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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

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TERI University-Ph.D. Thesis, 2007

14

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



16

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



17

(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



19

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



20

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



21

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



23

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



25

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.



26

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



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.



28

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



29

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



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



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



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



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.



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.



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.



36

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



37

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



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.



39

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



40

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



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



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



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



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.



45

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



46

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



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



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



49

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.



50

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



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-



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



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



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



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.



56

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