Review of Literature
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 ethanol are in the form of industrial solvent and
beverages.
The world wine production rate is approximately 2.65 × 108 hectolitres (hL) year-1 of
which 63% comes from the European Union (Duarte et al., 1997). This increased to
2.5 × 109 hL year-1 in 2005 (Research and Markets, 2006). There were 285
distilleries in India in 1999 producing 2.7 x 109 L of alcohol and generating 4 × 1010
L of wastewater each year (Joshi, 1999). This number has gone up to 319, producing
3.25 × 109 L of alcohol and generating 40.4 × 1010 L of wastewater annually (Uppal,
2004). Over the years, the sizes and number of distilleries have grown and small
units have made way for large distilleries. Consequently, bigger conventional
aerobic-treatment plants have been built to deal with the constantly increasing
effluent volumes. Space and money to construct these installations are the biggest
hindrances for such investments (Fumi et al., 1995). Therefore the wastewaters
from sugar and distillery industries are generally stored in large unlined lagoons,
which cause groundwater contamination (Kannan et al., 2004).
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 clarification) and molasses
(final residue from sugar crystallization section). Bagasse is used in paper
manufacturing and as fuel in boilers; molasses as raw material in distillery for
alcohol production while pressmud has no direct industrial application (Nandy et
3
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al., 2002). The effluents from molasses based distilleries contain large amounts of
dark brown colored molasses spentwash (MSW). In the distillation process, ethanol
ranges from 5-12% by volume, hence it follows that the amount of waste varies from
88-95% by volume of the alcohol distilled. An average molasses based distillery
generates 15 L of spentwash L-1 of alcohol produced (Beltran et al., 2001). MSW is
one of the most difficult waste products to dispose because of low pH, high
temperature, dark brown color, high ash content and high percentage of dissolved
organic and inorganic matter (Beltran et al., 1999b). The BOD and COD, the index
of its polluting character, typically range between 35,000-50,000 and 100,000-
150,000 mg L-1, respectively (Nandy et al., 2002).
As the MSW contain huge amounts of putriciable organics, the wastewater that is
disposed in canals or rivers even after treatment produces obnoxious smell
wherever it is stagnant. The unpleasant odour due to the presence of skatole, indole
and other sulphur compounds, which are not effectively decomposed by yeast or
methanogenic bacteria during distillation, is an issue of grave public concern
(Mahimaraja and Bolan, 2004).
Worldwide environment regulatory authorities are setting strict norms for
discharge of wastewaters from industries. 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, Charter on corporate responsibility for environmental protection,
CREP, 2003). It further says that till 100% utilization of spentwash is achieved,
controlled and restricted discharge of treated effluent from lined lagoons during
rainy season will be allowed by state pollution control boards (SPCBs)/CPCB in
such a way that the perceptible coloring of river water bodies does not occur. At
present, 107 distilleries have reported complete compliance with the CREP norms to
become zero-discharge units (Tewari et al., 2007).
In the following text, the physical, chemical and biological treatment technologies
adopted so far for the treatment of distillery wastewater, both at lab and field scale
has been discussed. The overall objective of this chapter is to present a literature
review on the state of the art in this field and address the issues requiring further
research.
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Pollution and toxicity profile of distillery effluent The production and characteristics of spentwash are highly variable and depend
upon 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 condensers 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 fertilizers
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).
Highly colored effluents can have negative environmental impacts if released into
surface waters, where they may disrupt the growth of normal aquatic flora (Wilkie
et al., 2000). Undiluted effluent has toxic effect on fishes and other aquatic
organisms. The estimated LC50 for distillery spentwash was found to be 0.5% using
a bio-toxicity study on fingerlings of a fresh water fish species viz., Cyprinus carpio
var. communis (Mahimaraja and Bolan, 2004). Similarly, the distillery effluent has
been proven toxic for catfish, Channa punctatus (Kumar and Gopal, 2001). The
minimum concentration that caused 100% mortality was recorded to be 16%
confirming the toxic nature of these effluents. Impacts of distillery effluent on
carbohydrate metabolism of freshwater fish, Cyprinus carpio were studied recently
by Ramakritinan et al. (2005). The respiratory process in C. carpio under distillery
effluent stress was affected resulting in a shift towards anaerobiosis at organ level
during sublethal intoxication. In addition to the effects on aquatic organism,
spentwash leads to significant levels of soil pollution in the cases of inappropriate
land discharge. When MSW is disposed in soil, it acidifies the soil and thereby
affects agricultural crops. It is reported to inhibit seed germination, reduce soil
alkalinity, cause soil manganese deficiency and damage agricultural crops
(Kannabiran and Pragasam, 1993; Agrawal and Pandey, 1994; Ramana et al.,
2002b). However, effect of distillery effluent on seed germination is governed by its
concentration and is crop-specific. In a study by Ramana et al. (2002a) the
germination percent in five crops decreased with increase in concentration of the
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effluent. The germination was inhibited in all the five crops studied with
concentration exceeding 50%.
At the same time, organic wastes contained in distillery effluent are valuable source
of plant nutrients especially N, P, K and organic substrates if properly utilized
(Pathak et al., 1999). Impact of long and short-term irrigation of a sodic soil with
distillery effluent in combination with bioamendments such as farm yard manure,
rice husk and Brassica residues were studied by Kaushik et al. (2005). Application
of 50% post-methanation effluent (PME) along with bioamendments proved to be
the most useful in improving the properties of sodic soil and also favoured
successful germination and improved seedling growth of pearl millet. 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 purified
and decolorized effluents. Recently enhanced production of oyster mushrooms
(Pleurotus sp.) using distillery effluent as substrate amendments have been
reported (Pant et al., 2006).
Colorants in distillery wastewaters
The molasses wastewater from alcoholic fermentation has large amount of brown
pigment and high oxygen demand. The color 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 Maillard reaction of sugars (carbohydrates) with
proteins (amino groups), caramels from overheated sugars, and furfurals from acid
hydrolysis mainly contribute to the color of the effluent (Kort, 1979). During heat
treatment, the Maillard reaction (non enzymatic reaction) takes place accompanied
by formation of a class of compounds knows as Maillard products. The chemistry of
the Maillard reaction is very complex, encompassing a whole network of
consecutive and parallel chemical reactions (Coca et al., 2004). Maillard reaction
proceeds effectively at >50 °C and it is favoured at pH 4-7 (Morales and Jiménez -
Perez, 2001). Melanoidins are one of the final products of the Maillard reaction.
They are complex compounds with their structures not fully understood.
Melanoidins also contribute to the browning and sensorial properties of food
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(Wedzicha and Kaputo, 1992). The composition of melanoidins depends on the
reaction conditions, mainly temperature, heating time, pH, water content and the
nature of reactants. An increase in pH or temperature leads to an increase in the
reactivity between the sugar and the amino group (Martins et al., 2001).
Melanoidins resemble humic substances in their chemical properties, being acidic,
polymeric and highly dispersed colloids which are negatively charged due to the
dissociation of carboxylic and hydroxylic groups (O’Melia, 1972). Indeed, humic
acid substances can be synthesized from a standard melanoidin preparation such as
a glucose/glycine mixture, and this is considered to be one way for the formation of
the humic acids in the environment (Stevenson, 1982). It 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 Spanish
sweet wines were studied by Rivero-Perez et al. (2002) and they reported that high
molecular weight brown pigments (mostly hypothesized to be melanoidins) isolated
by dialysis were correlated with the color of the wines but not to their antioxidant
activity. Romero et al. (2007) studied the humic acid-like (HAL) fractions in winery
and distillery wastes. The HAL fraction initially characterized by aliphatic character,
small acidic functional group content, presence of proteinaceous materials and
polysaccharide-like structures, after vermicomposting turns into typical soil humic
acid.
Melanoidins or related formation products can occur in different processes of
beverage manufacture, such as heat concentrated juices and musts, beers or wines
(Kroh, 1994). From studies using 13C and 15N CP-NMR spectrometry, Hayase et al.
(1986) confirmed the presence of olefinic linkages and conjugated enamines; these
unsaturated bonds were suggested to be important for the structure of the
chromophores in melanoidin. The decrease in intensity of absorption for the
ozonated sample indicates a cleavage of the –C=C- on ozonation. This cleavage
could be a major reaction in the decolorization process.
In spite of intensive research in the field of melanoidins, there is not much
generalized knowledge about their structural composition. 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
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Cammerer et al. (2002). They indicated that oligo- and polysaccharides reacted in
the Maillard reaction preferentially as complete molecules at the reducing 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, C27H31N4O13) was isolated from the reaction mixture of D-xylose and
glycine in 60% ethanol (starting pH 8.1) 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
nondialyzable 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. Low molecular weight colored substances with a molecular weight
smaller than 3500 Da has been reported. In the glucose/glycine Maillard reaction,
they account for 88% of the colored substances, whereas the high molecular weight
substances account for 12% (Martins and Van Boekel, 2003).
Recently, the empirical formula of melanoidin has been suggested as C17–18H26–
27O10N. The molecular weight distribution is between 5,000 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).
Treatment of Distillery wastewater Physico-chemical treatment
Physical methods for distillery effluent are incineration, vinasses (spentwash)
concentration and valorization, potash recovery and production of organic fertilizer.
All of these have been discussed in detail by Pathade (1999). The most widely
accepted industrial treatment is the vinasse valorization as fertilizer in the field after
concentration. However, the final solids are limited because of the sulphate
potassium crystallization and precipitation in evaporator tubes, storage tanks and
fertilizer sprayers. The partial mineralization of distillery effluent has been studied
using conventional anion and cation exchange membranes (Wilde, 1987). Low
current efficiency was obtained due to the nature of the liquor. Removal of 50-60%
of the potassium was achieved at a current efficiency of 50-55% and a D.C. power
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consumption of 0.75-0.85 kWh kg-1 of potassium removed. Electrodialysis was
attempted by Decloux et al. (2002) to reduce the potassium concentration in
vinasse and a decrease of potassium from 10 to 2.5 g L-1 was achieved.
In general, the chemical treatments use several reactives with the main objective of
oxidizing refractory organic pollutants. A variety of treatment methods and
strategies like thermal pretreatment, wet air oxidation, concentration–incineration
etc. have been suggested or tested for the treatment of the distillery wastewater.
Some of the physico-chemical methods adopted for treatment of distillery effluent
are listed in Table 2.1.
Advanced oxidation technologies (AOT) have been investigated for the treatment of
distillery wastewater. The oxidant species of these AOTs is the hydroxyl radical,
which can be generated in water through different combinations of oxidants, like
ozone and hydrogen peroxide, or of a single oxidant and ultra violet (UV) radiation.
The very high oxidizing capacity of the hydroxyl radical in all of these systems
enable them to react very rapidly with most of the organic and inorganic
compounds in water (Maston and Davies, 1994). Ozone is a powerful oxidant and is
used in industrial wastewater treatment due to its ability to convert biorefractory
compounds into less toxic and more readily biodegradable compounds. This leads
to significant decrease in the time required for bioremediation (Scott and Ollis,
1995). The recalcitrant compounds present in spentwash are very reactive towards
ozone, soluble in water and readily available. It is very reactive towards compounds
incorporating conjugated double bonds, often associated with color and functional
groups with high electron densities (Coca et al., 2007). Ozonation of pure vinasses
require high ozone dose to achieve significant efficiency for removing the organic
matter (Beltran et al., 1999a). It was observed that ozonation is effective even at
high concentration of vinasses (low dilution). This is due to high reactivity of several
organic pollutants present in the vinasses (e.g., phenols) towards ozone. However,
to obtain significant organic matter removal from high vinasses concentration, the
required ozone dose is equally high. Here the dilution of vinasses with domestic
sewage is an attractive option to avoid excessive consumption of ozone. This would
further bring down the cost and make it an acceptable pretreatment step.
Degradation of four phenolic acids, namely caffeic, p-coumaric, syringic and
vannilic, which are major pollutants present in wine distillery effluent by single UV
radiation and by advanced oxidation processes through combination of ozone and
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UV radiation, was conducted by Benitez et al. (1997). The later resulted in a slight
increase of the oxidation rate of phenolic acids compared with the results obtained
in the single ozonation, due to the action of hydroxyl radicals generated from the
synergistic effect of both oxidant agents. Very recently, Coca et al. (2007)
determined the physico-chemical parameters corresponding to the decolorization
reaction between ozone and melanoidins in molasses fermentation effluents. It was
shown that ozone reacts with colorants in molasses wastewater through direct
reactions proceeding within the film in a fast and pseudo-first-order kinetic regime.
Therefore, reactors with high specific interfacial areas are highly recommended for
decoloring molasses processing effluents with ozone.
Integrated aerobic biological oxidation and ozonation has been shown to be an
efficient process to treat distillery wastewater. The most important fraction of
pollutants is eliminated by biological oxidation (i.e., BOD and COD removals higher
than 95% and 80% respectively) to make the combined treatment economically
acceptable. However, the biological process has been shown to be unable in
removing poly phenols and other UV-absorbing compounds. Here, ozone has been
proved as an efficient treatment with the purpose of removing the biorefractory
compounds. Nevertheless, the high alkalinity accumulated in the wastewater due to
biological oxidation of organic matter made ozonation a non-refractive treatment to
further decrease total organic carbon (TOC) and COD (Beltran et al., 2001). In
another study Benitez et al. (2003) carried out treatment of wine vinasses by a
combined process of an ozone oxidation followed by an aerobic degradation step.
Due to the absence of microorganisms required for the aerobic degradation in wine
vinasses, an activated sludge taken from a municipal wastewater treatment plant
was acclimatized to this substrate. The combined process of an ozonation step
followed by an activated sludge step provided an enhancement in the substrate
removal 27.7 to 39.3% compared to what is obtained in the single aerobic treatment
of wastewater without ozone pretreatment.
Peña et al. (2003) investigated chemical oxidation of biologically pretreated
molasses wastewater with ozone and obtained color removal values over 80% and
COD reduction of 25% in 20 minute of reaction at an ozone dosage greater than 4.2
g h-1. However, there was no change in TOC. A study comparing the efficiency in
decolorizing biologically pre-treated molasses wastewater of different oxidation
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processes using ozone, single H2O2, Fenton’s reagent and ozone combined with
H2O2 was performed by Coca et al. (2005a). Ozone treatment was able to reduce
about 76% of color. A combination of ozone with a low concentration of H2O2 was
able to increase the color removal efficiency up to 89%. Molasses wastewater
contains a great concentration of alkalinity (about 900 mg L-1 as CaCO3) due to the
presence of bicarbonate ions. These ions have been described as strong inhibitors of
reactions between hydroxyl radicals and organic matter (Glaze and Kang, 1989).
Hayase et al. (1984) found that under alkaline conditions H2O2 was able to
decolorize synthetic melanoidins dissolved in deionized water. The oxidative action
of H2O2 is due to the perhydroxyl ion (O2H-), which attach nucleophilically the
chromophores groups of melanoidins, reducing color. The main operational
parameters affecting ozonation efficiencies of wastewater from beet molasses
alcoholic fermentation such as pH, bicarbonate ion, temperature and stirring rate
have been studied (Coca et al., 2005b). Efficiencies were unaffected by pH but
elimination of bicarbonate ion, a strong inhibitor of hydroxyl radical reactions,
yielded an improvement in both color and COD reduction efficiencies. The highest
efficiencies were achieved at 40 °C. Color and COD reductions were about 90% and
37%, respectively. It was revealed that the direct oxidation pathway predominates
over radical reactions in the first stage of ozonation, when organics with structural
elements, which are a prerequisite for light absorption at 475 nm, were present at
high concentrations. Chromophoric groups are directly attacked by ozone very
probably accompanied by hydrogen peroxide formation. UV radiation combined
with hydrogen peroxide or ozone has been tried for treatment of distillery
wastewater. The application of 254 nm UV radiation had no effect on COD and TOC
of the wastewater. A combination of UV radiation and hydrogen peroxide was not
found suitable as a single step treatment step for distillery wastewaters because of
the high concentration of hydrogen peroxide needed and the low TOC conversion
achieved. Also from practical viewpoint, the lamp cost, maintenance and energy
consumption hinder such type of chemical oxidation (Beltran et al., 1997a). The
combination of ozone and hydrogen peroxide was not found to be an appropriate
method for degrading distillery wastewater since results obtained were similar to
those from ozonation alone. The O3/UV radiation system gave the highest
degradation rates in distillery wastewaters, although there was only a 10% increase
over ozonation alone. However, the economic feasibility depends on cost of energy
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for generation of ozone and UV radiation. Also, the cost associated with these AOTs
and small difference achieved over ozonation implies that ozonation alone remain
the best alternative (Beltran et al., 1997b). Recently, Srivastava et al. (2006)
reported that ozonation of the anaerobically treated distillery effluent at an ozone
dose of 2.08 mg mg-1 initial TOC and subsequent aerobic biodegradation resulted in
87.4% COD removal as compared to 66% removal when ozonation was not used. In
yet another study, Kumar et al. (2006) showed that insertion of an intermediate
ozonation step during treatment of distillery spentwash by anaerobic-aerobic
biodegradation resulted in increase of percentage of COD removal from 70% when
no ozonation was used to greater than 95% at ozone doses of approximately 5.3 mg
ozone absorbed mg-1 initial TOC. Besides, when ozonation was carried out after pH
reduction and inorganic carbon removal, it resulted in more efficient ozone
absorption by anaerobically treated spentwash. Supercritical water oxidation where
organic materials are oxidized to CO2, H2O and N2 has been applied to molasses
distillery wastewater (Goto et al., 1998). Color and odour were completely removed
at supercritical temperature and pressure with sufficient amount of hydrogen
peroxide, which was used as an oxidant and was the most influential factor on the
destruction. Sangave et al. (2007b) extensively studied the effect of ozone as pre-
aerobic treatment and post-aerobic treatment for the treatment of the distillery
wastewater. Ozone was found to be effective in bringing down the COD (up to 27%)
during the pretreatment step itself. In the combined process, pretreatment of the
effluent led to enhanced rates of subsequent biological oxidation step, almost 2.5
times increase in the initial oxidation rate. The integrated process (ozone–aerobic
oxidation–ozone) achieved 79% COD reduction along with decoloration of the
effluent sample.
Activated carbon is utilized as an adsorbent to remove color from the clarified juice
in sugar refineries. It has been widely used for the removal of color from
wastewater. However, activated carbon adsorption is an expensive process owing to
the high cost of the carbon. Activated carbon prepared from cane bagasse was used
by Bernardo et al. (1997) to decolorize molasses wastewater. It was found that
activated carbons have high adsorptive capacities that favourably compare with a
commercial activated carbon. Chemically modified bagasse was also used by Mane
et al. (2006) for color removal from distillery spentwash. 50% reduction in color
was obtained, which was attributed to chemical sorption and the conventional ion
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exchange mechanism. It could be very promising technology for color removal from
synthetic melanoidins, especially in Southeast Asia, where bagasse is readily
available and cheap. Activated carbon containing micropores, mesopores and a
significant amount of macropores have good adsorption efficiency for compounds
such as tannic acid and melanoidins (Figaro et al., 2006). Adsorption capacity of
three different adsorbents namely, activated charcoal, fly ash and wood ash, was
tested and compared for removal of various pollutants and heavy metals from
distillery spentwash (Tewari et al., 2006). Activated charcoal was found to be the
best adsorbent due to its organophillic character followed by fly ash and wood ash
in terms of reduction in physico-chemical parameters. Two polymer based
adsorbents namely, cellulose acetate phthalate (CAP) and poly vinyl chloride (PVC)
were used to decolorize the distillery effluent, as these are water insoluble, easily
available and cheap (Ravi et al., 2006). CAP was found to be a better absorbent than
PVC. Complete color and odour removal was achieved in a 5% diluted distillery
effluent solution when treated with 2 g of calcium hypochlorite that acted as an
oxidizing agent (Vasanthy and Thamaraiselvi, 2006). Diluted spentwash treated
with calcium oxide showed complete deodorization and slight reduction in color.
Very recently, the removal of molasses-derived color and chemical oxygen demand
from the biodigester effluent of a molasses-based alcohol distillery effluent
treatment plant was studied using inorganic coagulants—FeCl3, AlCl3 and
polyaluminium chloride (PAC) (Chaudhari et al., 2007). 55, 60 and 72% COD
reductions and about 83, 86 and 92% color reductions were obtained with the use of
60 mM L-1 AlCl3, 60 mM L-1 FeCl3 and 30 ml L-1 of PAC, respectively, at their
optimum initial pH. The COD and color reduction was found to increase
considerably with an increase in the dosage of coagulants up to their optimum
values.
The catalytic thermal pretreatment (or catalytic thermolysis, CT) of distillery
wastewater using CuO catalyst as a pretreatment process to recover the majority of
its energy content with consequent COD and BOD removal has been explored
recently (Chaudhari et al., 2008). At 140 °C with 3 kg m-3 catalyst loading and pH0
2, a maximum of 60% COD could be reduced. The CT process results in the
formation of settleable solid residue and the slurry obtained after the thermolysis
exhibited very good filtration characteristics. Sangave et al. (2007a) used a
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combination of different treatment techniques for treatment of distillery spentwash.
Initially the effluent samples were subjected to thermal pretreatment (TPT-DW)
and anaerobic treatment (ANA-DW). Advanced oxidation techniques, viz.,
Ultrasound (US) and Ozone were then used for further COD reduction followed by
the conventional aerobic oxidation using mixed microbial consortium. Ozone
pretreatment was observed to be better as compared to US for both TPT-DW and
ANA-DW as the extent of mineralization of the pollutant for the pre-treated effluent
was greater as compared to the untreated effluent.
Apart from these treatment methods, wastewater recycling has also been tried
industrially. Adoption of clean technologies for ethanol production which could
eliminate all the conventional biological treatment systems, thus establishing a
zero-discharge system is the goal of physical treatment processes. A zero-discharge
system for the alcohol fermentation industry was developed by recycling distillery
waste (Kim et al., 1997). With an aim of eliminating all end of pipe technology being
used for wastewater, stillage was able to be recycled as cooking water for the next
fermentation after treating it with a membrane separation processes. The new
process gave a little longer fermentation time, but the average ethanol production
yield (8.8%) was similar to that in the conventional process (9.0%). Still the
message for the ethanol industry is that ‘zero effluent’ plants (those which do not
release significant volumes of water with high BOD or COD or high concentration of
salts) are not easily designed-especially when the prime mode of water recycle is via
the fermentation system (Ingledew, 2003). The pollution profile of alcohol
distilleries treating beet sugar molasses was investigated by Eremektar et al. (1995).
It was observed that recycle application reduces the amount of wastewater by
approximately 25% without affecting the COD content. Vinasses were used instead
of water in the preparation of the fermentation medium and repeatedly recycled
(Navarro et al., 2000). A decrease of 66% in nutrients addition, 46.2% in fresh
water and 50% in sulfuric acid requirement was achieved together with an
improvement in the efficiency of the fermentation. The problem of build-up of yeast
by-products and compounds that inhibit yeast fermentation can be overcome by
recycling a certain percentage of the total vinasse in order to keep the concentration
of undesirable compounds below the level of toxicity that appears in a vinasse with
26% solids content. Since the amount of water required for the preparation of the
fermentation medium in an alcohol producing plant is about 77% of the total water
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consumption, the re-use of 60% vinasse reduces 46.2% of the quantity of water
required.
Kumaresan et al. (2003) treated odorous distillery effluent for the removal of acetic
acid using emulsion liquid membrane in a batch process. Each emulsion globule
consists of droplets of an aqueous internal stripping phase encapsulated in an
organic membrane phase containing a surfactant as micelle interfacial layer.
Minimum values of BOD and COD of 96 and 927 mg L-1, respectively was obtained.
Membrane ultrafiltration was used for clarification as well as for decolorization of
raw brown sugar obtained from the Indian sugar industry since the color here is
also because of melanoidins only (Hamachi et al., 2003). It was found that even
with the membrane of molecular weight cut off (MWCO) of 1 kDa, the maximum
color removal was limited to 58.67%. A calefactive aerobic membrane bioreactor
(MBR) equipped with a stainless steel membrane, 0.2 mm pore size was used by
Zhang et al. (2006) to treat simulated distillery wastewater of about 1000 mg L-1
COD at 30-45 °C. With a hydraulic retention time (HRT) of 10-30 h and a
volumetric loading rate (VLR) of 0.6-2.8 kg COD.m-3.h-1, mean COD and total
nitrogen (TN) removal efficiencies were 94.7% and 84.4%, respectively.
Usefulness of reverse osmosis (RO) in the treatment of condensates arising from the
concentration of distillery vinasses has also been studied (Couallier et al., 2006).
RO is a promising process for the treatment of effluents arising from the
concentration of vinasses in beet distilleries and the elimination of some of the
compounds inhibiting alcoholic fermentation they contain. Recycling purified
condensates would thus enable to spare a major part of the ground water used in
the distilleries at the fermentation stage and insure a better quality of dilution
water. A hybrid nanofiltration (NF) and RO pilot plant was used to remove the color
and contaminants of the distillery spentwash (Nataraj et al., 2006). Color removal
by NF and a high rejection of 9.8% total dissolved solids (TDS), 99.9% of COD and
99.9% of potassium was achieved from the RO runs, by retaining a significant flux
as compared to pure water flux, which shows that membranes were not affected by
fouling wastewater run. The absence of heat application and a high rate of mass
transfer generated by RO shows that a large amount of water can be permeated
economically instead of being vaporized by the energy-intensive evaporation
processes or steam distillation using tall towers.
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In recent years electrochemical oxidation have emerged as yet another method to
treat concentrated wastewater from distilleries. Using electrochemical treatment in
presence of sodium chloride as supporting electrolyte, complete decolorization of
distillery effluent with 93.5% BOD reduction and 85.2% COD reduction was
reported by Manisankar et al. (2004). Without electrolytes when the treated
effluent was kept at room temperature for a week, the turbidity/coloration
reappeared. It was suggested that melanoidin may be adsorbed on the electrode
surface and may decrease the efficiency of the electrode. Piya-areetham et al. (2006)
studied the color and COD reduction in distillery wastewater by using
electrochemical processes. Among graphite particles and titanium sponge used as
anodes, the later showed a high potential to reduce color in wastewater. The
optimum condition for treating effluent with 10 times dilution was found at the
current intensity of 9 A at initial pH of 1 with a titanium sponge anode in presence
of 1.0 M NaCl.
A treatment process for reducing COD content and copper concentration
simultaneously from semiconductor wastewater and distillery slops was evaluated
by Navarro et al. (2005). The process known as ‘waste exchange’ utilized the
complementary properties of the positively charged copper ions (which served as
coagulant for slops) in semiconductor wastewater and net negative charge of
melanoidin (organic chromophoric pollutant) in distillery slops which served as
precipitant for copper) to mutually neutralize each other. At optimum conditions,
average removals of COD and copper were 86% and 92% respectively, in an actual
and undiluted system. Decolorization efficiency using distillery slops was 89%.
Electrocoagulation (EC) technique with addition of indigenously prepared activated
areca nut (Areca catechu) carbon was used for distillery effluent treatment (Kannan
et al., 2006). Except for turbidity, where the removal was same with or without
areca nut carbon, all other water quality parameters fared better in the presence of
areca nut carbon.
EC in conjunction with electroflocculation (EF) is one of the methods, which is
simple, but very effective for treating many turbid waters and wastewaters. Very
recently, electrochemical treatment of alcohol distillery wastewater using iron
electrode with and without the presence of H2O2 was investigated (Yavuz, 2007).
Two electrochemical methods, namely, EC and EF were used to treat pre-treated
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27
distillery wastewater. As compared to EC, EF process was found to be very efficient
even for those distillery wastewaters, which has high concentration of refractory
organic matters. The optimum working conditions were; current density of 60 mA
cm-2, supporting electrolyte concentration of 0.3 M Na2SO4, H2O2 concentration of
60000 mg L-1, pH 4 and gradual addition of H2O2. It was also observed that step-by-
step addition of H2O2 instead of initial addition was an easy and effective way of
rising removal efficiency both for COD and TOC.
In general, physico-chemical treatments of distillery wastewater require high
reagent doses and generate a large amount of sludge besides their high operational
cost and fluctuation of color removal efficiency (Migo et al., 1993; Sirianuntapiboon
et al., 2004). These schemes therefore appear either incomplete or economically
unviable when used as sole treatment methods. However their application remains
important because the conventional anaerobic-aerobic treatments are not able to
remove the color causing compounds significantly (González et al. 2000). Therefore
it becomes imperative to continue research into alternative biotechnological
processes to achieve color removal from these recalcitrant vinasses.
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Table 2.1 Physico-chemical methods employed for distillery effluent treatment
S. No.
Chemical/ Coagulant Dosage COD Removal
(%)
Color Removal
(%)
pH Reference
1. Chitosan 10 mg L-1 99 98 6 Lalov et al.,
1999
2 I
a)
b)
c)
II
Ferric Hydroxyl Sulphate
Fresh Slops (FS)
Biodigested Effluent
(BDE)
Lagoonated (LE)
Activated Charcoal
57 mM
21(TOC removal)
73
73
32
87
94
99
Migo et al.,
1993
3 Ferric chloride
Ferric sulphate
Aluminium sulphate
Calcium oxide
Calcium chloride
57 mM
57 mM
57 mM
57 mM
536 mM
- 96
97
96
83
78
2-7
3-4.5
3-4.5
4-6
>13
Migo et al.,
1997
4 a)
b)
Lime
Ozone
10 mg L-1
0.9 mg L-1
82.5
68
68
12.3 Inanc et al.,
1999
5 Ozone 4.2 g h-1 15-25 80 - Peña et al.,
2003
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6 a)
b)
UV Radiation
UV Radiation+H2O2
- Nil
3.8
Nil
- Beltran et al.,
1997a
7 a)
b)
O3+H2O2
O3+UV Radiation
- Nil
21.5
Nil
- Beltran et al.,
1997b
8 Ozone 50 mg h-1 16 80 4 Alfafara et al.,
2000
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30
Biological treatment
Biological treatments have been recognized as effective methods of treatment for
highly polluted industrial wastewaters. 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 specific microbial activity (pure bacteria and fungi) for the
decolorization and mineralization of spentwash. There are several reports citing the
potential of microorganisms for use in this process. Moreover, the biologically
treated effluent could be used safely and effectively to increase the soil productivity.
This section is discussed in detail as anaerobic and aerobic treatments.
Anaerobic treatment
Direct aerobic treatment of raw spentwash appear to be unsatisfactory because of
high initial cost, large continuing energy demand, acidity of the waste, its high
temperature and production of a large amount of activated sludge (Athanasopoulos,
1987). Therefore anaerobic digestion is widely accepted as the first treatment step in
distilleries. Wilkie et al. (2000) have reviewed the role of anaerobic digestion in
stillage (spentwash) treatment. Anaerobic digestion can convert a significant
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 effluents is widely
applied as an effective step in removing 90% of the COD in the effluent stream
(Wolmarans and de Villiers, 2002). During this stage, 80-90% BOD removal takes
place and biochemical energy recovered is 85-90% as biogas.
Thus, anaerobic treatment processes appear to be ideally suited as primary
treatment for these types of waste as they are net energy producing, operate most
efficiently at high COD values, produce small quantities of excess sludge and the
methane produced is used as an energy source in the same distillery. Anaerobic
processes also require much lower additions of nutrients, nitrogen and phosphorus,
than would be necessary for aerobic processes. The treated effluents have
solubilised organic matter, which is amenable to quick subsequent aerobic
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31
treatment. The presence of biodegradable components in the effluents coupled with
the advantages of anaerobic processes over other treatment methods makes it an
attractive option (Rajeshwari et al., 2000).
Common types of anaerobic bioreactors are open lagoon, covered lagoon, fluidized
bed reactor, contact reactor, UASB, anaerobic filter, hybrid reactor and
thermophilic reactor (Nandy et al., 2002). A list of common types of anaerobic
reactors used for distillery effluent treatment is given in Table 2.2.
The highest BOD removal is possible in open lagoon whereas highest biomethane
produced is in UASB type bioreactor. The UASB system has become the most widely
applied reactor technology for high rate anaerobic treatment of industrial effluents.
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 methanogenic 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 thermophilic (50-55 °C)
anaerobic digestion of wastewater from an alcohol distillery was reported by
Vlissidis and Zouboulis (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 influent concentrations of 3526-52126 mg L-1.
When the organic loading rates (OLRs) of 15 kg m-3 day-1 and above were used, the
COD removal efficiency dropped to less than 20%, in one of the duplicate reactors.
Anaerobic digestion of natural and beet molasses alcoholic fermentation wastewater
previously fermented with Penicillium decumbens to achieve reduction of its
phenolic content was attempted (Jiménez and Borja, 1997). The pretreatment with
P. decumbens considerably enhanced the rate of subsequent anaerobic degradation
of the molasses thus significantly reducing the treatment time. A mesophilic two-
stage system consisting of an anaerobic filter (AF) and an UASB reactor was found
suitable for anaerobic 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 2-4 kg COD
m-3 day-1 at pH 6.0 and for the methanogenic stage, organic loading of 1-2 kg COD
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TERI University-Ph.D. Thesis, 2007
32
m-3 day-1 at pH 7.6. Haun et al. (1997) reviewed the operation of anaerobic
treatment plants in Germany. The distilleries producing alcohol from molasses
slops have a problem of discontinuous production because the production is from
autumn to spring.
An advanced version of UASB system was reported by 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 effluent circulation. This system could handle high upflow liquid
and gas velocities making possible treatment of low strength effluents at short
hydraulic retention times as well as treating high strength effluents such as from
brewery at very high volumetric loading rates up to 35 kg COD m-3. A laboratory-
scale hybrid UASB reactor, which combined an UASB in the lower part and a filter
in the upper part, was used for the treatment of distillery spentwash (Shivayogimath
and Ramanaujam, 1999). COD removal efficiency was 80% even at a high OLR of 36
kg COD m-3 with a short HRT of 6 hours. Also using anaerobically digested sewage
sludge as seed and distillery spentwash as the substrate, the granulation was
achieved in three months on hybrid UASB. Akunna and Clark (2000) studied the
performance of a granular bed anaerobic baffled reactor (GRABR) in the treatment
of a whisky distillery wastewater having COD and BOD concentrations of 16600-
58000 and 8900-30000 mg L-1, respectively. The removal of total BOD and COD
from the wastewater were 80-92% and 90-96%, respectively with a HRT of 4 days
and at a loading rate of 2.37 kg COD m-3 day-1. García-Calderón et al. (1998)
reported the application of the down-flow fluidization 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.
Kumar et al. (2007) studied anaerobic biodegradation of distillery spentwash in a
lab-scale continuous anaerobic hybrid (combining sludge blanket and filter) reactor.
The optimum COD removal efficiency was found to be 79% corresponding to
optimum HRT and OLR of 5 days and 8.7 kg COD m-3 d-1. The decline in
performance of reactor at higher HRT was attributed to the reduction of sulfate into
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TERI University-Ph.D. Thesis, 2007
33
sulfide, which might have inhibited the metabolism of methanogenic bacteria and
reduced the COD removal and yield.
Most recently, two distinct full-scale UASB reactors treating alcohol distillery
wastewaters were investigated in terms of performance, acetoclastic methanogenic
activity and archaeal composition (Akarsubasi et al., 2006). Predominant archaeal
sequences in both reactors belonged to Methanobacterium formicicum and
Methanosaeta soehngenii. Effect of addition of macronutrients and micronutrients
in the distillery effluent was investigated on the performance in simulated UASB
system by Sharma and Singh (2001). Calcium and phosphate were found to be
detrimental to treatment efficiency. Uzal et al. (2003) investigated the biochemical
methane potential (BMP) of malt whisky distillery wastewater both with and
without basal medium to observe the effect of nutrient supplementation. When a
COD concentration of 20,920 mg L-1 was maintained in the influent to the first
stage, the effluent quality of the first stage began to deteriorate. Furthermore, a
significant color 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 effect of the wastewater on granular biomass, thus increasing oxidation-
reduction potential. The authors concluded that two stage UASB reactor
configuration is an efficient system for malt whisky wastewater treatment until up
to 33,866 mg L-1 influent COD concentration. For the overall sequential system
(anaerobic/aerobic) treatment COD and BOD removal efficiencies were 99.5% and
98.1%, respectively, for the treatment of malt whisky wastewater. In aerobic phase,
the effluent of anaerobic bioreactor is exposed to atmospheric oxygen in a tank with
homogenizers for proper mixing of the effluents. BOD is reduced to 200 and
effluent diluted with wastewater from bottling and washer sections and disposed off
after clarification (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 fermentation industry in
Turkey producing baker’s yeast from sugarbeet molasses, the biogas recovery is
worth $300.
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34
Immobilization of bacteria in biofilm and on bioflocs is a crucial step in anaerobic
degradation because of advantages 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 anaerobic digestion, long
incubation periods are required for the start-up stage. Other operational problems
are the low growth rate of anaerobic bacteria and the loss of biomass in systems
with high hydraulic rates. Thus conventional anaerobic digestion frequently does
not achieve a satisfactory purification of vinasses (Beltran et al., 1999b; Benitez et
al., 2003). The formation of hydrogen sulphide in anaerobic reactors is the result of
the reduction of oxidized sulphur compounds and of the dissimilation of sulphur
containing amino acids such as cysteine. Methanogenic bacteria can tolerate
sulphide concentration up to 1000 mg L-1 total sulphide and a complete loss of
methane production occurred at 200 mg L-1 of un-ionized H2S during digestion of
flocculent sludge. Anaerobic contact process incorporating an ultrafiltration (UF)
unit was used to treat distillery wastewater characterized by high and low carbon to
nitrogen concentrations. The UF unit was employed to thicken the sludge without
washing out the microbes in the process. This treatment system showed methane
yields of up to 0.6 m3 kg-1 volatile solids (VS) and removed up to 80% of the volatile
acids (Kitamura et al., 1996). Two-phase anaerobic digestion of cane-molasses
alcohol stillage proved to be superior to the single-phase process in terms of
substrate loading rate and methane yield, without affecting the treatment
performance (Yeoh, 1997). While maintaining BOD and COD and reduction of 85%
and 65% respectively, the two-phase achieved methane yield three times that of
single-phase system. Recently, Yu et al. (2006) reported on the performance of two
upflow anaerobic filters, one multi-fed and another single-fed for the treatment of
winery wastewater. The multi-fed reactor removed over 82% of COD even at an
OLR of 37.68 COD g L-1 d-1 and a short HRT of 8 h. The multi-fed reactor was found
to be more efficient than the single-fed one in terms of COD removal efficiency and
stability against hydraulic loading shocks.
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TERI University-Ph.D. Thesis, 2007
35
A number of environmental factors affect the activity of wastewater microbial
populations and the rate of biochemical 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 affecting the microbiological activity
in the biological process. The pH of wastewater increases from 4.0 to 7.5 after
anaerobic digestion. This is due to the oxidation of organic acids to carbon dioxide
and the reaction between the carbon dioxide and basic compounds to form
carbonates and bicarbonates (Beltran et al., 1999b). Because of its high organic
load, the distillery wastewater is often diluted with tap water to simulate the
concentration of a typical industrial effluent entering a wastewater treatment plant.
However, spentwash even after anaerobic treatment does not meet the stringent
effluent standards laid down by CPCB, India, in terms of very high levels of BOD,
COD, solids etc. (Asthana et al., 2001). Also, the secondary spentwash produced by
the anaerobically digested primary molasses spentwash (DMSW) effluent is darker
in color, needing huge volumes of water to dilute it and is currently used in
irrigation water causing gradual soil darkening. The effluent therefore is released
after diluting with fresh water which is a very dear commodity to industries.
Besides, sometimes failure of the anaerobic digestion threatens the criteria for
discharge 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 additional costs for coagulants (Kim et al., 1997). The very
low BOD/COD ratio of 0.049 indicates that m0lasses spentwash after anaerobic
digestion is poorly biodegradable and contains mostly refractory compounds for
which a physico-chemical post-treatment is required (Figaro et al., 2006). Hence
aerobic treatment is necessary for anaerobically treated final effluent.
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Table 2.2 Anaerobic methods employed for distillery effluent treatment
Reactor type Organic Loading Rate
(OLR) (kg COD m -3 day-1)
COD
Removal (%)
BOD
Removal
(%)
Retention
Time
(Days)
Reference
Downflow fixed-film
reactor
- 60-73 85-97 - Bories and
Ranyal, 1988
Granular bed
anaerobic baffled
reactor (GRABR)
2.4 90-96 80-92 4 Akunna and
Clark, 2000
Hybrid anaerobic
baffled reactor
20 70 - - Boopathy and
Tilche, 1991
Upflow anaerobic
sludge blanket (UASB)
reactor
28 39-67 80 - Harada et al.,
1996
Istanbul UASB reactor 6–11 90 - - Akarsubasi et
al., 2006 Tekirdag UASB reactor 2.5–8.5 60-80 - -
Upflow anaerobic
sludge blanket at
Tekirdag (TUASB)
2.5-8.5 60-80 - - Ince et al.,
2005
Upflow anaerobic
sludge blanket at
(Istanbul)
1-4.5 70-80 - -
Diphasic fixed-film
reactor with granular
activated carbon (GAC)
as support media
21.3 67.1 - 4 Goyal et al.,
1996
Anaerobic contact filter 19,000 mg L-1 (influent
COD concentration)
73-98 - 4 Vijayaraghavan
and
Ramanujam,
2000
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Aerobic treatment
Bacterial treatment
Microbial treatments employing pure bacterial culture have been reported
frequently in past and recent years. A detailed list of bacteria tried by different
researchers for decolorization of distillery effluent is given in Table 2.3.
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 4 to 5 days without any aeration. The major products
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 wastewater. The highest COD removal rate was
observed 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. However,
immobilization of activated sludge did not lead to any improvement in treatment
performance. The only advantage observed was in terms of less space required for
treatment plant. Pseudomonas fluorescens, decolorized melanoidin wastewater
(MWW) up to 76% under non-sterile conditions and up to 90% in sterile samples
(Dahiya et al., 2001a). The difference in decolorization might be due to the fact that
melanoidin stability varies with pH and temperature and at higher temperature
during sterilization melanoidin-pigments decompose to low molecular weight
compounds (Ohmomo et al., 1988b). The effect of immobilization on the
decolorization of a melanoidin solution may be explained by the fact that
Lactobacillus hilgardii requires a small amount of oxygen for the decolorization
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 decomposition of melanoidins. Cibis et
al. (2002) achieved biodegradation 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 melanoidin due to
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TERI University-Ph.D. Thesis, 2007
38
production of H2O2, hydroxyl, perhydroxyl and active oxygen radicals, resulting in
the decolorization of the effluent (Kalavathi et al., 2001). 96%, 81% and 26%
decolorisation of distillery effluent through bioflocculation by Oscillatoria sp.,
Lyngbya sp. and Synechocystis sp. respectively was reported by Patel et al. (2001).
Distillery spentwash, despite carrying high organic load contains little readily
available carbon. Isolation of bacterial strains capable of degrading recalcitrant
compounds of anaerobically digested spentwash from soil of effluent discharge site
was reported by Ghosh et al. (2004). These were Pseudomonas, Enterobacter,
Stenotrophomonas, Aeromonas, Acinetobacter and Klebsiella, all of which could
carry out degradation of some component of spentwash. Maximum 44% COD
reduction was achieved using these bacterial strains either singly or collectively.
Sirianuntapiboon et al. (2004) used an acetogenic bacterium to obtain a
decolorization 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 decolorization of MWWs. The decolorization activity might
be due to a sugar oxidase.
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Table 2.3 Bacteria employed for the decolorization of distillery effluent
S. No. Name Comments Color Removal (%) Reference
1 Xanthomonas fragariae
All the three strains needed glucose as carbon source and NH4Cl as
nitrogen source. The decolorization efficiency of free cells was better
than immobilized cells
76 Jain et al., 2002
2 Bacillus megaterium 76
3 Bacillus cereus 82
4 Bacillus smithii Decolorization occurred at 55 °C in 20 days under anaerobic
conditions in presence of peptone or yeast extract as supplemental
nutrient. Strain couldn’t use MWW as sole carbon source.
35 Kambe et al., 1999
5 Lactobacillus hilgardii Immobilized cells of the heterofermentative lactic acid bacterium
decolorized 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
decolorization of MWWs
76 Sirianuntapiboon et
al., 2004
7 Pseudomonas fluorescens This decolorization was obtained with cellulose carrier coated with
collagen. Reuse of decolorized cells reduced the decolorization
efficiency
94 Dahiya et al.,
2001a
8 Pseudomonas putida The organism needed glucose as a carbon source, to produce
hydrogen peroxide which reduced the color
60 Ghosh et al., 2002
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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
21 Bacillus sp. 27
22 Pseudomonas aeruginosa The three strains were part of a consortium which decolorized the
anaerobically digested spentwash in presence of basal salts and
glucose
67 Mohana et al.,
2007 23 Stenotrophomonas maltophila
24 Proteus mirabilis
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TERI University-Ph.D. Thesis, 2007
41
Fungal treatment
In the recent years, several basidiomycetes and ascomycetes type fungi have been
used in the decolorization of natural and synthetic melanoidins in connection with
color reduction of wastewaters from distilleries. The aim of fungal treatment is to
purify the effluent by consumption of organic substances, thus, reducing its COD
and BOD, and at the same time to obtain some valuable product, such as fungal
biomass for protein-rich animal feed or some specific fungal metabolite.
White-rot fungi are filamentous fungi that inhabit the wood of dead and dying trees
and are so called because of the white appearance of the rotted wood, caused partly
by the absence of lignin and partly oxidized aromatic lignin derivatives. The brown
rot fungi remove cellulose and hemicellulose, but leave the lignin as a brown
residue. Filamentous fungi have lower sensitivity to variations 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 decolorize
melanoidins and MSW (Table 2.4).
Coriolus sp. No. 20, in class basidiomycetes was the first strain for the application
of its ability to remove melanoidins from MWW (Watanabe et al., 1982). This
isolate did not show any decolorization activity when molasses pigment was used as
carbon source but it showed the activity when sorbose or glucose was added. A lot of
work employing fungi for distillery effluent decolorisation was done by Ohmomo
and co-workers. As early as 1985, Ohmomo et al. used Coriolus versicolor Ps4a for
molasses wastewater decolorization and obtained 80% decolorization 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 influenced by the kind of sugars used for
cultivation. The wine distilleries produce large volumes 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 decolorized molasses melanoidin medium and a synthetic
melanoidin medium by 87.5% and 84.5% respectively, under experimental growth
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42
conditions. Mycelia grown in solutions of melanoidin turned dark brown. Electron
microscopy revealed that the mycelia absorbed melanoidin pigment, which was in
the form of electron dense material in the cytoplasm. However, melanoidin could be
eluted from the mycelia by washing in a solution of NaOH and the relative amount
of melanoidin eluted from the mycelia increased with increase in the concentration
of NaOH (Sirianuntapiboon et al., 1995). Aspergillus 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 Ni2+ and Co2+. Also, NH4+, accumulated in the anaerobically treated
wastewater, was efficiently removed by utilization of residual volatile fatty acids
(VFA) as electron donors during biological denitrification and nitrification, and the
residual organic matter could be removed simultaneously.
Color elimination from MSW using Aspergillus niger was studied by Miranda et al.
(1996). Under optimal nutrient concentration 83% of the total color removed was
eliminated biologically and 17% by adsorption on the mycelium. Ohmomo et al.
(1988a) concluded that microbial decolorization of melanoidins is due to two
decomposition mechanisms; in the first the smaller molecular weight melanoidins
are attacked and in the second the larger molecular weight melanoidins are
attacked.
Under nutrient limiting conditions, fungal cells generally 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 biofilm. The immobilization
of the fungus offers advantages such as short retention 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,
1999a). Miyata et al. (2000) suggested an inhibitory effect of organic nitrogen on
melanoidin decolorization by fungus Coriolus hirsutus. At the same time glucose
was also required for enhancing decolorization as the peroxidases require H2O2,
which is generated by glucose oxidation, to decolorize melanoidin. Chopra et al.
(2004) reported that absence of additional nitrogen could not inhibit activity of
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43
fungus C. versicolour sp. No. 20 considerably, as significant decolorization and
COD reduction occurred even in the absence of it.
Color removal from distillery effluent using a marine fungus, Flavodon flavus has
been reported by Raghukumar and Rivonkar (2001). This fungus was more effective
in decolorizing raw MSW than was the molasses wastewater collected either after
anaerobic treatment or after aerobic treatment. This might be due to changes in the
chemical structure of melanoidin pigments during anaerobic and aerobic treatment.
However, the oxygen demand of the fungus was quite high. P. chrysosporium JAG-
40 decolorized synthetic and natural melanoidins present in spentwash up to 80%
(Dahiya et al., 2001b). The larger molecular weight fractions of melanoidin were
decolorized rapidly, while the small molecular weight fractions remained in solution
and were metabolized slowly. Also, the decolorization was less in sterilized
spentwash than in non-sterile solution. This observation is completely opposite of
the one when Pseudomonas fluorescens was used by same authors (Dahiya et al.,
2001a). Kahraman and Yesilada (2003) reported molasses decolorization 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. versicolour decolorized 48% of 30% diluted vinasse
without any additional carbon source which increased to 71% on addition of cotton
stalks. Aspergillus niveus, a litter degrading fungi was used by Angayarkanni et al.
(2003) for the treatment of distillery effluent using paddy straw, sugarcane bagasse,
molasses and sucrose as carbon source for growth of fungus in the effluent.
Sugarcane bagasse at 1% (w/v) concentration resulted in maximum removal of color
(37%) and COD (91.68%). The decrease in color removal in this study might be due
to the fact that the effluent taken for study was alkaline (pH 9.0) and the
melanoidins responsible for color 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 decolorization of
anaerobically digested (UASB) and aerobically treated distillery wastewater. With
diluted wastewater at optimum values of supplemented materials 75%
decolorization was achieved which reduced to 40% on using undiluted wastewater.
It was suggested that decolorization by fungi takes place due to the destruction of
colored molecules and partially because of sorption phenomena. A longer aeration
period causes the adsorbed color molecules to be released as a result of endogenous
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44
respiration and cell death, hence reducing decolorization efficiency. In Iran, where
distilleries use beet molasses for ethanol production, the decolorization of
anaerobically treated distillery wastewater by Aspergillus fumigatus using response
surface methodology has been reported (Mohammad et al., 2006). It was shown
that initial maltose concentration, pH and mycelia mass, all affected decolorization
efficiency. Pre-treatment of vinasses with Penicillium decumbens reduced 67.7% of
the initial content of phenolic compounds, decreasing considerably its biotoxicity
(Jiménez et al., 2006). This fungal pre-treatment also resulted in 43% more
volumetric methane production.
Yeast Citeromyces was used for treating MWW and high and stable removal
efficiencies in both color 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 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 conditions, respectively. Moriya et al. (1990)
used two flocculant strains of yeast, Hansenula fabianii and Hansenula anomala
for treatment of wastewater from beet molasses-spirits production 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.
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Table 2.4 Fungi employed for the decolorization of distillery effluent
S. No. Name Comments Color Removal
(%)
Reference
1 Phanerochaete chrysosporium Both the fungi required a readily available carbon source for
melanoidin decolorization while N source had no effect. Maximum
decolorization was observed in 6.25% (v/v) spentwash.
53
Kumar et al., 1998 2 Coriolus versicolor 71
3 Trametes versicolor COD and N-NH4 removal observed in presence of sucrose and
KH2PO4 as nutrient source
82 Benito et al., 1997
4 Geotrichum candidum Fungus immobilized on polyurethane foam showed stable
decolorization of molasses in repeated-batch cultivation
80 Kim and Shoda, 1999a
5 Coriolus hirsutus A large amount of glucose was required for color removal but
addition of peptone reduced the decolorizing ability of the fungus.
80 Miyata et al., 2000
6 Penicillium sp. All fungi produced decolorization from first day of incubation, with
maximum being shown by P. decumbens at fourth day with a
reduction of 70% of the phenolic content of the wastewater
30
Jiménez et al., 2003 7 Penicillium decumbens 41
8 Penicillium lignorum 28
9 Aspergillus niger 25
10 Aspergillus niger UM2 Decolorization was more by immobilized fungus and it was able to
decolorize up to 50% of initial effluent concentrations
80 Patil et al., 2003
11 Aspergillus fumigatus G-2-6 Thermophilic strain tried for molasses wastewater decolorization but
coloring compounds hardly degraded
56 Ohmomo et al., 1987
12 Mycelia sterilia Organism required glucose for the decolorizing activity 93 Sirianuntapiboon et al.,
1988
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13 Aspergillus niger Maximum color removal was obtained when MgSO4, KH2PO4,
NH4NO3 and a carbon source was added to wastewater
69 Miranda et al., 1996
14 Flavodon flavus MSW was decolorized 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 decolorization 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
decolorized 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 decolorizing 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 decolorized 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 decolorization 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 decolorization
56 Angayarkanni et al., 2003
21 Trametes sp. I-62 No color observed associated with either fungal mycelium or
polysaccharides secreted by the fungus and therefore color removal
was attributed to fungal degradation and not to a simple physical
binding
73 González et al., 2000
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47
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 decolorization activity in 0.5% melanoidin when
sorbose or glucose was added as carbon source.
80 Watanabe et al., 1982
32 Williopsis saturnus strain CBS
5761
Yeast isolates from a 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 spentwash was used with glucose @ 2% added as
carbon source
34 Chopra et al., 2004
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48
39 Phanerochaete chrysosporium Sugar refinery effluent was treated in a RBC using polyurethane
foam and scouring web as support
55 Guimarães et al., 2005
40 Pycnoporus coccineus Immobilized mycelia removed 50% more color than free mycelia 60 Chairattanamanokorn et
al., 2005
41 Coriolus versicolor Cotton stalks were added as additional carbon source which
stimulated the decolorization 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 spentwash 100 D’Souza et al., 2006
47 Phanerochaete
chrysosporium
NCIM 1073 Molasses medium decolorization was checked in stationary and
submerged cultivation conditions
0 Thakkar et al., 2006
NCIM 1106 82
NCIM 1197
76
48 Citeromyces sp. WR-43-6
Organism required glucose, Sodium nitrate and KH2PO4 for maximal
decolorization
69 Sirianuntapiboon et al.,
2003
49 Hansenula fabianii The flocculant strains could reduce 28.5% TOC from wastewater
without dilution
Not checked in
this study
Moriya et al., 1990
50 Hansenula anomala
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Mixed consortium treatment
During last two decades, several attempts have been made to investigate the
possibility of using cell immobilization in the technology of aerobic wastewater
treatment (Fedrici, 1993; Sumino et al., 1985). Early experiments were restricted to
the use of selected pure cultures immobilized on solid supports for the degradation
of specific 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 different carriers and used for wastewater treatment (Shah et al., 1998). Jet loop
reactors (JLR), the efficiency 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 dm3 working volume was used for the aerobic treatment of
winery wastewater (Petruccioli et al., 2002). COD removal efficiency higher than
90% was achieved with an organic load of the final effluents 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, Eusebio et al. (2004) reported the operation
of a JLR for more than one year treating winery wastewater collected in different
seasons and achieved an average COD removal efficiency 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 decolorization of molasses spentwash in absence of any additional carbon or
nitrogen source using soil as inoculum. A decolorization of 69% was obtained using
10% (w/v) soil and 12.5% (v/v) MSW after 7 days incubation. Tertiary treatment of
distillery waste previously treated by a combined anaerobic filter-aerobic trickling
filter system has been evaluated in a laboratory stabilization pond (Travieso et al.,
2006). An increase of the HRT determined an increase of total COD and BOD5
removal efficiencies for all the influent substrate concentrations. An increase in the
influent strength determined a decrease of the BOD5 removal efficiency. The
increase of the HRT was favourable for the development of photosynthetic
organisms while the increase of the influent concentration favoured the growth of
heterotrophic microorganisms.
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Phytoremediation approach
Algal growth potential bioassay is a standard and reproducible method. Also, it
constitutes an economic assay to determine the potential of water bodies, natural
waters and wastewaters, to support or inhibit the microalgae growth. Algae growth
potential is based on the relation of a maximum biomass yield concerning the
biologically used nutrients for microalgae growth. Algae growth potential was
determined in distillery wastewater pretreated by anaerobic processes and by a
combined anaerobic-aerobic system. The biologically treated distillery wastewater
provided satisfactory conditions for microalgae growth (Travieso et al., 1999).
Billore et al. (2001) used Phragmites kharka in a constructed wetland for treatment
of wastewater from an Indian distillery and obtained 36% removal of total Kjeldahl
nitrogen and 48% removal of total suspended solids (TSS). Enhanced decolorization
was achieved by phytoremediation of distillery effluent 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 effluent (Singh et al., 2005).
Increasing concentration of the effluent greatly reduced the biomass of the plant
with maximum accumulation of Fe being recorded in plants growing in 100%
effluent. In another study Trivedy and Nakate (2000) employed Typha latipholia
for distillery effluent 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 treatment with Lemna minuscula and Chlorella vulgaris, Valderrama
et al. (2002) reported 52% color removal from distillery effluent. The microalgal
treatment removed nutrients and organic matter from wastewater and produced
oxygen for other organisms. The macrophyte removed organic matter and
eliminated the microalgae from treated wastewater. However, despite the potential
of aquatic macrophytes in cleaning wastewaters, the use of these plants in designing
a low cost treatment system is still at experimental stage and is considered to be a
potentially important area of environmental management. Very recently a
combination of bacterial pretreatment followed by free water surface flow through
wetland plants has been investigated to determine its effect on removal of heavy
metals in bioremediation of post-methanated distillery effluent (Chandra et al.,
2007). The biphasic treatment of the effluent with Typha angustata and Bacillus
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51
thuringiensis removed large quantities of various heavy metals at a range of effluent
concentrations. At 30% effluent concentration, color, BOD, COD, phenol and TN
decreased by 98.33%, 98.89%, 98.5%, 93.75% and 82.39% respectively after 7 days
of free water surface flow treatment.
Role of enzymes in effluent decolorization
With a necessity of improvement in biological remediation techniques, enzyme
technology has been receiving increased attention (Whiteley and Lee, 2006).
According to Aitken (1993), enzymes were first proposed for the treatment of waste
in the 1930s, but it was not until the 1970s that enzymes were used to target specific
pollutants in waste. Although the enzymatic system related with decolorization of
melanoidins is yet to be completely understood, it seems greatly connected with
fungal ligninolytic mechanisms. The white-rot fungi has a complex enzymatic
system which is extracellular and non-specific, and under nutrient-limiting
conditions is capable of degrading lignolytic compounds, melanoidins, and
polyaromatic compounds that cannot be degraded by other microorganisms (Benito
et al., 1997). A large number of enzymes from a variety of different 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 decolorization of melanoidins by
Trametes (Coriolus) strain was reported during the 1980s. Several reports claimed
that intracellular sugar-oxidase- type enzymes (sorbose-oxidase or glucose-oxidase)
had melanoidin-decolorizing activities. It was suggested that melanoidins were
decolorized by the active oxygen (O2; H2O2) produced by the reaction with sugar
oxidases (Watanabe et al., 1982). Decolorization by microbial methods includes the
enzymatic breakdown of melanoidin and flocculation by microbially secreted
substances. Ohmomo et al. (1985) used C. versicolour Ps4a, which decolorized
molasses wastewater by 80% in darkness under optimum conditions.
Decolorization activity involved two types of intracellular enzymes, sugar-
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52
dependent and sugar-independent. One of these enzymes required no sugar and
oxygen for appearance of the activity and could decolorize MWW up to 20% in
darkness and 11-17% of synthetic melanoidins. Thus, the participation of these H2O2
producing enzymes as a part of the complex enzymatic system for melanoidin
degradation by fungi should be taken into account while designing any treatment
strategy. One of the more complete enzymatic studies regarding melanoidin
decolorization was reported by Miyata et al. (1998). Color removal of synthetic
melanoidin by C. hirsutus involved the participation of peroxidases, MnP and
manganese–independent peroxidase (MIP) and the extracellular H2O2 produced by
glucose-oxidase, without disregard of a partial participation of fungal laccase.
Mansur et al. (1997) obtained a maximum decolorization of around 60% on day 8
after inoculating with fungus Trametes sp. I-62. Here effluent was added at a final
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 melanoidin mineralizing 47 kDa extracellular protein
corresponding to the major mineralizing enzyme system from T. versicolour was
isolated by Dehorter and Blondeau (1993). This Mn2+ dependent enzyme system
required oxygen and was described to be as peroxidase. Uniform, small and spongy
pellets of the fungus T. versicolour were used as inoculum for color removal using
different nutrients such as ammonium nitrate, manganese phosphate, magnesium
sulphate and potassium phosphate and also sucrose as carbon source (Benito et al.,
1997). Maximum color removal of 82% and 36% removal of N-NH4+ was obtained
on using low sucrose concentration and KH2PO4 as the only nutrient. Some studies
have identified the lignin degradation related enzymes participating in the
melanoidin decolorization. Intracellular H2O2 producing sugar oxidases have been
isolated from Coriolus strains. Also, C. hirsustus have been reported to produce
enzymes that catalyze melanoidin decolorization directly without additions of sugar
and O2. Miyata et al. (1998) used C. hirsutus pellets to decolorize a melanoidin-
containing medium. It was elucidated that extracellular H2O2 and two extracellular
peroxidases, MIP and MnP were involved in decolorization activity.
Lee et al. (2000) investigated the dye-decolorizing peroxidase by cultivating
Geotrichum candidum Dec1 using molasses as a carbon source. Components in the
molasses medium were found to have two different effects on enzyme production
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53
and its activity. They stimulated the production of decolorizing peroxidase but
inhibited the decolorizing activity of the purified enzyme. The decolorizing activity
increased with increase in dilution ratio and reached about 7 times as high as that of
nondiluted culture broth when the dilution ratio was 25. This implies that the
inhibitory effect of molasses can be eliminated at dilution ratios of more than 25.
The activity with 50 g L-1 molasses was the highest. Further studies on the topic are
necessary to elucidate the degradation mechanisms and hence to allow an
improvement in color-removal efficiency, which has great potential in
biotechnological and environmental applications (Gonzalez et al., 2000). Recently
D’Souza et al. (2006) reported 100% decolorization of 10% spentwash 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
spentwash. The enzyme cellulase was used for the pretreatment step with an
intention of transforming the complex and large pollutant molecules into simpler
biologically assimilable smaller molecules. It was suggested that enzymatic
pretreatment of the distillery effluent leads to in situ formation of the hydrolysis
products, which have different physical properties 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. Even though the pretreatment
step did not reduce the COD of the effluent, it altered the metabolic value of the
effluent by the microbes used in the aerobic oxidation step by generating
intermediate hydrolysis products from the parent cellulosic compounds present in
the spentwash. In another study, Sangave and Pandit (2006b) used irradiation and
ultrasound combined with the use of an enzyme as pretreatment technique for
treatment of distillery wastewater. The combination of the ultrasound and enzyme
yielded the best COD removal efficiencies as compared to the processes when they
were used as stand-alone treatment techniques.
Apart from distillery effluent, enzymatic decolorization of molasses has also been
tried. Molasses medium decolorization by P. chrysosporium was studied by
Thakkar et al. (2006). Under stationary cultivation conditions, none of the strains
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54
could decolorize molasses nor produce enzymes LiP, MnP and laccase. All of them
could produce LiP and MnP when cultivated in flat bottom glass bottles which
increased the surface area under stationary cultivation conditions.
Conclusion
For industries which are guzzlers of 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 technologies. In 1980, Sheehan and Greenfield reviewed the problem of
stillage production and various disposal techniques prevalent till that time. Much
wastewater has flown out of distilleries worldwide since then and many new
methods experimented with to treat this recalcitrant wastewater. 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 decolorizing wastewater. But as discussed above, these processes are not
efficient enough to treat these large volumes of colored wastewaters. A combination
of different treatment processes including a decolorization step could result in an
effective bioremediation of the molasses wastewaters.
In general, microbial decolorization is an environment-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 effluent 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. In all cases, where decolorization was applied to anaerobically digested
stillage compared to raw stillage, the level of decolorization was enhanced. Most of
these microbial decolorization studies required effluent dilution for optimal activity
and, in cases where aerobic fermentation is required; the energy demand was
significant. Decolorization technology has not been applied at full-scale and cannot
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55
yet be considered a developed technology. While using microorganisms, use of
media supplement pose extra burden on overall effluent treatment process.
Development of such a system where pollutants are biodegraded without addition of
any additional chemical amendment will be highly desired.
The use of cellulose carriers for microbial treatment offers 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 has technological advantages and yet is in its infancy,
requiring economical considerations (for process development) in order to apply it
on the plant scale. The cost of the enzyme is also an important factor to be
considered before using this technique on a large scale. Capital 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. A technology is only acceptable to industry when it requires less capital,
less land area and is more reliable compared to the existing well-established
options. Nevertheless, the feasibility of application of the process to full-scale would
need further research in this continuous culture set-up, in order to minimize the
added nutrients and extend the biomass activity for a longer period. An
understanding of complete profile of the effluent and the structures of coloring
compounds would also be helpful in achieving the appropriate treatment solutions.
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