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Chapter 13

Bioremediation, Bioconversion and Detoxification of Organic Compounds in Pulp and Paper Mill Effluent for Environmental Waste Management Monika Mishra and Indu Shekhar Thakur

Abstract Pulp and paper industry is one among the 11 most polluting industry in India are utilizing natural resources as lignocellulose, inorganic and organic materials, and large volume of water in pulping and bleaching stages of the paper manufacturing. In manufacturing processes, 1 tonne of paper generates about 150 m3 of waste water contains lignin and its degradation products, resin acids, fatty acids and lignosulphonics. In bleaching stages, waste water contains nascent chlorine, chlorinated organic compounds, dioxin, furan, peroxides etc. The coloured compounds and adsorbable organic halogens formed due to environmental factors in waste water exhibit strong mutagenic effects, physiological impairment and they are ecoestrogens. In addition they are recognized as an excellent source of food, fuel, feed, chemicals, vitamins and bioactive compounds after biodegradation and bioconversion of biowaste. This potential is being realized as data from research in the areas of the physiology and chemistry of microorganisms which have been used for treatment of effluent by aerobic and anaerobic methods in different types of bioreactors. The major enzymes, xylanases and ligninases, have been used and commercialized successfully for biopulping and biobleaching purposes for production of ecofriendly pulp and paper, recovery of products, and environmental waste management. Their commercial role as value added products has been established after evaluation of detoxification of toxicants. In this review, the pulping and bleaching processes of pulp and paper, formation of organic compounds, use of microorganisms in degradation and bioconversion of biowaste in different types of bioreactors are discussed. The methods for detection of toxic compounds in the effluent and evaluation of detoxification are also described so that value added products recovered from effluent can be used commercially.

M. Mishra (*) • I.S. Thakur School of environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India e-mail: [email protected]; [email protected]

T. Satyanarayana et al. (eds.), Microorganisms in Environmental Management: Microbes and Environment, DOI 10.1007/978-94-007-2229-3_13, © Springer Science+Business Media B.V. 2012

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M. Mishra and I.S. Thakur

Keywords Pulp and paper mill effluent • Decolorization • Detoxification • Biobleaching • Biopulping • Enzymatic degradation • DGGE • SEM/TEM • GC-MS

13.1

Introduction

The rapid increase in population and the increased demand for industrial establishments to meet human requirements have created problems such as overexploitation of available resources, leading to pollution of the land, air and water environments. The pulp and paper industry is the sixth largest polluter (after oil, cement, leather, textile, and steel industries) discharging a variety of gaseous, liquid, and solid wastes into the environment (Ali and Sreekrishnan 2001). Wood is the major raw material for the forest based industries. One of nature’s most important biological processes is the degradation of lignocellulosic materials such as wood and agricultural wastes to carbon dioxide, water, and humic substances through the natural detoxification processes. The virtue of biotechnology lies in its potential to supply more specific reactions, to provide less environmentally deleterious processes, to save energy, and to be used where non-biological chemistry is unfeasible. There are, at present, about 515 units engaged in the manufacture of paper, paper boards and newsprint in India. The heavy demand for paper has led to the rapid expansion of the paper industries. At present about 60.8% of the total production is based on non-wood raw material and 39.2% on woody material. Annual paper production in India in the year 2009 has been estimated at 5.39 million metric ton. During the production of 1 ton of paper about 150 m3 of effluent is generated (www. indiastat.com). Manufacturing of paper is an elaborate process involving mainly two steps: pulping and bleaching. The wood is chopped into small pieces mixed with sodium hydroxide and sodium sulphite (kraft pulping) or acids (sulphite pulping) and heated at very high temperatures (~200°C) and pressure for 1–3 h. The lignin and hemicellulose present in it degrades and cellulose is left as pulp. This pulp is washed with water. The effluent generated at this stage is called black liquor as its dark brown in color due to the presence of lignin, hemicellulose, their degradation products, resin acids, lignosulphonics and phenols. Further the pulp is bleached as it has traces of lignin and hemicellulose which impart it yellow tinge. For bleaching nascent chlorine, hydrogen peroxide and ozone are used. The effluent generated at this stage has adsorable organic halides, chlorophenols, dioxin and furans. The bleached pulp is white in color and is used to make sheets of paper. The whole process of making paper generates extremely toxic effluent. Use of biological approaches like effluent treatment, biobleaching and biopulping can be used to reduce the toxicity of the effluent. It has been observed that fungi are the

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Bioremediation, Bioconversion and Detoxification...

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main degraders of lignocellulosic materials, particularly wood. Apart from fungi there are some bacteria, Bacillus and Pseudomonas, are also capable of wood degradation (Bourbonnais and Paice 1987). A mixed culture of algae was able to decolorize pulp and paper mill effluent (Lee et al. 1978).

13.2 13.2.1

Critical Review Process of Making Paper

The pulp and paper production is an elaborate process having high demand for water and electricity. The raw material can be wood, bagasse, wheat straw, rice straw and similar agricultural wastes having high fiber content. The steps involved in pulp and papermaking are (Fig. 13.1): 1. Raw material is cleaned and cut in small pieces. 2. Separation of cellulose fibers from lignin and hemicellulose is known as pulping. Pulping can be mechanical or chemical. In chemical pulping two basic methods, Kraft pulping (using sodium hydroxide and sodium sulphite) and Sulphite pulping (using sulphuric acid and bisulphite ions) are used. Pulping contributes maximum to the pollution load. 3. For making low quality paper used for wrapping pulp is directly fed into paper making machines where it is turned into sheets, dried with steam, cut into the desired sizes and packed. 4. For making good quality writing paper, the pulp is bleached to remove residual lignin to make it white. For bleaching chlorine, hydrogen peroxide, oxygen, ozone and similar bleaching agents are used. The effluent generated in digester house, i.e. pulping stage is dark brown in color due to lignin, its degradation products, lignosulphonics, hemicelluloses, resin acid and phenols (Chuphal et al. 2005). The main problem at this stage is degradation of lignin. It is an amorphous, polyphenolic complex polymer formed from dehydrogenative polymerization of three phenylpropaniod monomers, coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol (Lin and Dence 1992). Though lignin is resistant to microbial attack, still it is degraded to humus, water, and carbon dioxide following the death of the plant tissues which indicates that in nature a number of microorganisms exist which are capable of lignin degradation. This property of microorganism is exploited by biotechnology to make the manufacturing process eco-friendly. The pulping process can be made environmental friendly in two ways: treatment of effluent generated at this stage and treatment of raw material, i.e. biopulping.

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M. Mishra and I.S. Thakur The soils, dirt’s, and barks are removed from the wood and Wood preparation

chips are separated from the barks and water is used to clean the wood. Thus the watewater form this source contains suspended solids, BOD, dirt, grit, fibres etc. The wastewater generated from digester house is called ‘‘black liquor’’. Kraft spent cooking ‘‘black liquor’’ contains the

Digester house

cooking chemicals as well as lignin and other extractives from the wood. The wastewater contains resins, fatty acids, color, BOD, COD, AOX, VOCs (terpenes, alcohols, phenols, methanol, acetone, chloroform etc. The watewater from the pulp washing contains high pH,

Pulp washing

BOD, COD, and suspended solids and dark brown in color.

The wastewater generated from bleaching contains dissolved lignin, carbohydrate, color, COD, AOX, inorganic chlorine compounds such as Pulp bleaching

chlorate ClO3-, Organo chlorinc

compounds such as dioxins, furans, chlorophenols, VOCs such as acetone, methylene chloride, carbon disulfide, chloroform, chloromethane, trichloroethane etc. The wastewater generated from papermaking contains particulate waste, organic compounds, inorganic dyes, COD,

Paper making acetone etc.

Fig. 13.1 Pollutants from various sources of pulping and paper making (US EPA 1995)

The effluent generated at the pulping stage in pulp and paper mill is highly colored, alkaline and toxic. It has high COD, BOD and TSS. It contains lignosulphonics, resin acid, phenols, lignin and its breakdown products and hemicellulose (Pokhrel and Viraraghavan 2004). These are complex organic compounds, when released in environment without treatment; reacts with a wide variety of other chemicals in presence of light and heat to form highly toxic and recalcitrant compounds (Kinae et al. 1981; Zacharewski et al. 1995). Thus it is obligatory to treat the effluent before disposal into the environment.

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13.2.2

267

Characterization of the Toxic Organic Compounds

Four different families of organic compounds that can be found in pulp and paper mill waters and sludge sediments are:

13.2.2.1

Biocides

Biocides are often used for wood preservation and during paper-making to avoid problems associated with microbial, fungal and algal growth. Biocides used in paper-mills can be of different types: 2,2-dibromo-3-nitrilpropionamide (DBNPA), 2-(thiocyanomethylthio)-benzotiazole (TCMTB) etc. The fate of biocides is as follows: a fraction will degrade (chemically or biologically); a fraction will remain in circulating waters; and, finally, a fraction will be present in the effluent or remain in the solid matter. An additional problem is that, because of their physico-chemical properties, some biocides may retain fibres that can accumulate in the final paper product (Abrantes et al. 1998).

13.2.2.2

Resin and Fatty Acids

Wood extractives include lipophilic (fatty and resin acids (RAs), sterols, steryl esters and triglycerides) and hydrophilic (lignans, low-molecular-mass lignins, lignin-like substances and hemicelluloses) compounds that dissolve in waters during paper production. Among the wood extractives, resin and fatty acids have a great tendency to form pitch deposits and stickies that hamper the machine functioning and decrease the physical properties of the paper i.e. tensile strength, opacity, brightness, etc. (Gutierrez et al. 2001). Resin and fatty acids are not removed by primary flocculation, whereas a decrease of 50% or more is observed after biological treatment (Rigol et al. 2003). Resin and fatty acids are of different types: linoleic acid, stearic acid, palmitic acid, margaric acid, isopimaric acid, dehydroabietic acid, dichlorodehydroabietic acid etc. which are toxic to aquatic life, causing jaundice in rainbow trout.

13.2.2.3

Surfactants and Plasticizers

Surfactants, such as linear alkylbenzene sulfonates and alkylphenol ethoxylates, are present in effluent because of their use as cleaning agents or as additives in antifoamers, deinkers, dispersants, etc. The anionic surfactants, linear alkylbenzene sulfonates (LASs), represent 25% of total consumption. The non-ionic surfactants, alkylphenol ethoxylates (APEOs), degrade to nonylphenol (NP) or, to a lesser extent, to octylphenol (OP), which are considered persistent environmental pollutants (PEPs).

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LASs have been detected in waste waters of paper-mills at concentrations up to 5,000 mg/L (Rigol et al. 2002) and concentrations of 0.3–10 mg/L have been detected for APEOs such as NP and OP (Rigol et al. 2003). NP and OP are known to cause aquatic toxicity and endocrine disruption in animals. Laboratory experiments, using rat models have shown NP and OP to have negative impact on the hormonal development of mammals, e.g., underdeveloped testis.

13.2.2.4

Chlorinated Compounds

The various chlorinated compounds that are utilized or produced during the bleaching process and are detectable in the effluents and sludge are chlorolignin compounds, chlorophenols such as pentachlorophenol (PCP), chlorobenzenes, chlorinated acetic acids, chlorinated thiophenes, chloroguaiacols, chlorosyringol, chlorovanillin, chlorocatechol, polychlorinated dibenzo-para-dioxin (PCDD), polychlorinated dibenzofuran (PCDF), polychlorinated biphenyl (PCB). Raw wood material is often treated with pentachlorophenol (PCP) and other chlorophenols that act as wood preservatives. Chlorophenols are highly lipophilic and its benefits are low degradability in outdoor conditions. They have been encountered in water (Virkki et al. 1994) and sediments (Judd et al. 1996) of several paper-mills. In paper-making, chlorine and chlorinated compounds are also sources of dioxins and furans, which have been detected in sediments in the vicinity of a pulp and paper-mill (Munawar et al. 2000) and in effluents, along with polychlorinated dibenzothiophenes (Sinkkonen et al. 1992). Chlorinated thiophenes act as weak mutagens. Chlorinated acetones are Ames test positive and mutagenic. 1,3-dichloroacetone has been identified as one of most potent mutagens in effluent. Chloroguaiacols are thought to be precursors of tetrachloro-dibenzo-para-dioxin (TCDD). Chloroveratoles and anisoles are thought to be extremely toxic constituents of effluent with bioaccumulation potential and possible presence in sludge. Chlorophenols and trichloroacetic acid have tested toxic (50% growth inhibition) to a range of plants. Chlorolignins when degrade probably become chlorocatechols, chloroguaiacols and chloroveratoles, which are more toxic. Bacteria in soil are able to “o-methylate” chlorophenols and chlorolignins creating chloroveratoles. Exposure to chlorinated dioxins and dibenzofurans causes chloracne which is mostly seen on the cheeks, behind the cheeks, in the armpits and groin region. Chloracne persists for more than 10 years. Abnormal reproductive effects such as decreased testosterone, reduced sperm count, male feminization are seen among males while females experience decreased fertility, miscarriage and endometriosis. Other effects that result from exposure to chlorinated dioxins include immune suppression, liver enzyme changes, nervous system damage and thymus, spleen and bone marrow damage (Mandal 2005). Both PCBs and dioxins are known carcinogens (Fig. 13.2).

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O N

N

N

Br O

S

NH2

H OH

S

S

Br DBNPA

TCMTB

COOH DHA

PALMITIC ACID

BIOCIDES

FATTY AND RESIN ACIDS

H–(CH2)x–CH–(CH2)y–CH3

CH2COOH

O

OH

CH2 x + y = 10-13 x ; y = 0-13 LAS

C9H19

C9H19 NP1EC

SO3-

NP

O

PCDD

O

CHLOROPHENOL Cln

PCDF

OH Cln

OH H

Cln O

CHLOROGUAIACOL

Cln

Cln

Cln Clm

Clm

OH

DETERGENTS O

OH CHLOROCATECHOL

OH

O O CHLOROVANILLIN

CHLORINATED COMPOUNDS

Fig. 13.2 Chemical structure of organic compounds identified in paper-mill effluents (Lacorte et al. 2003)

13.2.3

Biodegradation of Persistant Xenobiotic Contaminants Present in Pulp and Paper Mill Effluents

13.2.3.1

Biodegradation of Chlorinated Dioxins

Lower chlorinated dioxins can be degraded by aerobic bacteria from the genera of Sphingomonas, Pseudomonas and Burkholderia (Field and Sierra-Alvarez 2008). Most studies have evaluated the co-metabolism of monochlorinated dioxins with unsubstituted dioxin as the primary substrate. The degradation is usually initiated by unique angular dioxygenases that attack the ring adjacent to the ether oxygen. Chlorinated dioxins can also be attacked co-metabolically under aerobic conditions by white-rot fungi that utilize extra cellular lignin degrading peroxidases. Recently, bacteria that can grow on monochlorinated dibenzop-dioxins as a sole source of carbon and energy have also been characterized (Pseudomonas veronii). Higher chlorinated dioxins are known to be reductively dechlorinated in anaerobic sedi-

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ments. Similar to PCB and chlorinated benzenes, halo respiring bacteria from the genus Dehalococcoides are implicated in the dechlorination reactions.

13.2.3.2

Biodegradation of Chlorophenols

There are three main pathways involved in the biodegradation of chlorophenols. The first pathway is the monooxygenase catalysed hydroxylation of chlorophenols to form hydroquinones. The second pathway involves the hydroxylation of higher chlorinated phenols to chlorinated hydroquinones. The third pathway is the initial reductive dechlorination of chlorophenols under anaerobic conditions. Biodegradation of phenol by Pseudomonas putida (NICM 2174) and Pseudomonas pictorum (NICM 2074) mixed culture exhibited as a versatile inexpensive and potential result to turn a toxic material into harmless products (Annadurai et al. 1999). Pseudomonas aeruginosa strain is capable of degrading pentachlorophenol (PCP) (Susella et al. 1991). Anaerobic dechlorination of 2,4-dichlorophenol has been observed in fresh water sediments in presence of sulphate (Madsen and Aamand 1991). Anaerobic biodegradation of chlorophenols such as 2,3,4,6-tetrachlorophenol and 2,4,6-trichlorophenolin fresh and acclimated sludge has been reported (Boyd et al. 1983). Phenolic compounds such as ortho, meta and para isomers of chlorophenols are converted into phenols by dechlorination under anaerobic condition in digested sludge (Boyd and Shelton 1984). It has been found that Basidiomycetes such as Grammathels fuligo and Phanerochaete crassa has capability to degrade chlorinated phenols and pentachlorophenol. At appropriate temperature and pH, G. fuligo and P. crassa showed superior mycelial growth. Reduction in chlorophenols was due to adsorption.

13.2.4

Removal of Color

Numerous bacterial, fungal and algal cultures are known to decolorize pulp and paper mill effluents (Table 13.1).

13.2.4.1

Decolorization of Effluent by Algae

It has been reported that some algae can decolorize diluted bleach kraft mill effluents (Lee et al. 1978; Tarlan et al. 2002). It was found that pure and mixed algal cultures removed up to 70% of color within 2 months of incubation. All cultures exhibited a similar color reduction pattern consisting of a phase with rapid and accelerating removal rate and a phase with declining rate. Color removal was most effective during the first 15–20 day of incubation, and then gradually dropped off. Complete removal of color did not occur. Color removal by algae is caused by metabolic transformation of colored molecules to noncolored molecules with limited assimilation or degradation of molecular entities. Adsorption is not a major color removal mechanism.

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Table 13.1 Cultures used for decolorization of pulp and paper mill effluents Cultures Reference Bacteria Pseudomonas ovalis Kawakami (1975) Pseudomonas aeruginosa Blair and Davis (1980) Bacillus cereus Bourbonnais and Paice (1987) Bacillus sp. Mishra and Thakur (2010)

13.2.4.2

Algae Microcystis sp. Chlorella, Chlamydomonas

Lee et al. (1978) Dilek et al. (1999)

Fungi Trametes versicolor Phanerochaete chrysossporium Tinctoporia borbonica Schizophyllum commune Aspergillus niger Gloephyllum trabeum Trichoderma sp. Paecilomyces variotti Phlebia radiatta Bjerkandera sp. Cryptococcus sp.

Kirk et al. (1976) Eaton et al. (1980) Fukuzumi (1980) Belsare and Prasad (1988) Kannan (1990) Galeno and Agosin (1990) Prasad and Joyce (1993) Calvo et al. (1991) Moreira et al. (1999) Palma et al. (2000) Singhal and Thakur (2009)

Decolorization of Effluent by Fungi

Published papers report the use of wide variety of fungi like Merulius aureus syn. Phlebia sp. and Fusarium sambucinum Fuckel MTCC 3788 (Malaviya and Rathore 2007), Trametes versicolor (Pedroza et al. 2007), Paecilomyces sp (Singh and Thakur 2006; Chuphal et al. 2005), Coriolus versicolor and Rhizomucor pusillus strain RM7 (Driessel and Christov 2001) for decolorization of pulp and paper mill effluent. The decolorization of the pulp and paper mill effluent by fungi involves two main mechanisms; first use of various enzymes like lignin peroxidase, manganese peroxidaes, laccase, xylanase and second adsorption. 13.2.4.3

Decolorization of Effluent by Bacteria

Along with these fungi, numerous bacteria such as Pseudomonas sp., Flavobacteria, Xanthomonas sp., Bacillus sp., Aeromonas sp., Cellulomonas sp., etc. have been reported to decompose lignin and its derivatives (Kirk et al. 1977; El-Bestawy et al. 2008; Mishra and Thakur 2010). The contributions of bacteria have been reported for utilization of low-molecular weight lignin oligomers as the sole source of carbon and energy that produce enzymes and cleave intermonomeric linkages of lignin (Vicuna et al. 1993). Bacteria play a pivotal role in depolymerizing lignin in aquatic ecosystem because wood degrading bacteria have a wider tolerance of temperature, pH and oxygen limitations than fungi (Vicuna 1988).

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Few bacteria have been reported for treatment of pulp and paper mill effluent (Thakur et al. 2001; Chuphal et al. 2005; Thakur 2004). Although many groups of bacteria are able to metabolize the monomeric constituents of the aromatic lignin polymer, their activity on polymeric lignin substrates is limited and low rates of conversion of radiolabelled lignin substrates to CO2 were observed with most of the bacteria tested (Zimmermann 1990). In pulp and paper industry, bacteria are generally used for degrading chlorinated phenols (Chuphal et al. 2005). Bourbonnais and Paice (1987) tested Bacillus cereus and two strains of Pseudomonas aeruginosa for decolorization of bleach kraft effluent. Color was primarily removed by adsorption with little depolymerization.

13.2.5

Treatment of Effluent Generated at Pulping Stage

Various physiochemical methods like sedimentation, flotation, screening, adsorption, coagulation, oxidation, ozonation, electrolysis, reverse osmosis, ultra-filtration, and nano-filtration technologies have been used for treatment of suspended solids, colloidal particles, floating matters, colors, and toxic compounds (Pokhrel and Viraraghavan 2004). However, they have disadvantages like high cost and sludge generation. Sludge has to be landfill. This further increases the cost of treatment. Sometimes the sludge is burned to save cost of disposal. On burning, huge quantities of volatile organic toxic compounds are formed. These include dioxins, furfurals and other volatile organic compounds. Thus in general, these processes only change the state of pollutants from liquid to solid then to gas rather than treating them. On the other hand biological methods involve degradation of pollutants, solving the problem permanently. Biological treatment can be divided into aerobic and anaerobic depending on the availability of oxygen. Aerobic treatment involves activated sludge treatment, aerated lagoons and aerobic biological reactors. Anaerobic filter, upflow sludge blanket (UASB), fluidized bed, anaerobic lagoon, and anaerobic contact reactors are anaerobic processes, that are commonly used to treat pulp and paper mill effluents. Among these treatments one thing is common, use of microbes (Pokhrel and Viraraghavan 2004). A number of fungi, bacteria and algae have been reported to have effluent treatment capabilities.

13.2.6

Use of Bioreactor for Decolorization

Many studies have reported high removals of organic pollutants of kraft mill wastewater by sequential batch reactor (SBR) treatment (Franta and Wilderer 1997; Milet and Duff 1998). Substantial removal of COD, TOC, BOD, lignin and resin acids of TMP wastewater using high rate compact reactors (HCRs) at a retention time of 1.5 h had been reported (Magnus et al. 2000a, b). Removal of COD in a moving bed biofilm reactor (MBBR) had been demonstrated (Jahren et al. 2002; Borch-Due

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273

et al. 1997). Berube and Hall (2000) showed that approximately 93% removal of TOC could be achieved by a membrane bioreactor. These studies show the importance of bioreactors in treatment studies. Recently some studies have reported the use of integrative approach. An integrated or hybrid system is designed to take advantage of unique features of two or more processes. A combination of coagulation and wet oxidation removed 51% of COD, 83% of color and 75% of lignin (Verenich et al. 2001; Verenich and Kallas 2001). A combination of ozone and biofilm reactor removed 80% COD (Helble et al. 1999). A combination of activated sludge and with ozonation (as tertiary treatment) removed 87–97% COD, and 97% BOD (Schmidt and Lange 2000). However, all these techniques have high cost of treatment.

13.2.7

Biopulping

Apart from effluent treatment modification in process i.e. use of biopulping as a pretreatment before chemical and mechanical pulping is one economically viable alternative to make the manufacturing process cleaner and greener. Biopulping involves the biotreatment of lignocellulosic material by fungus having lignolytic enzyme system and the subsequent processing by mechanical or chemical pulping (Saad et al. 2008). In wood, cellulose fibers are embedded in hemicellulose and lignin. Paper is made from cellulose thus hemicellulose and lignin is waste. These cellulose fibers need to be separated from hemicellulose and lignin to make paper. Pulping process involves separation of cellulose from lignin and hemicellulose. Also pulping is most polluting step (Pokhrel and Viraraghavan 2004). Pulping technologies have undergone constant improvements due to market demands and new developments in research. The need for sustainable technologies has also brought biotechnology into the realm of pulp and papermaking. Enzymatic processes are being developed to increase pulp brightness, to reduce troublesome pith, to improve paper quality and to purify the effluent (Messner and Srebotnik 1994). Efforts have been made to improve pulp-producing process by using isolated enzymes. These efforts have limited success as lignin, which is the major problem, lacks the regular and ordered repeating units found in other natural polymers.

13.2.8

Biobleaching

Biobleaching of pulps is performed with either hemicellulolytic enzymes, in particular xylanases (Jeffries 1992) or lignin-degrading fungi and their enzymes (Reid and Paice 1994) while xylanases hydrolyze hemicellulose (xylan) in pulp thereby enabling the bleaching chemicals an easier access to lignin (Paice et al. 1992), white-rot fungi and their ligninolytic enzymes directly attack and depolymerize lignin in pulp (Kondo et al. 1994). However, in both cases the aim is to enhance

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delignification and therefore facilitate the subsequent bleaching of pulp by applying reduced amounts of bleaching chemicals, especially chlorine and chlorine-containing compounds (Senior et al. 1992). The interest in xylan degrading enzyme and their application in the pulp and paper industries have advanced significantly over the past few years (Bajpai et al. 1994; Garg et al. 1998; Srinivasan and Rele 1999). In kraft pulping, hemicelluloses and lignin are dissolved and partially degraded during the heating process. In a subsequent phase of the process the pH drops sharply because of the discharge of xylan side groups and xylan precipitates with readsorption of lignin on top of the cellulosic microfibrils. Lignin is colored during kraft pulping and as a consequence, cellulosic fibers become darkly stained. Usually one or more bleaching sequences are needed to remove the dark color caused by the deposition of lignin. Organic chlorine compounds are formed during the chemical bleaching of pulp. These compounds arise mainly from the reaction between residual lignin present in wood fibers, and the chlorine used for bleaching. Chlorinated organic compounds produced during chemical bleaching technologies are harmful to the environment and need to be substituted by environment compatible procedures. One environmentally safe technique is the use of xylanases. Xylanases cleave and solubilize reprecipitated xylan and lignin located on the surface of the microfibrils. This facilitates pulp bleaching and lowers chlorine consumption thereby reducing discharge of toxic organo-chlorine compounds in the environment (Senior et al. 1992; Tolan and Canovas 1992). The effectiveness of xylanase treatments has been evaluated in at least two aspects: first by determining the amount of sugars after enzyme incubations, where 0.5–1.0% of the pulp carbohydrate content is liberated and second, by observing increased bleachability with conventional methods after xylanase treatments ( Viikari et al. 1993). Furthermore, xylanase treatment of kraft pulp releases the lignin-carbohydrate complexes (Yang and Eriksson 1992). Moreover, xylanase treatment helps in increasing brightness of pulp which is very important to develop chlorine free bleaching process.

13.2.9

Mechanism Involved in Decolorization of Pulp and Paper Mill Effluent

There are mainly two major mechanisms involved in decolorization of pulp and paper mill effluent. (1) Enzymatic process and (2) Biosorption.

13.2.9.1

Enzymatic Processes for Lignin and Hemicelluloses Degradation

In pulp and paper industry, cellulose is used for paper production while lignin and hemicellulose end up in effluent. The bacteria capable of degrading lignin and hemicellulose can be used for treatment of effluent. The degradation process

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involves use of number of enzymes collectively called ligninase. Ligninase is a generic name for a group of isozymes that catalyze the oxidative depolymerization of lignin. They include lignin peroxidase, manganese peroxidase and laccase, aryl alcohol oxidase, glucose oxidase and NAD(P)H:quinone oxidoreductase etc. Another enzyme xylanase plays a crucial role in hemicellulose degradation. These enzymes are extracellular, are non-substrate specific and aerobic in nature. This is an essential requirement for lignin degradation as it is a randomly synthesized biopolymer that cannot enter inside the cell and degradation involves the cleavage of carbon-carbon or ether bond, that link various sub-units, in oxidative environment (Breen and Singleton 1999). The mechanism of action of these enzymes is as follows: Xylanase Xylan, a major constituent of hemicellulose, is composed of b-1, 4-linked xylopyranosyl residues which can be substituted with arabinosyl and methylglucuronyl sidechains. Xylanases (endo-1, 4- b -D-xylan xylanohydrolase; E.C. 3.2.1.8) are a group of enzymes that hydrolyse xylan backbone into small oligomers (Kiddinamoorthy et al. 2008). The xylanolytic enzyme system carrying out the xylan hydrolysis is usually composed of a repertoire of hydrolytic enzymes: b-1,4-endoxylanase, b-xylosidase, a-L-arabinofuranosidase, a-glucuronidase, acetyl xylan esterase, and phenolic acid (ferulic and p-coumaric acid) esterase (Fig. 13.3). The presence of such a multifunctional xylanolytic enzyme system is quite widespread among fungi, actinomycetes, and bacteria (Beg et al. 2001). Due to xylan heterogeneity, the enzymatic hydrolysis of xylan requires different enzymatic activities. Two enzymes, b-1,4-endo-xylanase (EC 3.2.1.8) and b-xylosidase (EC 3.2.1.37), are responsible for hydrolysis of the main chain, the first attacking the internal main-chain xylosidic linkages and the second releasing xylosyl residues by endwise attack of xylooligosaccharides (Subramaniyan and Prema 2002). These two enzymes are the major components of xylanolytic systems produced by biodegradative microorganisms such as Trichoderma, Aspergillus, Schizophyllum, Bacillus, Clostridium and Streptomyces sp. (Bedard et al. 1987; Valenzuela et al. 1997; Yang et al. 1992). However, for complete hydrolysis of the molecule, side-chain cleaving enzyme activities are also necessary. Xylanases have several different industrial applications including Kraft pulp bleaching in the paper industry, biodegradation of lignocellulose in animal feed, foods, and textiles, as well as biopulping in the paper and pulp industry (Madlala et al. 2001; Schwien and Schmidt 1982).

Lignin Peroxidase (LiP) Lignin peroxidase is a heme-containing glycoprotein which requires hydrogen peroxide as an oxidant. Fungi secrete several isoenzymes into their cultivation

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M. Mishra and I.S. Thakur β-XYLOSIDASE

β-1,4-D-xylopyranose linkage H

H H O D-xylopyranose ring

O H OAc H

H

H OH

O

H H OH ENDOXYLANASE

O

H

O

H

H

O

O

H OH

O

H

H

CH3O α-0-methy1-Dglucuronic acid ring H

H

O

H

α-GLUCURONIDASE H

O C

OH

H

O H

OH

ACETYL XYLAN ESTERASE α-1,3-L-arabinofuranose linkage

H

CH2O

H OH

OH

O

O H OH

O

H

H OH H α-1,2-4-0-methy1-Dglucuronic acid linkage

O

H

H

O

H

COOH H

H

H

OH

α-ARABINOFURANOSIDASE R

O

CH Ac: Acetyl group R--H:p-coumeric acid R--OCH3:ferrulic acid

FERORYL and p-COUMAROYL CH ESTERASES

OH

Fig. 13.3 A hypothetical plant xylan structure showing different substituent groups with sites of attack by microbial xylanases (Beg et al. 2001) Fig. 13.4 Mechanism of action for Lignin peroxidase LiP. ox stand for oxidized state of enzyme (Breen and Singleton 1999)

Glyoxal oxidase Glyoxylic acid

Glyoxal O2

H2O2

Veratryl alcohol

LiP LiPox

Ligninox

Lignin

medium, although the enzymes may also be cell-wall bound (Lackner et al. 1991). LiP oxidizes non-phenolic lignin substructures by abstracting one electron and generating cation radicals which are then decomposed chemically (Fig. 13.4). Reactions of LiP using a variety of lignin model compounds and synthetic lignin have thoroughly been studied, catalytic mechanisms elucidated and its capability for C~−C~bond cleavage, ring opening and other reactions has been demonstrated (Eriksson et al. 1990; Higuchi 1989). LiP is secreted during secondary metabolism as a response to nitrogen limitation. They are strong oxidizers capable of catalyzing the oxidation of phenols, aromatic amines, aromatic ethers and polycyclic aromatic hydrocarbons (Breen and Singleton 1999).

13

Bioremediation, Bioconversion and Detoxification... Lignin

Mn(III)

Malonate

Mn(III)

277 MnP

H2O2

MnPox

H2O

Malonate

Ligninox

Mn(III)

Fig. 13.5 Mechanism of action for Manganese peroxidase MnP. ox stands for oxidized state of enzyme (Breen and Singleton 1999)

Manganese Peroxidase (MnP) Manganese peroxidase is also a heme-containing glycoprotein which requires hydrogen peroxide as an oxidant. MnP oxidizes Mn(II) to Mn(III) which then oxidizes phenol rings to phenoxy radicals which lead to decomposition of compounds (Fig. 13.5). Evidence for the crucial role of MnP in lignin biodegradation are accumulating, e.g. in depolymerization of lignin (Wariishi et al. 1991) and chlorolignin (Lackner et al. 1991), in demethylation of lignin and delignification and bleaching of pulp (Paice et al. 1993), and in mediating initial steps in the degradation of high-molecular mass lignin (Perez and Jeffries 1992). Laccase (Lac) Laccase (EC No. 1.10.3.2. benzenediol: oxygen oxidoreductase) is a true phenoloxidase with broad substrate specificity. It is a copper containing glycoproteins widely reported in fungi and plants. Most famous are rot fungi like Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Coriolus versicolor var. antarcticus, Pycnoporus sanguineus, Trametes elegans, Bjerkandera adusta, Pleurotus eryngii, Phlebia radiata, etc. (Baldrian 2006). It has also been reported in some plants like Acer pseudoplantanus, Aesculus parviflora, Populus euramericana etc. In plants laccase participates in the radical-based mechanisms of lignin polymer formation (Sterjiades et al. 1992), whereas in fungi laccases probably have more roles including morphogenesis, fungal plant-pathogen/host interaction, stress defense and lignin degradation (Thurston 1994). The presence of laccase has been reported in bacteria; however, such reports remain controversial (Diamantidis et al. 2000). The reactions catalysed by laccases proceed by the monoelectronic oxidation of a suitable substrate molecule (phenols and aromatic or aliphatic amines) to the corresponding reactive radical. The redox process takes place with the assistance of a cluster of four copper atoms that form the catalytic core of the enzyme (Fig. 13.6); they also confer the typical blue color to these enzymes because of the intense electronic absorption of the Cu–Cu linkages (Piontek 2002). Lignin is formed via the oxidative polymerization of monolignols within the plant cell wall matrix. Peroxidases, which are abundant in virtually all cell walls,

278


a

Fig. 13.6 Catalytic action of laccases (Riva 2006)

His 452

H2O Cu His 396

His 400

His 64 His 454

His 111

Cu

b

CuI

His 458

Phe 463

His 109

T3 4 Sub

Cys 453 Cu

Cu

His 66

T2

His 305 OH

T1 4 Sub-

CuI

CuII CuI II Cu

CuI

CuI CuI

Fully oxidized copper cluster 2H2O

Fully reduced copper cluster O2

have long been held to be the principal catalysts for this reaction. Recent evidence shows, however, that laccases secreted into the secondary walls of vascular tissues are equally capable of polymerizing monolignols in the presence of O2. The role of laccases in lignification has often been debated. Laccase from Acer pseudoplantanus was able to polymerize monolignols, in the complete absence of peroxidase (Sterjiades et al. 1992). This shows that laccase was involved in the early stages of lignification, while peroxidases were involved later. Laccases are able to catalyze electron transfer reactions without additional cofactors, hence their use has been studied in biosensors to detect various phenolic compounds, oxygen or azides. Moreover, biosensors for detection of morphine and codeine (Bauer et al. 1999), catecholamines (Ferry and Leech 2005), plant flavonoids (Jarosz-Wilkołazka et al. 2005) and also for electroimmunoassay Kuznetsov et al. (2001) have been developed. An enzyme electrode based on the co-immobilisation of an osmium redox polymer and a laccase from T. versicolor on glassy carbon electrodes has been applied to ultrasensitive amperometric detection of the catecholamine neurotransmitters dopamine, epinephrine and norepinephrine, attaining nanomolar detection limits (Ferry and Leech 2005). Laccase can also be immobilized on the cathode of biofuel cells that could provide power, for example, for small transmitter systems (Chen et al. 2001; Calabrese et al. 2002). Aryl Alcohol Oxidase (AAO) Aryl-alcohol oxidase (EC 1.1.3.7) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO partici-

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279

pates in degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases (Ferreira et al. 2009). Thus, the two substrates of this enzyme are aromatic primary alcohol and O2, whereas its two products are aromatic aldehyde and H2O2. This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-OH group of donor with oxygen as acceptor. The systematic name of this enzyme class is aryl-alcohol: oxygen oxidoreductase. Other names in common use include aryl alcohol oxidase, veratryl alcohol oxidase, and aromatic alcohol oxidase. Aromatic primary alcohol + O2 an aromatic aldehyde + H 2 O2

13.2.9.2

Role of Biosorption in Effluent Decolorization

There are two main processes acting during biological decolorization. One is enzymatic action and second adsorption. Biosorption is mainly a physio – chemical process.

Adsorbate (1)

Adsorbent Adsorbent (Microbial (Microbial cell) cell)

(8)

+ -(2) + -

(3)

(7)

- + -

(6)

+

(5)

Fig. 13.7 Diagrammatic representation of different mechanisms of biosorption

+ (4) +

280


Microbial cell (adsorbent) is shown in light blue color and effluent, metal or/and dye (adsorbate) are shown in black color circles. (1) Precipitation, on and outside surface, (2) physical adsorption e.g. electrostatic force, (3) active diffusion in cell, (4) ion-exchange, (5) chemical adsorption by bond formation (6) complexation, production of organic acids to form complex with adsorbate, (7) deposition on surface and (8) entrapment in the surface structures of cell involving a biological entity like live or dead biomass of fungi or bacteria (biosorbent) and some chemicals, metals or dyes (sorbate). The biosorption process involves a solid phase (sorbent or biosorbent; biological material) and a liquid phase (solvent, normally water) containing a dissolved species to be sorbed (sorbate, chemicals present in effluent, metal ions). Due to higher affinity of the sorbate for the sorbent species, the latter is attracted and bound by different mechanisms. The process continues till equilibrium is established between the amount of solid-bound sorbate species and its portion remaining in the solution. The degree of sorbate affinity for the sorbate determines its distribution between the solid and liquid phases. A wide variety of biological materials are used as biosorbents. For example the waste mycelia available from fermentation processes, olive mill solid residues (Pagnanelli et al. 2002), activated sludge from sewage treatment plants (Hammaini et al. 2003), biosolids (Norton et al. 2003), live fungi and bacteria (Srivastava and Thakur 2006 a, b, 2007). The mechanism of biosorption is complex, involving ion exchange, chelation, adsorption by physical forces, entrapment in inters and intrafibrilliar capillaries and spaces of the structural polysaccharide network as a result of the concentration gradient and diffusion through cell walls and membranes. There are several chemical groups that would attract and sequester the sorbate in biomass: acetamido groups of chitin, structural polysaccharides of fungi, amino and phosphate groups in nucleic acids, amido, amino, sulphhydryl and carboxyl groups in proteins, hydroxyls in polysaccharide and mainly carboxyls and sulphates in polysaccharides of marine algae that belong to the divisions Phaeophyta, Rhodophyta and Chlorophyta.

13.3 13.3.1

Analysis Contribution of Instrumentation Techniques in Lignocellulose Degradation

To study the degradation of lignin by microbes, a number of instruments are used. They include electron microscopy (EM) and gas chromatography and mass spectrophotometer (GC-MS). 13.3.1.1

Electron Microscopy (EM)

Electron microscopy (EM) (scanning (SEM), scanning-transmission (STEM) and transmission (TEM)) and ancillary techniques (e.g. X-ray microanalysis, electron

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281

diffraction) are now routine procedures which have been successfully applied to an array of problems in lignocellulose biotechnological research. These problems range from conventional studies on morphological aspects of wood cell wall ultrastructure (Fengel and Wegener 1984), biodegradation (Blanchette et al. 1990) and biopulping (Sachs et al. 1989), to enzyme interactions with pulp fibers (Mora et al. 1986) and more recently pitch problems in paper mills (Blanchette et al. 1992). Conventional SEM and TEM have been used essentially to confirm the ability of various microbes to modify and degrade wood cell walls and to visualize these events in time and in space. SEM gives the picture of surface view while TEM gives the idea of the changes taking place inside the wood.

13.3.1.2

Gas-Chromatography Mass Spectrophotometery

Analytical techniques such as pyrolysis-gas chromatography mass spectrometry (Py-GC-MS) are useful for the chemical characterization of lignin-containing materials, providing data on the relative amounts of different types of lignin units (Calvo et al. 1995a, b). Curie-point Py-GC-MS is a rapid microanalytical method for the structural analysis of lignin polymers on a molecular level. The technique requires minimum sample preparation and preserves side-chain information of the phenylpropane structural units. Py-GC-MS has been applied to pulp mill effluents, chlorolignins in xylan (Erik et al. 1993). The main disadvantage of GC relies in the fact that derivatization is necessary and the life of some derivatives is reduced to 12–24 h (Latorre et al. 2003). GC-MS studies helps in understanding the process of degradation. If samples of different durations are analyzed then it is possible to study the process of degradation as the intermediates formed in between can be detected. Detection of intermediates or end products of degradation gives the conclusive evidence of lignin degradation. Recently, instead of using pyrolysis GC-MS, in which sample is suddenly heated at very high temperature to make it volatile, silylation is done. Samples are treated with silylating agents [BSTFA (N, O-bis (trimethylsilyl) trifluoroacetamide) and TMCS (trimethylchlorosilane)] and trimethyl silyl derivatives are analyzed (Raj et al. 2007).

13.3.2

Denaturing Gradient Gel Electrophoresis (DGGE)

Bioremediation, the use of microbes to degrade environmental contaminants is receiving increased attention as an effective biotechnology to clean up polluted environments as it offers several advantages over the traditional chemical and physical treatments for diluted and widely dispersed contaminants. The establishment of methods to monitor microbes and their genes in the natural environment is desirable because it is necessary to understand the dynamics of microbes that degrade pollutants in order to carry out bioaugmentation efficiently and safely (Tani et al. 2002). To achieve this goal denaturing gradient gel electrophoresis can be used.

282


In DGGE, DNA fragments of the same length but with different base-pair sequences can be separated. Separation in DGGE is based on the electrophoretic mobility of a partially melted DNA molecule in polyacrylamide gels, which is decreased, compared with that of the completely helical form of the molecule. The melting of fragments proceeds in discrete so-called melting domains: stretches of base pairs with an identical melting temperature. Once the melting domain with the lowest melting temperature reaches its melting temperature at a particular position in the DGGE gel, a transition of helical to partially melted molecules occurs, and migration of the molecule will practically halt. Sequence variation within such domains causes their melting temperatures to differ. Sequence variants of particular fragments will therefore stop migrating at different positions in the denaturing gradient and hence can be separated effectively by DGGE (Muyzer et al. 1993). By comparing the pattern of bands or amplifying the separated bands and then sequencing them, it is possible to track the presence or distribution of microbes of interest.

13.3.3

Detoxification Studies

Bioremediation using microorganisms is very attractive option but it is not always the case, it might increase toxicity (van de Wiele et al. 2005). The measurement for toxicity after microbial treatment suggests whether we should take the microbe for bioremediation further. There are many techniques reported so far to measure the toxicity. Effect of toxicity can be many ways such as genotoxity, carcinogenesis, teratogenesis, mutagenesis and stress caused by toxicant on physological activity. Genotoxic effect can be measured by many ways such as comet assay, end labeling of DNA to visualize the nicking in DNA. For carcinogenesis studies, a comprehensive study is required to established relationship. For mutational effect one can choose Salmonella mutagenesis test. The physiological stress can be measured by many ways depending on type of stress such as metabolic, neuronal, reproductive stress etc. Some specific biochemical and cell based assays are very much popular due to rapid and reproducible results. After biodegradation of any compound, it forms an array of secondary metabolites which may cause toxicity. In case, pulp and paper mill effluent has not been classified as potent carcinogen, teratogenic or genotoxic compounds. However, compounds present in it may bind AhR and hence caused the toxicity in terms of CYP activity and apoptosis. Mitochondria play a major role in apoptotic pathway as it is responsible for a variety of key events in apoptosis such as changes in electron transport, loss of mitochondrial trans-membrane potential (Δy), failure of Ca2+ control, generation of reactive oxygen species (ROS), and involvement in pro (Bax) and anti apoptotic Bcl-2 family proteins. These events have been proven to very useful indicator in toxicity evaluation of any compound including heterocycles (Ding et al. 2006). The immortal cell lines of human are able to mimic the toxic response in the in vivo system with fair reproducibility.

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The cancerous cell lines are being used for toxicity evaluation, widely, because it retains the inherency of the organ, tissue, or cell specific responses by cancerous cell lines for long time unlike to normal cell lines. Parameters of toxicity may be morphological deformation, membrane integrity, cell viability, mitochondrial membrane potential (ΔYm) etc. (Rikans and Yamano 2000). These toxic parameters can be assessed by both biochemical assay and microscopic observations. Compounds present in this effluent disrupt the endocrine system and produced stress responses like ROS, O2−2. The toxicity pathways of dioxin are now well established and a dioxin responsive element (DRE) has been identified but unfortunately, the binding of dibenzofuran to the DRE has not been established. However, it shows binding to aromatic hydrocarbon receptors (AhR) and induces cytochrome P-450 monooxygenases especially CYP1A1 and CYP1A2 (Chaloupka et al. 1994). It is well established that many of these contaminants are acute or even chronic toxins. Chlorinated organic compounds, which include dioxins and furans, have the ability to induce genetic changes in exposed organisms (Nestmann and Lee 1985).

13.4

Future Perspectives

The high polluting potential of pulp and paper industry wastewaters can no longer be ignored. Microbial decolorization and degradation of colored effluents is a costeffective and promising green technology for treatment of such effluents. Reports of white-rot fungi that show lignin-degrading ability in saline conditions are very few. Industrial effluents are mostly alkaline and rich in carbonates, chlorides, and sulfates. In light of this, the marine fungi and bacteria hold good promise for the application of bioremediation of colored effluents under saline conditions. In the early 1990s, it was believed that the substitution of elemental chlorine with chlorine dioxide would eliminate the formation of furans and dioxins and reduce adsorbable organically bound halogens levels by almost 90%. However, it has been realized lately that, despite the use of Elemental Chlorine Free (ECF) processes, organochlorines have not been eliminated from discharges, just reduced. The debate between ECF and Total Chlorine Free (TCF) may not be resolved soon, but it is clear that TCF technology has many advantages over ECF and is more eco-friendly in the long run.

13.5

Conclusions

For any reasonable measure of success in treating pulp and paper mill effluents, future abatement programs should include a bilateral strategy for the use of alternate, cleaner technologies (e.g. the replacement of chlorine for bleaching, oxygen delignification and prolonged cooking) on one hand, and the development of economically viable and efficient technologies to treat these effluents on the other.

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Pollution from pulp and paper mill effluents is a complex environmental problem; its permanent solution will require comprehensive system considerations as well as multidisciplinary and holistic approaches.

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