups (Manisankar et al., 2004).
4. Spent wash treatment
Biological treatments have been recognized as eective
methods of treatment for highly polluted industrial waste-
waters. Both anaerobic and aerobic systems are commonly
used to treat the wastewaters from agro-industrial plants
including distilleries as well. In the recent years, increasing
attention is also being directed towards utilizing microbial
activity (pure bacteria and fungi) for the decolourization
and mineralization of spent wash. There are several reports
citing the potential of microorganisms for use in this pro-
cess. Moreover, the biologically treated euent could be
used safely and eectively to increase the soil productivity.
This section is discussed in detail as anaerobic and aerobic
treatments.
4.1. Anaerobic treatment
Anaerobic digestion is widely accepted as the rst treat-
ment step in distilleries. Wilkie et al. (2000) have reviewed
the role of anaerobic digestion in stillage (spent wash)
treatment. Anaerobic digestion can convert a signicant
portion (>50%) of the COD to biogas, which may be used
as an inplant fuel, and also saves the energy that would be
required for aeration using aerobic treatment. At present,
the anaerobic biological treatment of distillery euents is
widely applied as an eective step in removing 90% of the
COD in the euent stream (Wolmarans and de Villiers,
2002). During this stage, 8090% BOD removal takes place
and biochemical energy recovered is 8590% as biogas.
A list of common types of anaerobic reactors used for
distillery euent treatment is given in Table 1. Akunna
and Clark (2000) studied the performance of a granular
bed anaerobic baed reactor (GRABR) in the treatment
of a whisky distillery wastewater having COD and BOD
concentrations of 16,60058,000 and 890030,000 mg L
1
,
respectively. The removal of total BOD and COD from
the wastewater were 8092% and 9096%, respectively with
a HRT of 4 days and at a loading rate of 2.37 kg COD
m
3
day
1
.
The highest BOD removal is possible in open lagoon
whereas highest biomethane produced is in upow anaero-
bic sludge blanket (UASB) type bioreactor. The UASB
system has become the most widely applied reactor
technology for high rate anaerobic treatment of industrial
euents. Its relative high treatment capacity compared to
other systems permits the use of compact and economic
wastewater treatment plants. Compared to aerobic system,
it has slow growth rate, mainly associated with methano-
genic bacteria. Therefore, it requires a long retention time,
and also only a small portion of the degradable organic
waste is being synthesized to new cells. Full-scale thermo-
philic (5055 C) anaerobic digestion of wastewater from
an alcohol distillery was reported by Vlissidis and Zoubou-
lis (1993). More than 60% removal of COD was achieved
with 76% of biogas comprising of methane thus making
it a valuable fuel.
Goodwin and Stuart (1994) studied two identical UASB
reactors operated in parallel as duplicates for 327 days for
the treatment of malt whisky pot ale and achieved COD
reductions of up to 90% for inuent concentrations of
352652126 mg L
1
. When the OLRs of 15 kg m
3
day
and above were used, the COD removal eciency dropped
to less than 20%, in one of the duplicate reactors. A meso-
philic two-stage system consisting of an anaerobic lter
(AF) and an UASB reactor was found suitable for anaero-
bic digestion of distillery waste, enabling better conditions
for the methanogenic phase (Blonskaja et al., 2003). The
optimum conditions for the stable work of reactor are:
for the acidogenic stage, organic loading of 24 kg
COD m
3
day
1
at pH 6.0 and for the methanogenic stage,
organic loading of 12 kg COD m
3
day
1
at pH 7.6.
An advanced version of UASB system was reported by
D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334 2323
Driessen and Yspeert (1999), wherein they used an internal
circulation (IC) reactor characterized by biogas separation
in two stages within a reactor with a high weight/diameter
ratio and the gas driven internal euent circulation. This
system could handle high upow liquid and gas velocities
making possible treatment of low strength euents at short
hydraulic retention times as well as treating high strength
euents such as from brewery at very high volumetric
loading rates up to 35 kg COD m
3
. Eect of addition of
macronutrients and micronutrients in the distillery euent
was investigated on the performance of simulated UASB
system by Sharma and Singh (2001). Calcium and phos-
phate were found to be detrimental to treatment eciency.
Uzal et al. (2003) investigated the biochemical methane
potential (BMP) of malt whisky distillery wastewater both
with and without basal medium to observe the eect of
nutrient supplementation. When a COD concentration of
20,920 mg L
1
was maintained in the inuent to the rst
stage, the euent quality of the rst stage began to deteri-
orate. A signicant colour change was observed from black
to brownish black and then to brown, and this was thought
to be due to reduced metabolic activity owing to the toxic
eect of the wastewater on granular biomass, thus increas-
ing oxidation-reduction potential. It was concluded that
two stage UASB reactor conguration is an ecient system
for malt whisky wastewater treatment until up to
33,866 mg L
1
inuent COD concentration. For the over-
all sequential system (anaerobic/aerobic) treatment COD
and BOD removal eciencies were 99.5% and 98.1%,
respectively, for the treatment of malt whisky wastewater.
In aerobic phase, the euent of anaerobic bioreactor is
exposed to atmospheric oxygen in a tank with homogeniz-
ers for proper mixing of the euents. BOD is reduced to
200 and euent diluted with wastewater from bottling
and washer sections and disposed of after clarication
(Ramendra and Awasthi, 1992). The stabilized sludge
serves as a soil conditioner and plant nutrient.
Cost economics of biogas production by anaerobic
treatment was calculated by Ciftci and Ozturk (1995). They
suggested that for every $100 spent for the operation of
full-scale anaerobic aerobic treatment plants in a fermen-
tation industry in Turkey producing bakers yeast from
sugarbeet molasses, the biogas recovery is worth $300.
Garcia-Calderon et al. (1998) reported the application of
the down-ow uidization technology for the anaerobic
digestion of red wine distillery wastewater. The system
achieved 85% TOC removal, at an organic loading rate
of 4.5 kg TOC m
3
day
1
. Perlite was found to be a good
carrier for the anaerobic digestion as it allowed a high
biomass hold-up, with minimum particle wash out, because
of its density.
Immobilization of bacteria in biolm and on bioocs is
a crucial step in anaerobic degradation because of advanta-
ges such as higher activities, higher COD removal percent
at short hydraulic retention times and better tolerance to
disturbances such as toxic and organic shock loadings.
At the same time there are certain disadvantages as well
because in addition to some readily biodegradable matter,
vinasses contain compounds like phenols, which are toxic
to bacteria and inhibit the digestion. Also, due to seasonal
nature of many of these industries and the absence of
microorganisms in vinasses capable of carrying out anaer-
obic digestion, long incubation periods are required for
the start-up stage. Besides, other operational problems in
anaerobic digestion such as low growth rate of anaerobic
bacteria and the loss of biomass in systems with high
hydraulic rates frequently does not achieve a satisfactory
purication of vinasses (Beltran et al., 1999). The for-
mation of H
2
S in anaerobic reactors is the result of the
reduction of oxidized sulphur compounds. Methanogenic
bacteria can tolerate sulphide concentration up to
1000 mg L
1
total sulphide. A complete loss of methane
production occurred at 200 mg L
1
of un-ionized H
2
S dur-
ing digestion of occulent sludge. Anaerobic contact pro-
cess incorporating an ultraltration (UF) unit was used
to treat distillery wastewater characterized by high and
low carbon to nitrogen concentrations. This treatment sys-
tem showed methane yield of up to 0.6 m
3
kg
1
VS and
Table 1
Anaerobic methods employed for distillery euent treatment
Reactor type Organic loading rate (OLR)
(kg COD m
3
day
1
)
COD
removal
(%)
BOD
removal
(%)
Retention
time
(days)
Reference
Downow xed-lm reactor 6073 8597 Bories and Ranyal, 1988
Granular bed anaerobic baed
reactor (GRABR)
2.37 9096 8092 4 Akunna and Clark, 2000
Hybrid anaerobic baed reactor 20 70 Boopathy and Tilche, 1991
Upow anaerobic sludge blanket
(UASB) reactor
28 3967 80 Harada et al., 1996
Istanbul UASB reactor 611 90 Akarsubasi et al., 2006
Tekirdag UASB reactor 2.58.5 6080
Diphasic xed-lm reactor with
granular activated carbon (GAC)
as support media
21.3 67.1 4 Goyal et al., 1996
Anaerobic contact lter 19,000 mg L
1
(inuent COD concentration)
7398 4 Vijayaraghavan and
Ramanujam, 2000
2324 D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334
removed up to 80% of the volatile acids (Kitamura et al.,
1996). Two-phase anaerobic digestion of alcohol stillage
proved to be superior to the single-phase process in terms
of substrate loading rate and methane yield, without aect-
ing the treatment performance (Yeoh, 1997). While main-
taining BOD and COD reduction of 85% and 65%
respectively, the two-phase achieved methane yield three
times that of single-phase system.
A number of environmental factors aect the activity of
wastewater microbial populations and the rate of biochem-
ical reactions. Of particular importance are temperature,
pH, nutrients, and inhibiting toxic compounds. Due to
acidic nature of vinasses, pH is one of the most relevant
factors aecting the microbiological activity in the biolog-
ical process. The pH of wastewater increases from 4.0 to
7.5 after anaerobic digestion due to the oxidation of
organic acids to CO
2
and the reaction between the CO
2
and basic compounds to form carbonates and bicarbonates
(Beltran et al., 1999). Because of its high organic load, the
distillery wastewater is often diluted with tap water to sim-
ulate the concentration of a typical industrial euent enter-
ing a wastewater treatment plant. However, spent wash
even after anaerobic treatment does not meet the stringent
euent standards laid down by CPCB, India, in terms of
very high levels of BOD, COD, solids etc. (Asthana
et al., 2001). Also, the secondary spent wash produced by
the anaerobically digested primary molasses spent wash
(DMSW) euent is darker in colour, needing huge vol-
umes of water to dilute it and is currently used in irrigation
water causing gradual soil darkening. The euent there-
fore is released after diluting with fresh water which is a
very dear commodity to industries. Besides, sometimes fail-
ure of the anaerobic digestion threatens the criteria for dis-
charge limit. To overcome this problem either a large
amount of water is used to dilute the wastewater prior to
anaerobic digestion or chemical coagulants are added.
These actions require expansion of the anaerobic digester
volume, large amounts of water for dilution, and addi-
tional costs for coagulants (Kim et al., 1997). Hence aero-
bic treatment is necessary for anaerobically treated nal
euent.
4.2. Aerobic treatment
4.2.1. Bacterial treatment
Microbial treatments employing pure bacterial culture
have been reported frequently in past and recent years. A
detailed list of bacteria tried by dierent researchers for
decolourization of distillery euent is given in Table 2.
Kumar and Viswanathan (1991) isolated bacterial strains
from sewage and acclimatized on increasing concentrations
of distillery waste. These strains were able to reduce COD
by 80% in 45 days without any aeration. The major prod-
ucts left after treatment were biomass, carbon dioxide and
volatile acids. Petruccioli et al. (2000) used an air bubble
column reactor with activated sludge carrying self adapted
microbial population in both free and immobilized on
polyurethane particles for treating aerobic winery wastewa-
ter. The highest COD removal rate was with free activated
sludge in the bubble column reactor. The most prominent
bacterial species isolated from the reactor liquid belonged
to Pseudomonas while Bacillus was isolated mostly from
colonized carriers. Pseudomonas uorescens, decolourized
melanoidin wastewater (MWW) up to 76% under non-ster-
ile conditions and up to 90% in sterile samples (Dahiya
et al., 2001a). The dierence in decolourization might be
due to the fact that melanoidin stability varies with pH
and temperature and at higher temperature during sterili-
zation melanoidin-pigments decompose to low molecular
weight compounds (Ohmomo et al., 1988b). The eect of
immobilization on the decolourization of a melanoidin
solution may be explained by the fact that Lactobacillus hil-
gardii requires a small amount of oxygen for the decolou-
rization and immobilization within Ca-alginate gel leads
to suitably limited aeration, supplying a small amount of
oxygen continuously (Ohmomo et al., 1988a).
Acetogenic bacteria are capable of oxidative decomposi-
tion of melanoidins. Cibis et al. (2002) achieved biode-
gradation of potato slops (distillation residue) by a mixed
population of bacteria under thermophilic conditions up
to 60 C. A COD removal of 77% was achieved under
non-optimal conditions. Marine cyanobacteria such as
Oscillatoria boryna have also been reported to degrade mel-
anoidin due to production of H
2
O
2
, hydroxyl, perhydroxyl
and active oxygen radicals, resulting in the decolourization
of the euent (Kalavathi et al., 2001). 96%, 81% and 26%
decolorisation of distillery euent through bioocculation
by Oscillatoria sp., Lyngbya sp. and Synechocystis sp.
respectively was reported by Patel et al. (2001).
Distillery spent wash, despite carrying high organic
load contains little readily available carbon. Isolation
of bacterial strains capable of degrading recalcitrant com-
pounds of anaerobically digested spent wash from soil of
euent discharge site was reported by Ghosh et al.
(2004). These were Pseudomonas, Enterobacter, Steno-
trophomonas, Aeromonas, Acinetobacter and Klebsiella all
of which could carry out degradation of some component
of spent wash. Maximum 44% COD reduction was
achieved using these bacterial strains either singly or col-
lectively. Sirianuntapiboon et al. (2004) used an acetogenic
bacterium to obtain a decolourization yield of 76.4%
under optimal nutrient conditions. However, this value
was only 7.3%, by using anaerobic pond. Also, it required
sugar, especially glucose and fructose for decolourization
of MWWs. The decolourization activity might be due to
a sugar oxidase.
4.2.2. Fungal treatment
In recent years, several basidiomycetes and ascomycetes
type fungi have been used in the decolourization of natural
and synthetic melanoidins in connection with colour reduc-
tion of wastewaters fromdistilleries. The aimof fungal treat-
ment is to purify the euent by consumption of organic
substances, thus, reducing its COD and BOD, and at the
D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334 2325
same time to obtain some valuable product, such as fungal
biomass for protein-rich animal feed or some specic fungal
metabolite. Filamentous fungi have lower sensitivity to vari-
ations in temperature, pH, nutrients and aeration and have
lower nucleic acid content in the biomass (Knapp et al.,
2001). Several fungi have been investigated for their ability
to decolourise melanoidins and MSW (Table 3).
Coriolus sp. no. 20, in class basidiomycetes was the rst
strain for the application of its ability to remove melanoi-
dins from MWW (Watanabe et al., 1982). This isolate
did not show any decolourization activity when molasses
pigment was used as carbon source but it showed the activ-
ity when sorbose or glucose was added. In 1985, Ohmomo
et al. used Coriolus versicolour Ps4a for MWW decolouri-
zation and obtained 80% decolourization in darkness
under optimum conditions. Later, Ohmomo et al. (1988b)
used autoclaved mycelium of Aspergillus oryzae Y-2-32
that adsorbed lower weight fractions of melanoidin and
degree of adsorption was inuenced by the kind of sugars
used for cultivation. The wine distilleries produce large vol-
umes of wastewaters having phenolic compounds, which
give a high inhibitory and anti-bacterial activity to this
wastewater, thus slowing down the anaerobic digestion
process. Partial elimination of these phenolics compounds
was obtained by using Geotrichum candidum (Borja et al.,
1993). Rhizoctonia sp. D-90 decolourized molasses mela-
noidin medium and a synthetic melanoidin medium by
87.5% and 84.5% respectively, under experimental growth
conditions. Electron microscopy revealed that the mycelia
absorbed melanoidin pigment, which was in the form of
electron dense material in the cytoplasm. However, mela-
noidin could be eluted from the mycelia by washing in a
Table 2
Bacteria employed for the decolourization of distillery euent
S. no. Name Comments Colour Removal (%) Reference
1 Xanthomonas fragariae All the three strains needed glucose as carbon source and
NH
4
Cl as nitrogen source. The decolourization eciency of
free cells was better than immobilized cells
76 Jain et al., 2002
2 Bacillus megaterium 76
3 Bacillus cereus 82
4 Bacillus smithii Decolourization occurred at 55 C in 20 days under anaerobic
conditions in presence of peptone or yeast extract as
supplemental nutrient. Strain could not use MWW as sole
carbon source
35.5 Kambe et al., 1999
5 Lactobacillus hilgardii Immobilized cells of the heterofermentative lactic acid
bacterium decolourized 40% of the melanoidins solution within
4 days aerobically
40 Ohmomo et al., 1988a
6 Acetobacter acetii The organism required sugar especially, glucose and fructose
for decolourization of MWWs
76.4 Sirianuntapiboon et al.,
2004
7 Pseudomonas
uorescens
This decolourization was obtained with cellulose carrier coated
with collagen. Reuse of decolourized cells reduced the
decolourization eciency
94 Dahiya et al., 2001a
8 Pseudomonas putida The organism needed glucose as a carbon source, to produce
hydrogen peroxide which reduced the colour
60 Ghosh et al., 2002
9 Acinetobacter sp. All these organisms were isolated from an air bubble column
reactor treating winery wastewater after 6 months of
operation. Most isolates from the colonized carriers belonged
to species of the genus Bacillus
Not checked in this
study
Petruccioli et al., 2000
10 Aeromonas sp.
11 Alcaligens faecalis
12 Bacillus sp.
13 Flavobacterium sp.
14 F. meningosepticum
15 Pseudomonas sp.
16 P. paucimobilis
17 P. vescicularis
18 Sphingobacterium
multivorum
19 Bacillus thuringiensis Addition of 1% glucose as a supplementary carbon source was
necessary
22 Kumar and Chandra,
2006
20 Bacillus brevis 27.4
21 Bacillus sp. 27.4
22 Pseudomonas
aeruginosa
The three strains were part of a consortium which decolourized
the anaerobically digested spent wash in presence of basal salts
and glucose
67 Mohana et al., 2007
23 Stenotrophomonas
maltophila
24 Proteus mirabilis
2326 D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334
Table 3
Fungi employed for the decolourization of Distillery euent
S.
no.
Name Comments Colour
Removal (%)
Reference
1 Phanerochaete
chrysosporium
Both the fungi required a readily available carbon source for
melanoidin decolourization while N source had no eect. Maximum
decolourization was observed in 6.25% (v/v) spent wash
53.5 Kumar et al., 1998
2 Coriolus versicolor 71.5
3 Trametes versicolor COD and NNH
4
removal observed in presence of sucrose and
KH
2
PO
4
as nutrient source
82 Benito et al., 1997
4 Geotrichum
candidum
Fungus immobilized on polyurethane foam showed stable
decolourization of molasses in repeated-batch cultivation
80 Kim and Shoda, 1999
5 Coriolus hirsutus A large amount of glucose was required for colour removal but
addition of peptone reduced the decolourizing ability of the fungus
80 Miyata et al., 2000
6 Penicillium sp. All fungi produced decolourization from rst day of incubation,
with maximum being shown byP. decumbens at fourth day with a
reduction of 70% of the phenolic content of the wastewater
30 Jimnez et al., 2003
7 Penicillium
decumbens
41
8 Penicillium
lignorum
28
9 Aspergillus niger 25
10 Aspergillus niger
UM2
Decolourization was more by immobilized fungus and it was able to
decolourize up to 50% of initial euent concentrations
80 Patil et al., 2003
11 Aspergillus
fumigatus G-2-6
Thermophilic strain tried for molasses wastewater decolourization
but colouring compounds hardly degraded
56 Ohmomo et al., 1987
12 Mycelia sterilia Organism required glucose for the decolourizing activity 93 Sirianuntapiboon et al., 1988
13 Aspergillus niger Maximum colour removal was obtained when MgSO
4
, KH
2
PO
4
,
NH
4
NO
3
and a carbon source was added to wastewater
69 Miranda et al., 1996
14 Flavodon avus MSW was decolourized using a marine basidiomycete fungus. It also
removed 68% benzo(a)pyrene, a PAH found in MSW
80 Raghukumar and Rivonkar,
2001; Raghukumar et al., 2004
15 Rhizoctonia sp. D-
90
Mechanism of decolourization of melanoidin involved absorption of
the melanoidin pigment by the cells as a macromolecule and its
intracellular accumulation in the cytoplasm and around the cell
membrane as a melanoidin complex, which was then gradually
decolourized by intracellular enzymes
90 Sirianuntapiboon et al., 1995
16 Coriolus versicolor
Ps4a
Two types of enzymes, sugar-dependent and sugar-independent,
were found to be responsible for melanoidin decolourizing activity
80 Ohmomo et al., 1985
17 Aspergillus oryzae
Y-2-32
The thermophilic strain adsorbed lower molecular weight fractions
of melanoidin and required sugars for growth
75 Ohmomo et al., 1988b
18 Phanerochaete
chrysosporium
JAG-40
This organism decolourized synthetic and natural melanoidins when
the medium was supplemented with glucose and peptone
80 Dahiya et al., 2001b
19 Coriolus hirsutus
IFO4917
Melanoidins present in heat treatment liquor were subjected to
sequencing batch decolourization by the immobilized fungal cells
45 Fujita et al., 2000
20 Aspergillus niveus The fungus could use sugarcane bagasse as carbon source and
required other nutrients for decolourization
56 Angayarkanni et al., 2003
21 Trametes sp. I-62 No colour observed associated with either fungal mycelium or
polysaccharides secreted by the fungus and therefore colour removal
was attributed to fungal degradation and not to a simple physical
binding
73 Gonzalez et al., 2000
22 Aspergillus niger All these organisms were isolated from an air bubble column reactor
treating winery wastewater after 6 months of operation
Not checked
in this study
Petruccioli et al., 2000
23 Candia sp.
24 C. lambica
25 C. lypolitica
26 Fusarium sp.
27 Penicillium sp.
28 P. roquefortii
29 Saccharomyces
cerevisiae
30 Trichoderma
koningii
31 Coriolus sp. no. 20 First strain for the application of its ability to remove melanoidins
from MWW, showed decolourization activity in 0.5% melanoidin
when sorbose or glucose was added as carbon source
80 Watanabe et al., 1982
(continued on next page)
D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334 2327
solution of NaOH and the relative amount of melanoidin
eluted from the mycelia increased with increase in the con-
centration of NaOH (Sirianuntapiboon et al., 1995). Asper-
gillus awamori var. kawachi has been used for production
of single cell protein from Japanese distillery (Shochu)
wastewater after aerobic cultivation (Kida et al., 1995).
The supernatant after cultivation could be anaerobically
treated, at a high TOC loading rate, by the addition of
Ni
2+
and Co
2+
. Also, NH

4
, accumulated in the anaerobi-
cally treated wastewater, was eciently removed by utiliza-
tion of residual volatile fatty acids (VFA) as electron
donors during biological denitrication and nitrication,
and the residual organic matter could be removed simulta-
neously. Colour elimination from MSW using Aspergillus
niger was studied by Miranda et al. (1996). Under optimal
nutrient concentration 83% of the total colour removed
was eliminated biologically and 17% by adsorption on
the mycelium. Ohmomo et al. (1988a) concluded that
microbial decolourization of melanoidins is due to two
decomposition mechanisms; in the rst the smaller molecu-
lar weight melanoidins are attacked and in the second the
larger molecular weight melanoidins are attacked.
Under nutrient limiting conditions, fungal cells gener-
ally cannot remain active during a long-term cultivation.
Therefore, the continuous-culture method is not practical
and the semi-batch or repeated-batch method can be an
alternative for long-term cultivation. The immobilization
of the fungus on a solid support is an appropriate means
for controlling the thickness of the biolm. The immobili-
zation of the fungus oers advantages such as short reten-
tion time, easy recovery of the cells and increased activity.
Furthermore, in the presence of the foam matrix, pellet
size is restricted by the size and the physical properties
of the foam (Kim and Shoda, 1999). Miyata et al. (2000)
suggested an inhibitory eect of organic nitrogen on mel-
anoidin decolourization by fungus Coriolus hirsutus. At
Table 3 (continued)
S.
no.
Name Comments Colour
Removal (%)
Reference
32 Williopsis saturnus
strain CBS 5761
Yeast isolates from a rotating biological contactor (RBC) treating
winery wastewater. Only 43% COD removal could be achieved
Not checked
in this study
Malandra et al., 2003
33 Pichia
membranaefaciens
strain IGC 5003
34 Candia intermedia
JCM 1607
35 Eremothecium
gossyphi
36 Saccharomyces
cerevisiae strain J2
37 Hanseniaspora
uvarum
38 Coriolus versicolor
sp no. 20
10% diluted spent wash was used with glucose @ 2% added as carbon
source
34.5 Chopra et al., 2004
39 Phanerochaete
chrysosporium
Sugar renery euent was treated in a RBC using polyurethane foam
and scouring web as support
55 Guimaraes et al. (2005)
40 Pycnoporus
coccineus
Immobilized mycelia removed 50% more colour than free mycelia 60 Chairattanamanokorn et al.,
2005
41 Coriolus versicolor Cotton stalks were added as additional carbon source which stimulated
the decolourization activity of all fungi in 30% vinasses
63 Kahraman and Yesilada,
2003
42 Phanerochaete
chrysosporium
37
43 Funalia trogii 57
44 Pleurotus
pulmonarius
43
45 Aspergillus-UB2 This was with diluted wastewater with optimum values of
supplemented materials
75 Shayegan et al. (2004)
46 Marine
Basidiomycete
NIOCC # 2a
Experiment was carried out at 10% diluted spent wash 100 Dsouza et al. (2006)
47 Phanerochaete
chrysosporium
Molasses medium decolourization was checked in stationary and
submerged cultivation conditions
Thakkar et al., 2006
NCIM 1073 0
NCIM 1106 82
NCIM 1197 76
48 Citeromyces sp.
WR-43-6
Organism required glucose, Sodium nitrate and KH
2
PO
4
for maximal
decolourization
68.91 Sirianuntapiboon et al., 2003
49 Hansenula fabianii The occulant strains could reduce 28.5% TOC from wastewater
without dilution
Not checked
in this study
Moriya et al., 1990
50 Hansenula anomala
2328 D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334
the same time glucose was also required for enhancing
decolourization as the peroxidases require H
2
O
2
, which
is generated by glucose oxidation, to decolourize melanoi-
din. Chopra et al. (2004) reported that absence of addi-
tional nitrogen could not inhibit activity of fungus C.
versicolour sp no. 20 considerably, as signicant decolouri-
zation and COD reduction occurred even in the absence of
it.
Colour removal from distillery euent using a marine
fungus, Flavodon avus has been reported by Raghukumar
and Rivonkar (2001). This fungus was more eective in
decolourizing raw MSW than was the molasses wastewater
collected either after anaerobic treatment or after aerobic
treatment. The oxygen demand of the fungus was quite
high. P. chrysosporium JAG-40 decolourized synthetic
and natural melanoidins present in spentwash up to 80%
(Dahiya et al., 2001b). The larger molecular weight frac-
tions of melanoidin were decolourized rapidly, while the
small molecular weight fractions remained in solution
and were metabolized slowly. Also, the decolourization
was less in sterilized spentwash than in non-sterile solution.
This observation is completely opposite of the one when
Pseudomonas uorescens was used by same authors
(Dahiya et al., 2001a). Kahraman and Yesilada (2003)
reported molasses decolourization in semi solid state
(SSS) cultivation by fungi C. versicolour, Funalia trogii,
P. chrysosporium and Pleurotus pulmonarius with cotton
stalks being used as additional source of carbon. C. versi-
colour decolourized 48% of 30% diluted vinasse without
any additional carbon source which increased to 71% on
addition of cotton stalks. Aspergillus niveus, a litter degrad-
ing fungi was used by Angayarkanni et al. (2003) for the
treatment of distillery euent using paddy straw, sugar-
cane bagasse, molasses and sucrose as carbon source for
growth of fungus in the euent. Sugarcane bagasse at
1% (w/v) concentration resulted in maximum removal of
colour (37%) and COD (91.68%). The decrease in colour
removal in this study might be due to the fact that the eu-
ent taken for study was alkaline (pH 9.0) and the melanoi-
dins responsible for colour were more soluble in the
alkaline pH. In the acidic pH, the melanoidins might be
precipitated and removed easily. Shayegan et al. (2005)
used an Aspergillus species isolated from the soil for
decolourization of anaerobically digested (UASB) and
aerobically treated distillery wastewater. With diluted
wastewater at optimum values of supplemented materials
75% decolourization was achieved which reduced to 40%
on using undiluted wastewater. It was suggested that deco-
lourization by fungi takes place due to the destruction of
coloured molecules and partially because of sorption phe-
nomena. A longer aeration period causes the adsorbed col-
our molecules to be released as a result of endogenous
respiration and cell death, hence reducing decolourization
eciency.
Yeast Citeromyces was used for treating MWW and
high and stable removal eciencies in both colour intensity
and organic matter were obtained. However, the semi-pilot
and pilot-scale experiments are to be tested for checking
the stability of Citeromyces sp. (Sirianuntapiboon et al.,
2003). Malandra et al. (2003) studied the microorganisms
associated with a rotating biological contactor (RBC)
treating winery wastewater. One of the yeast isolates was
able to reduce the COD of synthetic wastewater by 95%
and 46% within 24 h under aerated and non-aerated condi-
tions, respectively. Moriya et al. (1990) used two occulant
strains of yeast, Hansenula fabianii and Hansenula anomala
for treatment of wastewater from beet molasses-spirits pro-
duction and achieved 25.9% and 28.5% removal of TOC
respectively from wastewater without dilution. Dilution
of wastewater was not favourable for practical treatment
of wastewater due to the longer treatment time and higher
energy cost.
4.2.3. Mixed consortium treatment
During last two decades, several attempts have been
made to investigate the possibility of using cell immobili-
zation in the technology of aerobic wastewater treatment
(Fedrici, 1993; Sumino et al., 1985). Early experiments
were restricted to the use of selected pure cultures im-
mobilized on solid supports for the degradation of specic
toxic compounds (Anselmo et al., 1985; Livernoche et al.,
1983). Later, immobilized consortia of two or more
selected strains were employed (Kowalska et al., 1998;
Zache and Rehm, 1989) but of late activated sludge has
been immobilized on dierent carriers and used for waste-
water treatment (Shah et al., 1998). Jet loop reactors
(JLR), the eciency of which has already been shown in
both chemical and biological processes have also been
evaluated for aerobic treatment of winery wastewater. A
JLR of 15 dm
3
working volume was used for the aerobic
treatment of winery wastewater (Petruccioli et al., 2002).
COD removal eciency higher than 90% was achieved
with an organic load of the nal euents that ranged
between 0.11 and 0.3 kg COD m
3
. Most isolates belong
to the genus Pseudomonas and the yeast Saccharomyces
cerevisiae. Later, Eusibio et al. (2004) reported the opera-
tion of a JLR for more than one year treating winery
wastewater collected in dierent seasons and achieved an
average COD removal eciency of 80%. JLR have higher
oxygen transfer rates at lower energy costs. They also
observed Bacillus apart from Pseudomonas and the yeast
Saccharomyces cerevisiae. Adikane et al. (2006) studied
decolourization of molasses spent wash in absence of
any additional carbon or nitrogen source using soil as
inoculum. A decolourization of 69% was obtained using
10% (w/v) soil and 12.5% (v/v) MSW after 7 days
incubation.
4.2.4. Phytoremediation approach
Algal growth potential bioassay is a standard assay to
determine the potential of water bodies, natural waters
and wastewaters, to support or inhibit the microalgae
growth. Algae growth potential was determined in distill-
ery wastewater pretreated by anaerobic processes and by
D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334 2329
a combined anaerobic-aerobic system. The biologically
treated distillery wastewater provided satisfactory condi-
tions for microalgae growth (Travieso et al., 1999). Billore
et al. (2001) used Phragmites kharka in a constructed wet-
land for treatment of wastewater from the same industry
and obtained 36% removal of total Kjeldahl nitrogen and
48% removal of total suspended solids (TSS). Enhanced
decolourization was achieved by phytoremediation of
distillery euent by a macrophyte, Spirodela polyrrhiza
(L.) Schliden pretreated with Bacillus thuringiensis (Kumar
and Chandra, 2004). Recently, macrophyte Potamogeton
pectinatus was used for bioaccumulating heavy metals from
distillery euent (Singh et al., 2005). Increasing concentra-
tion of the euent greatly reduced the biomass of the plant
with maximum accumulation of Fe being recorded in
plants growing in 100% euent. In another study Trivedy
and Nakate (2000) employed Typha latipholia for distillery
euent treatment in a constructed wetland. The system
resulted in 78% and 47% reduction in COD and BOD
respectively in a period of 10 days. Using a combined treat-
ment with Lemna minuscula and Chlorella vulgaris Valder-
rama et al. (2002) reported 52% colour removal from
distillery euent. The microalgal treatment removed nutri-
ents and organic matter from wastewater and produced
oxygen for other organisms. The macrophyte removed
organic matter and eliminated the microalgae form treated
wastewater. However, despite the potential of aquatic mac-
rophytes in cleaning wastewaters the use of these plants in
designing a low cost treatment system is still at experimen-
tal stage and is considered to be a potentially important
area of environmental management.
5. Role of enzymes in euent decolourization
Although the enzymatic systemrelated with decolouriza-
tion of melanoidins is yet to be completely understood, it
seems greatly connected with fungal ligninolytic mech-
anisms. The white-rot fungi have a complex enzymatic
system which is extracellular and non-specic, and under
nutrient-limiting conditions is capable of degrading ligno-
lytic compounds, melanoidins, and polyaromatic com-
pounds that cannot be degraded by other microorganisms
(Benito et al., 1997). A large number of enzymes from a
variety of dierent plants and microorganisms have been
reported to play an important role in an array of waste
treatment applications.
Several studies regarding degradation of melanoidins,
humic acids and related compounds using basidiomycetes
have also suggested a participation of at least one laccase
enzyme in fungi belonging to Trametes (Coriolus) genus.
The role of enzymes other than laccase or peroxidases in
the decolourization of melanoidins by Trametes (Coriolus)
strain was reported during the 1980s. Several reports
claimed that intracellular sugar-oxidase- type enzymes (sor-
bose-oxidase or glucose-oxidase) had melanoidin-decolou-
rizing activities. It was suggested that melanoidins were
decolourized by the active oxygen (O
2
; H
2
O
2
) produced
by the reaction with sugar oxidases (Watanabe et al.,
1982). Decolourization by microbial methods includes the
enzymatic breakdown of melanoidin and occulation by
microbially secreted substances. Ohmomo et al. (1985) used
C. versicolour Ps4a, which decolourized molasses wastewa-
ter 80% in darkness under optimum conditions. Decolouri-
zation activity involved two types of intracellular enzymes,
sugar-dependent and sugar-independent. One of these
enzymes required no sugar and oxygen for appearance of
the activity and could decolourize MWW up to 20% in
darkness and 1117% of synthetic melanoidins. Thus, the
participation of these H
2
O
2
producing enzymes as a part
of the complex enzymatic system for melanoidin degrada-
tion by fungi should be taken into account while designing
any treatment strategy. One of the more complete enzy-
matic studies regarding melanoidin decolourization was
reported by Miyata et al. (1998). Colour removal of syn-
thetic melanoidin by C. hirsutus involved the participation
of peroxidases (MnP and MIP) and the extracellular H
2
O
2
produced by glucose-oxidase, without disregard of a partial
participation of fungal laccase. Mansur et al. (1997)
obtained a maximum decolourization of around 60% on
day 8 after inoculating with fungus Trametes sp. I-62. Here
euent was added at a nal concentration of 20% (v/v)
after 5 days of fungal growth, the time at which high levels
of laccase activity were detected in the extracellular
mycelium.
The white-rot basidiomycete T. versicolour is an active
degrader of humic acids as well as of melanoidins. A mel-
anoidin mineralizing 47 kDa extracellular protein corre-
sponding to the major mineralizing enzyme system from
T. versicolour was isolated by Dehorter and Blondeau
(1993). This Mn
2+
dependent enzyme system required oxy-
gen and was described to be as peroxidase. Uniform, small
and spongy pellets of the fungus T. versicolour were used as
inoculum for colour removal using dierent nutrients such
as ammonium nitrate, manganese phosphate, magnesium
sulphate and potassium phosphate and also sucrose as car-
bon source (Benito et al., 1997). Maximum colour removal
of 82% and 36% removal of NNH

4
was obtained on using
low sucrose concentration and KH
2
PO
4
as the only nutri-
ent. Some studies have identied the lignin degradation
related enzymes participating in the melanoidin decolouri-
zation. Intracellular H
2
O
2
producing sugar oxidases have
been isolated from Coriolus strains. Also, C. hirsustus have
been reported to produce enzymes that catalyze melanoidin
decolourization directly without additions of sugar and O
2
.
Miyata et al. (1998) used C. hirsutus pellets to decolourize a
melanoidin-containing medium. It was elucidated that
extracellular H
2
O
2
and two extracellular peroxidases, a
manganese-independent peroxidase (MIP) and manganese
peroxidase (MnP) were involved in decolourization
activity.
Lee et al. (2000) investigated the dye-decolourizing per-
oxidase by cultivating Geotrichum candidum Dec1 using
molasses as a carbon source. Components in the molas-
ses medium stimulated the production of decolourizing
2330 D. Pant, A. Adholeya / Bioresource Technology 98 (2007) 23212334
peroxidase but inhibited the decolourizing activity of the
puried enzyme. It was found that the inhibitory eect of
molasses can be eliminated at dilution ratios of more than
25. Recently Dsouza et al. (2006) reported 100% decolou-
rization of 10% spent wash by a marine fungal isolate
whose laccase production increased several folds in the
presence of phenolic and non-phenolic inducers.
A combined treatment technique consisting of enzyme
catalyzed in situ transformation of pollutants followed by
aerobic biological oxidation was investigated by Sangave
and Pandit (2006a) for the treatment of alcohol distillery
spent wash. It was suggested that enzymatic pretreatment
of the distillery euent leads to in situ formation of the
hydrolysis products, which have dierent physical proper-
ties and are easier to assimilate than the parent pollutant
molecules by the microorganisms, leading to faster initial
rates of aerobic oxidation even at lower biomass levels.
In another study, Sangave and Pandit (2006b) used irradi-
ation and ultrasound combined with the use of an enzyme
as pretreatment technique for treatment of distillery waste-
water. The combination of the ultrasound and enzyme
yielded the best COD removal eciencies as compared to
the processes when they were used as stand-alone treatment
techniques. Enzymatic decolourization of molasses med-
ium has also been tried using P. chrysosporium (Thakkar
et al., 2006). Under stationary cultivation conditions, none
of the strains could decolourize molasses nor produce
enzymes lignin peroxidase, manganese peroxidase and lac-
case. All of them could produce lignin peroxidase and man-
ganese peroxidase when cultivated in at bottom glass
bottles under stationary cultivation conditions.
6. Conclusions
For industries using large quantities of water such as
distilleries, it is essential to treat and reuse their wastewater.
However, most of the times, the discharge standards
applied to most agro-industries, including distilleries are
often too stringent and below the levels that can be
achieved with appropriate biological treatment technolo-
gies. It has been observed and often reported that the use
of an individual process alone may not treat the wastewater
completely. A combination of these processes is necessary
to achieve the desirable goal. An anaerobic, or chemical
coagulation/oxidation pretreatment followed by aerobic
biological oxidation is a common technique used for decol-
orizing wastewater. But as discussed above, these processes
are not ecient enough to treat these large volumes of col-
oured wastewaters. A combination of dierent treatment
processes including a decolourization step could result in
an eective bioremediation of the molasses wastewaters.
In general, microbial decolourization is an environ-
ment-friendly and cost-competitive alternative to chemical
decomposition process. However, the problem still persists
because several organisms that have been shown to degrade
melanoidin are not best suited for treating MSW. This is
because they deplete oxygen in the euent and further,
higher fungi are not easily adopted for aquatic habitats.
The investigations so far can be seen as an initial step
toward solving the problem. Moreover, most of these
microbial decolourization studies required euent dilution
for optimal activity. While using microorganisms, use of
media supplement pose extra burden on overall euent
treatment process.
The use of cellulose carriers for microbial treatment
oers an alternative method for cell immobilization.
Gel entrapment has been conventional process of cell
immobilization. However, in processes like wastewater
treatment where large volumes are involved, entrapment
in gel beads is not as practical and economic when used
on an industrial scale. Further, the emerging treatment
methods like enzymatic treatment have technological
advantages and yet are in its infancy, requiring economical
considerations in order to apply it on the plant scale. Cap-
ital and operating costs of the available physicochemical
and biological treatment processes of distillery waste
stream are inevitably high thus making these processes less
lucrative to the industry. Nevertheless, the feasibility of
application of the process to full-scale would need further
research in this continuous culture set-up, in order to min-
imize the added nutrients and extend the biomass activity
for a longer period. An understanding of complete prole
of the euent and the structures of coloring compounds
would also be helpful in achieving the appropriate treat-
ment solutions.
Acknowledgements
Authors wish to thank Dr. R. K. Pachauri, Director-
General, TERI, New Delhi, India for support in research.
Financial assistance from University Grants Commission,
New Delhi in form of Senior Research Fellowship to the
rst author is duly acknowledged.
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