Distillery wastewater: bioremediation approaches

Abstract

The alcohol distilleries are growing extensively worldwide due to widespread industrial applications of alcohol such as in chemicals, pharmaceuticals, cosmetics, beverages, food and perfumery industry, etc. The industrial production of ethanol by fermentation results in the discharge of large quantities of high-strength liquid wastes. Distillery wastewater is one of the most polluted waste products to dispose because of the low pH, high temperature, dark brown colour, high ash content and high percentage of dissolved organic and inorganic matter with high biochemical oxygen demand (BOD) and chemical oxygen demand (COD) values. Its characteristics are depending on the feed stock and various aspects of ethanol production process. Spent wash polluted the water bodies into ways; first, the highly coloured nature which can block out sunlight, thus reducing oxygenation of the water by photosynthesis and hence becomes detrimental to aquatic life. Second, it has a high pollution load which would result in eutrophication of contaminated water sources. Distillery wastewater, without any treatment can result in depletion of dissolved oxygen in the receiving water streams and poses serious threat to the aquatic flora and fauna. This review presents an account of the problems associated with distillery wastewater and a detailed study of existing biological treatment approaches. The role of various microorganisms and their enzymes in the wastewater treatment has been discussed to develop a better understanding of the phenomenon.

Keywords:

• bioremediation
• distillery wastewater
• biological treatment

1. Introduction

Distilleries are one of the most polluting industries as 88% of its raw materials are converted into waste and discharged into the water bodies, causing water pollution. In the distillery, for every litre of alcohol produced, about 15 l of spent wash is released (Ravikumar et al. 2007). Alcohol serves as a basic chemical for a large number of chemical industries and therefore, demand for alcohol will see a great increase in future and distilleries are fulfilling this demand. The first distillery in India was set up at Kanpur (then Cawnpore) in 1805 for manufacturing rum for the army. At present, there are about 315 distilleries with a total capacity of 3250 million litres of alcohol per annum with 40.4 billion litres of effluent, annually (Mohana et al. 2009). Most of the distilleries co-exist with sugar mills and utilize the molasses from cane sugar manufacturing as the starting material for alcohol production.

A maximum number of distilleries is present in Maharashtra (67) with an annual capacity of about 625 million litres of alcohol per year and an effluent generation of about 9367 million liter per year, approximately. Annual capacity and effluent generation of distillery industry in various States of India is shown in Figure 1 (Chauhan and Dikshit 2007).

Figure 1. Annual capacity and effluent generation of distillery industry in various states of India.

2. Manufacturing process and wastewater generation

Alcohol (preferably ethanol) is mainly produced from cellulosic materials. Raw materials, mainly used in distilleries are sugarcane molasses, grains, grapes, sugarcane juice, and barley malt. Alcohol production in distilleries consists of four main steps viz. feed preparation, fermentation, distillation, and packaging. Figure 2 shows the various steps of manufacturing process in distillery industries. Diluted sugarcane molasses is inoculated with yeast and fermented in either batch or continuous mode to yield a broth containing 6–8% ethanol. In the continuous process, the cellulosic materials first delignified and hemi-cellulose and cellulose are subsequently acid hydrolyzed into simple sugars. These sugars are fermented in the presence of yeast to yield ethanol and carbon dioxide. The alcohol vapor is removed from the fermentation solution under reduced pressure and subsequently distilled. Carbon dioxide gas may be sparged throughout the fermenting solution in order to aid in the removal of the alcohol from the fermenting solution (Saha et al. 2005; Tewari et al. 2007; Satyawali and Balkrishnan 2008). The gaseous carbon dioxide is captured and utilized for the manufacture of additional quantities of ethanol or other basic chemicals.

Figure 2. Process description.

3. Characteristics of wastewater generated during manufacturing process

Alcohol distilleries are highly water intensive units generating large volumes of high strength wastewater which pose a serious environmental concern. The quantum and characteristics of wastewater generated at various stages in the manufacturing process is provided in Table 1 and their characteristics are shown in Table 2.

Table 1. Wastewater generation in various operations.

Distillery operations	Average wastewater generation (kLD/distillery)	Specific wastewater generation (kL wastewater/kL alcohol)
Spent wash (from distillation)	491.9	11.9
Fermenter cleaning	98.2	1.6
Fermenter cooling	355.1	2.0
Condenser cooling	864.4	7.9
Floor wash	30.8	0.5
Bottling plant	113.8	1.3
Others	141.6	1.2

Table 2. Typical characteristics of different wastewater streams.

Parameter	Spent wash	Fermenter cooling	Fermenter cleaning	Condenser cooling	Fermenter wash	Bottling plant
Colour	Dark brown	Colourless	Colourless	Colourless	Faint	Colourless
pH	4–4.5	6.26	5.0–5.5	6.8–7.8	6	7.45
Alkalinity (mg/L)	3500	300	Nil	–	40	80
Total solids (mg/L)	100,000	1000–1300	1000–1500	700–900	550	400
Suspended solids (mg/L)	10,000	220	400–600	180–200	300	100
BOD (mg/L)	45,000–60,000	100–110	500–600	70–80	15	5
COD (mg/L)	80,000–120,000	500–1000	1200–1600	200–300	25	15

The distilleries have been generating huge quantities of high toxic effluents. The production and the characteristics of the spent wash are highly variable and dependent on the raw material used and various aspects of the ethanol production process (Wedzicha and Kaputo 1992; Pant and Adholeya 2007a, 2007b). The characteristics of different types of distillery wastewater are given in Table 3.

Table 3. Chemical characteristics of distillery wastewaters (Melamane et al. 2007).

Parameters	Type of wastewater
	Wine distillery wastewater	Vinasse	Molasses wastewater	Lee stillage
pH	3.53–5.4	4.4	5.2	3.8
Alkalinity (meq/L)	30.8–62.4	–	6000	9.86
Phenols (g/L)	0.029–0.474	0.477	0.450	–
VFA (g/L)	1.01–6	–	8.5	0.248
CODT (g/L)	3.1–40	–	80.5	–
CODS (g/L)	7.6–16	97.5	–	–
BOD5 (g/L)	0.21–8.0	42.23	–	20
TOC (g/L)	2.5–6.0	36.28	–	–
VS (g/L)	7.34–25.4	–	79	–
VSS (g/L)	1.2–2.8	–	2.5	0.086
TS (g/L)	11.4–32	3.9	109	68
TSS (g/L)	2.4–5.0	–	–	–
MS (g/L)	6.6	–	30	–
MSS (g/L)	900	100	1100	–
TN (g/L)	0.1–64	–	1.8	1.53
NH^+ 4 (g/L)	0.140	–	–	0.0451
NO^− 3 (g/L)	–	–	–	–
TP (g/L)	0.24–65.7	–	–	4.28
PO4 ^3− (g/L)	130–350	–	–	–

The spent wash generated from distilleries has high chemical oxygen demand (COD) (80,000–100,000 mg/L) and biochemical oxygen demand (BOD) (40,000–50,000 mg/L), high temperature, is dark brown in colour having low pH (<4.0–4.5) (Central Pollution Control Board 1994). COD and BOD values of this spent wash are due to the presence of a number of organic compounds, such as polysaccharides, reduced sugars, lignin, proteins, melanoidin, waxes, etc. The amount of inorganic substances such as nitrogen, potassium, phosphates, calcium, and sulfate is also very high (Table 2) (Melamane et al., 2007). Spent wash contains about 2% melanoidin which has an empirical formula of C₁₇–18H₂₆–27O₁₀N and molecular weight between 5000 and 40,000 Da (Martin et al. 2002; Manisankar et al. 2004). These compounds have antioxidant properties, which render them toxic to many microorganisms such as those typically present in wastewater treatment processes (Kumar et al. 1997).

According to EPA Guidelines for Wineries and Distilleries (2004), some of the potential effects of the various constituents of liquid and solid waste by-products from the wine making process on the environment are summarized in Table 4.

Table 4. Potential environmental impacts of winery and distillery wastes.

Winery waste constituent	Indicators	Effect
Organic matter	Biochemical oxygen demand, total organic carbon, chemical oxygen demand	• Depletes oxygen when discharged into water leading to the death of fish and other aquatic organisms
		• Odours generated by anaerobic decomposition cause nuisance
Alkaline or acidic	pH	• Death of aquatic organisms at extreme pH ranges
		• Affects microbial activity in biological wastewater treatment processes
		• Affects the solubility of heavy metals in the soil and availability and/or toxicity in waters
		• Affects crop growth
Nutrients	Nitrogen, phosphorous, potassium	• Eutrophication or algal bloom when discharged to water or stored in lagoons; algal blooms can cause undesirable odours in lagoons
		• N as nitrate and nitrite in drinking water supply can be toxic to infants
		• Toxic to crops in large amounts
Salinity	Electrical conductivity, total dissolved Solids	• Imparts undesirable taste to water
		• Toxic to aquatic organisms
		• Affects water uptake by crops
Heavy metals	Cadmium, chromium, cobalt, copper, nickel, lead, zinc, mercury	• Toxic to plants and animals
		• Neurotoxicity
Solids	Total suspended solids	• Reduces soil porosity, leading to reduced oxygen uptake
		• Can reduce light transmission in water, thus compromising ecosystem health
		• Smothers habitats odour generated from anaerobic decomposition

Many technologies have been tried for the treatment of spent wash, however, none of these methods have been found effective and economically viable to achieve the standard norms (Table 5) set by the Central Pollution Control Board, Government of India.

Table 5. Effluent standards for distilleries, maltries, and breweries.

Sl. no.	Parameter	Values
1	pH	5.5–9.0
2	Colour and odour	Absent
3	Suspended solids mg/L	100
4	BOD (3 days 27°C) mg/L
	(a) Disposal into inland surface water/river/stream	30
	(b) Disposal on land for irrigation	10

4. Treatment options of distillery spent wash

Current treatment options used to treat distillery spent wash includes physical, chemical, physicochemical and biological methods before its disposal. The selection of treatment methods depends on various factors viz. treatment efficiency, treatment cost, local geography, climate, landuse, regulatory constraints, and public acceptance of the treatment.

4.1. Physical treatment

Physical treatment methods are used at the initial stage of effluent treatment. Various physical treatment methods currently being used in distillery wastewater treatment are screening, flow equalization mixing, flotation, and sedimentation (Thakur 2006). Adsorption is also a one of the most widely used physical method. Adsorption on activated carbon is widely employed for removal of colour and specific organic pollutants. Physical treatment is used to decrease suspended/settable solids from wastewater which may be removed inexpensively via sedimentation by using the force of gravity to separate suspended material, oil, and grease from the wastewater (Jayanti and Narayanan 2004). Hamoda et al. (2004) reported removal of total suspended solids (TSS) by passing wastewater through a fine granular media such as sand, the particles are captured in the fine pores and sorbed on the surface of the granular media. Nataraj et al. (2006) also reported that membrane based nano-filtration and reverse osmosis processes can be used to reduce the total dissolved solids (TDS), COD, and potassium (K⁺) content of distillery wastewater by 99.80, 99.90, and 99.99%, respectively.

4.2. Chemical treatment

During chemical wastewater treatment, compound like chlorine (Cl₂), oxygen (O₂), ozone (O₃), and permanganate (MnO₄) are added to the wastewater to oxidize the wastewater components into carbon dioxide (CO₂), water, inorganic matter, and other harmless products (Benitez et al. 2003). Coagulation and flocculation processes are used for rapid and economical removal of suspended, inert, or undesirable colloidal materials in industrial wastewater (Tatsi et al. 2003). Mohana et al. (2009) have reported that melanoidins can be decolourized by various physiochemical methods. Majority of these methods remove colour by either concentrating the colour into sludge or by partial or complete breakdown of the colour molecules. Adsorption is a physical process so discuss it in physical process paragraph. Adsorption on activated carbon is widely employed for removal of colour and specific organic pollutants.

Pikaev et al. (2001) reported that treatment of distillery wastewater using iron sulfate (Fe₂ (SO₄)₃) as a coagulant results, 40% removal of wastewater pollutants. Beltrain de Heredia et al. (2005) also achieved 55% reduction in COD by using integrated Fenton-coagulant/flocculation process in distillery wastewater treatment. In another report, addition of ferrous sulfate, aluminum chloride, calcium oxide, ferric chloride along with coagulants reduced the colour to 95%, 74.4%, 80.2%, and 83%, respectively, while COD was reduced as 78%, 61.3%, 39.8%, and 55%, respectively (Chaudhari et al. 2007).

4.3. Biotechnological/biological processes

All biological treatment processes depends upon the natural growth and selection of microorganisms in a suspended culture or fixed film (Droste 1997; Lettinga et al. 1997), during treatment process, these microorganisms utilize pollutants for growth and convert the organic substrate in the wastewater into simpler substances such as CO₂ and water, in the presence (aerobic) or absence (anaerobic) of O₂ (Henry and Heinke 1996). Distillery wastewater needs to undergo intensive treatment in order to meet stipulated guidelines. Physical/chemical methods are invariably costly and are less employed in industries. While in recent years, biological wastewater treatment systems have attracted the attention of workers throughout the world and have helped in developing efficient and low-cost waste treatment systems (Naik et al. 2008). Biotechnology treatment encompasses a range of scientific and engineering techniques for applying biological systems to the manufacture or transformation of valuable materials or the elimination of problematic, often poisonous, liquid, solid, or gaseous wastes. There are a number of waste treatment systems which are based on aerobic and/or anaerobic microbial action to the wastewater (Willmott et al. 1998).

This approach can remove most of the biologically removable organics, CODs, and colour. Identification and optimization of biotechnological treatment methods is a necessity of the present time (Thakur 2006). There are several reports citing the potential of biological treatment for distilleries wastewater also. Both aerobic and anaerobic systems are commonly used to treat distillery spent wash. In view of potential applications, this section is discussed in details as biological treatment.

Various biotechnical/biological processes are being practiced successfully to remove the high pollution load from the distillery wastewater.

4.3.1. Anaerobic treatment

Anaerobic digestion is a natural process in which various microbial species work together, in the absence of O₂, to transform organic wastes through a verity of intermediates into biogas (Mata-Alvarez 2003). During anaerobic digestion, biomass and biogas are produced, while pathogenic microorganisms and offensive organic matter are reduced. Therefore, anaerobic digestion can be used both as a depollution tool and to produce energy (Moletta 2005). Anaerobic digestion of high strength wastewater usually occurs in successive steps and is accomplished by four trophic groups of bacteria (Ranade et al. 1999). These bacteria groups function in a synergistic relationship and form a food chain, in which the final products are CH₄ and CO₂ (Figure 3). There are a number of microorganisms that are involved in the process of anaerobic digestion including acetic acid-forming bacteria (acetogens) and methane-forming archaea (methanogens). There are four key biological and chemical stages of anaerobic digestion: hydrolysis, acidogenesis, acetogenesis, and methanogenesis.

Figure 3. Schematic diagram of anaerobic digestion indicating the process steps and the four bacteria groups involved in the process (Garcia-Heras 2003).

Various anaerobic treatment devices have been used, viz. anaerobic lagoons, anaerobic digester, anaerobic contact reactor, anaerobic filter, up flow UASB, anaerobic fluidized, and expended bed reactor.

Anaerobic digestion is the most suitable option for the treatment of high strength organic effluent. Anaerobic treatment is an accepted practice and various high rate anaerobic reactor designs have been tried at pilot and full-scale operation (Lata et al. 2002). The major advantages of anaerobic treatment using up flow UASB is that besides treatment of the wastewater, methane gas is also generated in this process which can be used as a fuel specially in boilers, thus anaerobic treatment processes not only helps in wastewater treatment but also lowers fuel consumption cost. Some of the anaerobic systems being applied for treatment of distillery wastewater are summarized in Table 6.

Table 6. Anaerobic methods employed for distillery effluent treatment.

Reactor type	Organic loading rate (Kg COD/m^3day)	BOD removal (%)	COD removal (%)	Temp. (°C)	HRT (days)	Reference
Anaerobic digester	–	–	–	35	3	Martin et al. (2002)
Anaerobic digester	23–25	–	64	–	8	Acharya et al. (2008)
Anaerobic filter and UASB	3.0–5.4	–	90	37.5	1.3	Blonskaja et al. (2003)
Anaerobic granular sludge reactor	10.0	–	80–90	15.0–18.0	1	Collins et al. (2005)
Anaerobic up-flow fixed bed	0.2–18.0	–	–	36	–	Genovesi et al. (2000)
Down flow fluidized bed	1.8–4.5		>95	35	1.3	Garcia-Calderon et al. (1998)
Down flow fixed film reactor	14.2–20.4	60–73	85–97	–	3.3–2.5	Bories et al. (1988)
Two stage anaerobic bioreactor	7	86	71	–	–	Vlissidis and Zouboulis (1993)
UASB	5.46–20.0	–	70–90	–	2.1	Goodwin et al. (2001)
UASB	2.0–18.0	–	90	34–36	–	Wolmarans and De Villiers (2002)
UASB	6.1–18.0	–	>90	35	2	Laubscher et al. (2001)
Thermophilic UASB	Upto 28	>80	39–67	–	–	Harada et al. (1996)
UASB	5.1–10.21		90	35	2.2	Keyser et al. (2003)
Granular bed anaerobic baffled reactor	4.75	90	82–90	–	–	Akunna and Clark (2000)

The performance and treatment efficiency of anaerobic process can be influenced both by inoculum source and feed pre-treatment. Anaerobic lagoons are the simplest option for the anaerobic treatment of distillery spent wash. Rao (1972) reported that employing two anaerobic lagoons in series resulted in final BOD levels up to 600 mg/l. Rao (1972) carried out the pioneering research work in the field of distillery waste management by studying the application of anaerobic lagoon treatment in two pilot-scale lagoons in series, with overall BOD removal ranging from 82 to 92%. Treatment of winery wastewater was investigated using an anaerobic sequencing batch reactor (ASBR). The reactor was operated at an organic loading rate (OLR) of 8.6 kg COD m⁻³ d⁻¹ with soluble COD removal efficiency greater than 98%, hydraulic retention time (HRT) of 2.2 days (Ruiz et al. 2002).

Garcia-Calderon 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% total organic carbon (TOC) removal, at an organic loading rate of 4.5 kg TOC m³ day⁻¹. Up flow UASB reactor is the most popular high rate digester that has been utilized for anaerobic treatment of various types of industrial wastewaters (Akunna and Clark 2000). Treatment by a UASB reactor resulted in 75% COD removal in sugarcane molasses spent wash (MSW). Wolmarans and De Villiers (2002) have also reported COD removal efficiency of greater than 90% over three seasons in a UASB plant treating distillery wastewater. In another anaerobic treatment method, fluidized bed reactors contain an appropriate media such as sand, gravel, or plastics for bacterial attachment and growth. A two stage process with an anaerobic filter followed by a UASB reactor was investigated by Blonskaja et al. (2003). The acidogenic and methanogenic phases were clearly separated ensuring better conditions for the methanogens. COD reduction was 54% and 93% in the first and second stage, respectively. The highest BOD removal is possible in open lagoon, whereas highest biomethane produced is in up flow UASB bioreactor. 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 (Pant and Adholeya, 2007).

4.3.2. Aerobic treatment

Aerobic processes are biological treatment processes that occur in the presence of oxygen. The aerobic environment in the reactor is achieved by the use of diffused or mechanical aeration, which maintain the mixed liquor in a completely mixed regime. Aerobic digestion is an alternative method of treating the organic sludge's produced from various treatment operations. In the conventional aerobic digestion (Coverti et al. 1993), the wastewater is aerated for an extended period of time in an open, unheated tank using conventional air diffusers, or surface aeration equipment (Hsu and Shich 1993). According to Metcalf and Eddy Inc. (1995), there are two variations in the aerobic digestion process, namely, conventional and pure oxygen. Aerobic treatment systems are used mainly to remove the BOD of these wastes. Partial reduction of BOD and COD is achieved in many distilleries using biological treatment (Jawed and Tare 1999; Laubscher et al. 2001; Wolmarans and De Villiers 2002; Coetzee et al. 2004).

Jackson et al. (2007) used a bioreactor system to treat distillery wastewater with a two-week HRT. COD of the distillery wastewater was decreased from 2255 mg/l to a final value of < 150 mg/l. Some of the aerobic systems being applied for treatment of distillery wastewater are summarized in Table 7.

Table 7. Aerobic methods employed for distillery effluent treatment.

S. no.	Process	Treatment agents	Wastes treated	Reference
1.	Tricking filters	Packed bed (stones or synthetics) covered by microbial films	Acetaldehyde, benzene, chlorinated hydrocarbons, nylon, rocket fuel	Biesterfeld et al. (2003), Schubert and Gunthert (2001), Hammer and Hammer (1996)
2.	Activated sludge	Aerobic micro organisms suspended in wastewater	Refinery, petrochemical and biodegradable organic wastewaters	Nazaroff and Alvarez-Cohan (2001), Foresti (2001), Contreras et al. (2001), Biesterfeld et al. (2003) and Jimenez et al. (2003)
3.	Aerated lagoon	Surface impoundment plus mechanical aeration	Biodegradable organic chemicals	Ramana et al. (2002a, 2002b), Kivaisi (2001) and Bories et al. (2005)

The aerobic treatment of industrial wastewaters usually depends on the oxidative activities of microorganisms. Although, a large number of microorganisms, such as bacteria, cynobacteria, yeast, fungi, etc. have been used for treatment of spent wash. Filamentous fungi can be of important sources of phenolic-degrading organisms, as they frequently grow on wood, utilizing lignin as a carbon source (Benitez et al. 1999; Coulibaly et al. 2003; Mendonca et al. 2004).

4.3.2.1. Fungal treatment

Fungi have shown potential for the treatment of various specific pollutants and mixed wastewaters, including dark-coloured, phenolic wastewaters such as molasses (Jimenez et al. 2003) and olive mill waste (Perez et al. 1998; Aggelis et al. 2003; Fenice et al. 2003; Mendonca et al. 2004; Ruiz et al. 2006), which means that fungal treatment of these wastewaters could be used as a pre-treatment step for anaerobic digestion. According to Coulibaly et al. (2003), fungi can be used to treat wastewaters in a liquid environment, where bioreactors with wastewater can be exposed to the specific live fungus that is capable of degrading a single pollutant or preferably a mixture of pollutants. Another promising approach would be to use enzymes derived from fungi to treat the wastewater (Coulibaly et al. 2003). Some of the fungi applied for treatment of distillery wastewater are summarized below in Table 8.

Table 8. Fungi employed for the decolourization and COD removal of distillery effluent.

Fungi	COD removal (%)	Colour removal (%)	Reference
Aspergillus sps. Aspergillus fumigatus G-2-6, Aspergillus niger, A. niveus, A. fumigatus UB260	69–75	70–90	Ohmomo et al. (1987), Miranda et al. (1996), Shayegan et al. (2004), Angayarkanni et al. (2003) and Mohammad et al. (2006)
Penicillium spp., Penicillium decumbens, Penicillium lignorum	50	70	Sirianuntapiboon et al. (1995)
Coriolus sp. No. 20	NR	80	Watanabe et al. (1982)
Phanerochaete chrysosporium	NR	85 free (59 immobilized)	Fahy et al. (1997)
Trametes versicolour	75	80	Benito et al. (1997)
Phanerochaete chrysosporium	73	53.5	Kumar et al. (1998)
Coriolus versicolour	70	71.5	Miyata et al. (1998, 2000)
Coriolus hirsutus	NR	80
Coriolus hirsutus IF044917	NR	45	Fujita et al. (2000)
Flavodon flavus	NR	80	Raghukumar and Rivonkar (2001) and Raghukumar et al. (2004)
Coriolus versicolour	49	63	Kahraman and Yesilada (2003)
Funalia trogii	62	57	Pant and Adholeya (2007a, 2007b)
Phanerochaete chrysosporium	57	37
Pluereotus pulmonaris	34	43
Phanerochaete chrysosporium 1557	NR	75	Vahabzadeh et al. (2004)
Phanerochaete chrysosporium ATCC 24725	48	55	Guimaraes et al. (2005)
P. chrysosporium NCIM 1073	Nil	Nil	Thakkar et al. (2006)
NCIM 1106	NR	82
NCIM 1197	NR	76
Marine basidiomycetes NIOCC	NR	100	D'Souza et al. (2006)

Four white-rot fungal cultures were examined for their ability to decolourize and bioremediate anaerobically digested molasses spent wash (DMSW) generated by biomethanation plants. Two cultures Coriolus versicolourand Phanerochaete chrysospotium showed an ability to decolourize and reduce the COD of diluted DMSW (125% v/v). Maximum decolourization (71.5 and 53.5%) and COD reduction (90.0 and 73.0%) were achieved in 6.25% (v/v) DMSW medium by C. versicolourand P. chrysospotium, respectively (Kumar et al. 1998). Phanerochaete chrysosporium and Trametes versicolour are the most widely studied among these. Phanerochaete chrysosporium JAG 40 resulted in 80% decolourization of diluted synthetic melanoidin (absorbance unit of 3.5 at 475 nm), as well as with 6.25% anaerobically digested spent wash (Kumar et al. 1998; Dahiya et al. 2001). Flavodon flavus (Klotzsch) Ryvarden, a basidiomycete (NIOCC strain 312) isolated from decomposing leaves of a sea grass, decolourized pigments in MSW by 80% after 8 days of incubation, when used at concentrations of 10% and 50% (Raghukumar and Rivonkar 2001). Aspergillus fumigatus has been found to be effective for decolourization of anaerobically treated distillery wastewater (Mohammad et al. 2006). Fungal consortium was employed in fluidized film aerobic system (FFAS). The analyzed effluent at the end of FFAS treatment showed a reduction of 70% in BOD and 63% in COD without causing any colour change (Ravikumar et al. 2007).

The fungi Geotrichum candidumroman Coriolus versicolourroman Phanerochaete chrysosporium, and Mycelia sterilia were screened for their ability to decolourize spent wash and to reduce the COD level. A 10 day pretreatment with Geotrichum candidum at 30°C resulted in reducing the COD by 53.17% and total phenols by 47.82%, enabling other bioremediating organisms to grow. Coriolus versicolour immobilized in a packed-bed reactor reduced the COD of spent wash by a further 50.3%, giving an overall reduction in COD of 77% to 15,780 mg/l (Fitzgibbon et al. 2007).

4.3.2.2. Bacterial treatment

Bacterial cultures are capable of bioremediation of distillery spent wash. Some of the bacteria applied for treatment of distillery wastewater are summarized below in Table 9. Pioneering work on spent wash decolourizarion by bacteria was done by Kumar et al. (1997). They observed that two aerobic bacterial isolates LA-1 and D-2 brought about maximum decolourization (36.5% and 32.5%) and COD reduction (41% and 39%) under optimized conditions in eight days. The most prominent bacterial species isolated from the reactor liquid belonged to Pseudomonas, while Bacillus was isolated mostly from colonized carriers. Pseudomonas fluorescens, decolourized melanoidin wastewater (MWW) up to 76% under non-sterile conditions and up to 90% in sterile samples (Dahiya et al. 2001). 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 60⁰C. A COD removal of 77% was achieved under non-optimal conditions.

Table 9. Bacteria employed for the decolourization biodegradation of distillery effluent.

Bacteria	COD removal (%)	Colour removal (%)	Reference
Xanthomonas fragairae, B. megaterium, and B. cereus	55–68	38–58	Jain et al. (2002)
Pseudomonas, Enterobacter, Aeromonas, Stenotrophomonas, Acienatobacter, and Klebsiella	44	Nil	Ghosh et al. (2004)
Microbacterium hydrocarbonoxydans, Achromobacter xylosoxidans, Bacillus subtilis, B. megaterium, B. anthracis, B. licheniformis, A. xylosoxidans, Achromobacter sp., B. thuringiensis, B. licheniformis, B. subtilis, Staphylococcus epidermidis, Pseudomonas migulae, Alcaligens faecalis, and B. cereus	85–86	76	Chaturvedi et al. (2006)
Pseudomonas aeruginosa PAO1, Stenotrophomonas maltophila, and Proteus mirabilis	51	67	Mohana et al. (2009)
Bacillus sp.	21	30	Kaushik and Thakur (2009)
Microbacterium hydrocarbonoxydans (AJ880397), Achromobacter xylosoxidans (AJ880764), Bacillus subtilis (AJ880760), B. megaterium (AJ880767), B. anthracis (AJ880766) from upper zone; B. licheniformis (AJ880762), A. xylosoxidans (AJ880763), Achromobacter sp. (AJ880396), B. thuringiensis (AJ868359), B. licheniformis (AJ880758), B. subtilis (AJ880761) from middle zone; and Staphylococcus epidermidis (AJ880759), Pseudomonas migulae (AJ887999), and Alcaligens faecalis (AJ880765) B. cereus (AJ853737)	75.5	86	Chaturvedi et al. (2006)

4.3.2.3. Algal treatment

The prokaryotic, oxygen-evolving photoautotrophs (cyanobacteria) have been reported to be useful for treatment of solid wastes and wastewaters containing phenol (Shashirekha et al. 1997). Since they are able to metabolize and use these compounds as nitrogen, phosphorus, carbon, and sulfur sources, respectively. Kalavathi et al. (2001) reported degradation and metabolization of the melanoidin in distillery effluent by the marine cyanobacterium Oscillatoria boryana BDU 92181.They, the decolourization of pure melanoidin pigment (0.1% W/V) by about 75% and crude pigment in the distillery effluent (5% V/V) by about 60% in 30 days reported. It was observed that the strain Oscillatoria resulted in almost complete colour removal (96%), whereas Lyngbya and Synechocystis were less effective resulting in 81 and 26% colour reduction, respectively (Patel et al. 2001). The treatment of anaerobically treated 10% distillery effluent using the microalga Chlorella vulgaris followed by Lemna minuscula resulted in 52% reduction in colour (Valderrama et al. 2002).

4.3.2.4. Phytoremediation

Phytoremediation of effluents is a low cost technique is used to remediate sites, contaminated with heavy metals and toxic organic compounds. Phytoremediation takes advantage of plants, nutrients utilization processes transpire water through leaves, and act as transformation system to metabolize organic compounds such as oil and pesticides. They may also absorb and bioaccumulate toxic trace elements, such as the heavy metals like lead, cadmium, and selenium (Witten et al. 1997). Phytoremediation depends on the availability of plant species – ideally those native to the region of interest – able to tolerate and accumulate high concentrations of heavy metals (Baker and Whiting 2002). Some plants can accumulate remarkable levels of heavy metals: 100–1000-fold the levels normally found in most species. This striking phenomenon, known as metal hyperaccumulation (i.e. the ability to accumulate at least 0.1% of the leaf dry weight in a heavy metal), is only exhibited by <0.2% of angiosperms (Baker and Whiting 2002), making the selection of native species for phytoremediation efforts a difficult task. Many hyperaccumulating species, characterized by their tolerance to toxic levels of metals such as As, Co, Cu, Zn, Mn, Pb, Se, Ni, and Cd, are often endemic to metal-rich substrates and are rare in their distribution (Baker et al. 2000). There are about 420 species belonging to about 45 plant families recorded as hyperaccumulators of heavy metals (Cobbett 2003).

Aquatic plants have excellent capacity to reduce the level of toxic metals, BOD, and total solids from the wastewaters (Kumar and Chandra 2004). Aquatic weeds are known to accumulate metals and other pollutants from the contaminated water. Weeds like Hydrillaroman Agrostisroman Eichhorniaroman Pistia, etc. are known to remove considerable amounts of metals from the matter in which they are growing (Satyakala and Jamil, 1997). The water hyacinth (E. crassipes) has been found to be most effective an economical in removing many chemical pollutants, nutrients and heavy metals, from industrial wastewater (Rai et al. 1995).

Bioaccumulations of the four heavy metals Cr, Cu, Pb, and Zn in Lemna gibba (duckweed) as an environmental indicator of contaminated industrial wastewater were detected. Plant pigment contents (chlorophyll and carotenoids) were estimated. During the study, heavy metals were ranked according to the preference for bioaccumulation by L. gibba, Zn came in the first place followed by Cr, Pb, and Cu with bioaccumulation factors 13.9, 6.3, 5.5, and 2.5, respectively (Hegazy et al. 2009). The correlation between heavy metal contents and pigment contents in L. gibba fronds revealed negative relationships with chlorophyll contents and positive relationships with carotenoid content for all study heavy metals. The decrease in total chlorophyll content was mainly associated with the increase in contents of Zn, Cu, and Pb reaching correlation coefficients of −0.99, −0.97, and −0.92, respectively. Chlorophyll a degradation was mostly associated with the increase in Zn contents, while chlorophyll b degradation was influenced by the increase in contents of Cr, Cu, Zn, and Pb. The degreened stage was excluded from the correlation between heavy metal and carotenoid contents; otherwise, weak correlations were obtained. Apart from Zn and Pb, Cu and Cr contents, where significantly correlated with the increase in carotenoid content in L. gibba fronds attaining correlation coefficients of 0.99 and 0.98, respectively (Hegazy et al. 2009).

Many species of plants have been successful in absorbing contaminants such as lead, cadmium, chromium, arsenic, and various radionuclides from soils. One of phytoremediation categories, phytoextraction, can be used to remove heavy metals from soil using its ability to uptake metals which are essential for plant growth (Fe, Mn, Zn, Cu, Mg, Mo, and Ni). Some metals with unknown biological function (Cd, Cr, Pb, Co, Ag, Se, Hg) can also be accumulated (Cho-Ruk et al. 2006).

According to Sinha et al. (2004), the plants act both as “accumulators” and “excluders”, accumulators survive despite concentrating contaminants in their aerial tissues. They biodegrade or biotransform the contaminants into inert forms in their tissues. The excluders restrict contaminant uptake into their biomass.

4.3.2.5. Enzymatic treatment of wastewaters

The most recent research has been focused on the development of enzyme processes for the treatment of wastewater (Karam and Nicell 1999). 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 (Naik et al. 2008). Enzymes can act on specific recalcitrant pollutants by precipitation or transformation to other products. They can also change the characteristics of a given waste to render it more amenable to treatment or aid in converting waste material to value-added products (Karam and Nicell 1999). Various oxidative enzymes such as Peroxidases and/or phenoloxidases are mainly involved in biotransformation or bioremediation of recalcitrant compounds (Durán and Esposito, 2000).

Although the enzymatic system related with decolourization of melanoidins is yet to be completely understood, it seems greatly connected with fungal ligninolytic mechanisms. The white-rot fungi is reported to have 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). Miyata et al. (1998) used C. hirsutus pellets to decolourize a melanoidin-containing medium. It was elucidated that extracellular H₂O₂ and two extracellular peroxidases, a manganese-independent peroxidase (MIP) and manganese peroxidase (MnP) were involved in decolourization activity.

Fungi especially the white-rot fungi produce enzymes laccase, Mn peroxidase, and lignin peroxidase (LiP), which are involved in degradation of lignin in their natural lignocellulosic substrates. Lignin peroxidase is able to oxidize various aromatic compounds, while Manganese peroxidase oxidizes almost exclusively Mn(II) to Mn(III), which then degrades phenolic compounds. Laccases are copper-containing oxidases. They reduce molecular oxygen to water and oxidize phenolic compounds (Mester and Tien 2000). D'souza et al. (2006), cultured a marine fungal isolate, NIOCC # 2a from decaying mangrove wood and reported 100% decolourization of 10% spent wash by a fungal isolate whose laccase production increased several folds in the presence of phenolic and non-phenolic inducers.

Enzymatic decolourization of molasses medium has also been tried using P. chrysosporium (Thakkar et al. 2006). Under stationary cultivation conditions, none of the strains could decolourize molasses nor produce enzymes lignin peroxidase, manganese peroxidase, and laccase. A combined treatment technique consisting of enzymatic hydrolysis, followed by aerobic biological oxidation was investigated for the treatment of alcohol distillery spent wash. Sangave and Pandit (2006a) used cellulase enzyme for the pretreatment step with an intention of transforming the complex and large pollutant molecules into smaller molecules. 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.

Pant and Adholeya (2007) isolated two fungal strains producing ligninolytic enzymes and having the potential to decolourize distillery effluent from the soil of a distillery effluent contaminated site. These two isolates along with one isolate of Pleurotus florida EM 1303 were assessed for their ligninolytic enzyme activity in culture filtrate as well as after solid state fermentation on two substrates wheat straw and corncob powder. Both P. pinophilum TERI DB1 and A. gaisen TERI DB6 were found to produce laccase, manganese-dependent peroxidases (MnP) and lignin peroxidases (LiP). The immobilized fungal biomass was then used for decolourization of the post biomethanated distillery wastewater and observed the reduction in colour up to the magnitude of 86, 50, and 47% with P. florida, P. pinophilum, and A. gaisen respectively. Some of the enzymes produced from the different from fungi are summarized in Table 10.

Table 10. Enzymes produced by fungi employed for the decolourization of distillery wastewater.

Fungi	Colour removal (%)	Enzyme	Reference
Coriolus sp. No. 20	80	Sorbose oxidase	Watanabe et al. (1982)
Flavodon flavus	80	Glucose oxidase accompanied with hydrogen peroxide	Raghukumar and Rivonkar (2001) and Raghukumar et al. (2004)
Phanerochaete chrysosporium 1557	75	LiP and MnP	Vahabzadeh et al. (2004)
NCIM 1106	82	LiP and MnP
NCIM 1197	76	LiP and MnP
Marine basidiomycetes NIOCC	100	Laccase and exoploysaccharide produced by the fungus	D'Souza et al. (2006)
A. flavus TERI DB9	74.67		Pant and Adholeya (2007a, 2007b)
F. verticillioides ITCC 6140	68.33
A. niger TERI DB18	64.67	Laccase, MnP, LiP
A. niger TERI DB20	86.33
P. ostreatus (Florida) EM 1303

Bodalo et al. (2006) has compared the two enzymes, horseradish peroxidase (HRP) and soybean peroxidase (SBP), the two most widely used commercial peroxidases for the removal of phenol from wastewater. Both enzymes achieved maximal removal efficiency in a neutral pH medium although they were active in a pH range of between 6.0 and 8.0.

5. Conclusions

Various treatment technologies such as physico-chemical treatment, composting, and biological treatment have been investigated by the researchers. The physical and chemical treatment methods remove organic pollutants at low level; they are highly selective to the range of pollutants removed (colour, turbidity, TSS or foul odors and COD). Nevertheless, the disadvantages associated with these methods are excess use of chemicals, sludge generation with subsequent disposal problems, high operational costs, and sensitivity to variable water input.

The conventional biological treatment methods (anaerobic digestion, anaerobic lagoons, activated sludge process, etc.) are not effective for complete colour and other pollutants removal from this stream. Various researchers have demonstrated that anaerobic processes enabling recovery of biogas appear to be the most promising technology for the treatment of spent wash. The technologies currently used by distilleries for treatment of wastewater are biomethanation followed by two-stage biological treatment and disposal in water courses or for utilization on land for irrigation, composting with or without biomethanation. These technologies treat the wastewater up to a certain level. However, there are limitations posed by these technologies for full compliance of prescribed standards. These limitations are either due to high cost of treatment or inherent inability of technology to remove certain pollutants like TDS and color, to safe and acceptable limits for disposal into surface water or on land. The use of an individual process alone may not treat the wastewater completely. An anaerobic treatment followed by aerobic biological oxidation is a common technique used for decolourizing wastewater. But these processes are not efficient enough to treat these large volumes of coloured wastewaters. There is a need of an ideal, cost effective, and commercial treatment scheme which should comprise of biomethanation as the primary step followed by physicochemical treatment and concluding with aerobic treatment.

The emerging treatment methods like enzymatic treatment have technological advantages and yet are in its infancy, requiring economical considerations in order to apply it on the plant scale. There appears to be a great potential for enzymes in a large number of waste treatment areas. 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.

Acknowledgments

The authors gratefully acknowledge the University School of Environment Management, Guru Gobind Singh Indraprastha University, Kashmiri Gate, Delhi 110406, India, for providing the research grants to support this work.