Indian Journal of BiotechnologyVol 2, July 2003, pp 444-450

Waste Water Bioremediation in the Pulp and Paper Industry

L Christov'<" and B van Driessel1

'Sappi Biotechnology Laboratory, Department of Microbiology, Biochemistry and Food Science,University of the Free State, POBox 339, 9300 Bloemfontein, South Africa

2Sappi Forest Products Technology Centre, Sappi Management Services, POBox 3252, 1560 Springs, South Africa

Received 13 November 2002; accepted 20 February 2003

Effiuents from the pulp and paper industry contain chromophoric compounds and can be partly mutagenic andinhibitory to aquatic biosystems. The presence of various pollutants produced during pulp and paper manufacturingnecessitates the need for waste water pretreatment prior to discharge. Of all the methods investigated,bioremediation specifically holds promise in solving environmental problems in a cost-effective way. White-rot fungihave ability to process a variety of pollutants efficiently, however, development of suitable cultivation procedures hasdelayed industrial application. These and other issues affecting bioremediation of industrial wastewater, with specialreference to application thereof in the pulp and paper industry, are reviewed in this paper.

Key words: bioremediation, waste water, pulp and paper industry, white-rot fungi, decolourization

IntroductionThe release of the bleach plant waste waters with

high adsorbable organic halogen (AOX) levels intothe receiving waters has become one of the majorenvironmental problems for the pulp and paperindustry. Efforts are made to reduce the chloro-organic and chloride discharges by the substitution ofchlorine-based bleaching chemicals with other moreenvironment friendly agents. The waste waterscontaining chlorolignins can be highly colouredmainly due to the presence of solubilised lignin andits derivatives. Some of the chlorolignins of lowmolecular mass have mutagenic and toxic propertiesand are known to be highly resistant to biodegradationand to accumulate in aquatic organisms (Ander et al,1977). In addition, a high concentration of chloridesin the waste waters contribute to their corrosiveness.

In response to the environmental concerns andstringent emission standards, modifications of theproduction process at the pulping and bleachingstages include the cooking time for additional ligninremoval, introduction of oxygen de lignification as apretreatment step, and elemental chlorine-free (ECF)and totally chlorine-free (TCF) bleaching. Physicaland chemical methods of removing chloroorganics,although quite effective in decolourisation of pulp and

*Author for correspondance:Tel: (+27) 51401304; Fax: (+27) 51 401 3049E-mail: christol Osci.uovs.ac.za

paper mill waste waters, are unattractive for industrialapplications because of the high costs involved.However, biological methods of effluent treatmenthave the advantage of being cost-effective and inaddition to colour removal, they can also reduce boththe biological oxygen demand (BOD) and chemicaloxygen demand (COD) of the waste water (Eaton etal, 1980).

Bioremediation in the Pulp and Paper IndustryThe pulp and paper industry discharges waste

water, which usually contains halogenated organicmaterials, that can pose environmental problems(Wang et al, 1992; Kallas & Munter, 1994).Chlorinated organic compounds, produced during thechlorination bleaching phase, are made soluble indilute alkali and extracted from the pulp in thesubsequent extraction phase (Prasad & Joyce, 1991).Chloroorganics are often toxic to aquatic life, manyare genotoxic and have the potential to migrate widelythroughout the ecosystem, ultimately accumulating inthe fatty tissues of organisms (Suntio et al, 1988).During bleaching, chromophoric and highly oxidised,polymeric lignin derivatives are formed that give riseto a dark colourisation in the waste water (Livernocheet al, 1983; Bergbauer et al, 1991). The colour posesan aesthetic problem and contributes to BOD (Bajpai& Bajpai, 1994). Conventional biological treatmentscannot effectively remove the colour attributed to therecalcitrance of chlorolignins and the difficulty in

CHRISTO V & VAN DRIESSEL: WASTE WATER BIOREMEDIATION

mineralising these compounds (Boman & Frostell,1988; Prasad & Joyce, 1991; Mehna et al, 1995).White-rot fungi have the ability to degrade lignin andits chlorinated derivatives effectively (Eriksson, 1991;Bajpai & Baipai, 1994). However, lignin cannot serveas the sole carbon source for these fungi because itsdegradation is apparently a very energy-intensiveprocess (Lei sola et al, 1983; Boman & Frostell,1988). The specific cultivation conditions, necessaryfor white-rot fungi to degrade lignin (Boman &Frostell, 1988), have been devised such as thepatented Mycor process, continuous flow systems(Prasad & Joyce, 1991), immobilization of white-rotfungi (Livernoche et al, 1983; Kirkpatrick et al,1990), etc. Other organisms such as soil inhibitingfungi imperfecti (Rodriques et al, 1996), algae (Lee etal, 1978) and streptomyces strains (Hernandez et al,1994) have also been studied for their ability todecolourise chromophoric substances. White-rot fungiproduce a variety of ligninolytic enzymes, includingperoxidases and laccases (Lamar, 1992; Manzanareset al, 1995). These enzymes convert and biotransformphenolic or poly phenolic substances, and can be usedto treat waste waters (Paice & Jurasek, 1984; Roy-Arcand & Archibald, 1991; Al-Kassim et at, 1994;Limura et al, 1996). The use of membrane reactors forthe immobilization of biomass and/or enzymes leadsto increased stability and allows continuous use ofenzymes(Van der Walt et al, 1997). Adsorption ofcoloured compounds on biomass is reported to occurrduring cultivation (Livemoche et al, 1983; Royer etal, 1985; Feijoo et al, 1995). Oxidised phenoliccompounds have a high affinity for chitosan(Muzzarelli, 1994), which exhibits polycationiccharacteristics at acidic pH-levels that could facilitatesorption of anionic substances. Chitosan-coatedhollow-fibre membrane process have been used forthe bioremediation of a phenolic containing wastewater (Edwards et ai, 1997). Certain physico-chemical methods for the treatment/pre-treatment ofwaste water (Kallas & Munter, 1994) includeadsorption using ion exchange resins or activatedcarbon, ultra-filtration, chemical precipitation (Boman& Frostell, 1988), oxidation (ozonation, hydrogenperoxide, etc.) using chemical methods (Feijoo et al,1995), and photo-catalysis, involving irradiation oftitanium oxide (Sierka & Bryant, 1994). However, thede-colourisation of paper mill effluents has not beencompletely solved because the technical difficultiesencountered when cultivating white-rot fungi and the

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problems of setting up an effective and economicallyviable process for bioremediation of paper and pulpwaste waters (Manzanares et al, 1995).

Chemical Composition of Waste watersThe content of chlorinated substances in waste

water is usually measured as total organically boundchlorine (TOCl) or AOX (Eriksson, 1993).Chloroorganic material from pulp mill waste water isgenerally described in terms of their molecular mass.The molecular weight of these materials has seriousimplications in terms of biodegradability, toxicity andcolourisation (Martin et al, 1995; Jokela & Salkinoja-Salonen, 1992).

High Molecular Mass(HMM) CompoundsMore than one-half of the colour load from bleach

plant waste water originates from HMM lignin-derived material in the alkaline extraction stage (Paice& Jurasek, 1984). Waste water from the extractionstage accounts for 80% of the colour, 30% of theBOD and 60% of the COD of the mill pollution load(Prasad & Joyce, 1991). The HMM substances aregenerally believed to be stable and thereforebiologically inert (Jokela & Salkinoja-Salonen, 1992).However, HMM material in spent liquors from thechlorination and alkali extraction stages of bleacherysoftwood kraft pulp were chemically unstable underconditions prevailing in receiving water systems,because these materials slowly released chlorinatedcatechols and guaiacols (Eriksson et ai, 1985). Martinet al (1995) concluded conflicting results on thearomatic nature of HMM compounds, whereas bothassociation by adsorption and chemical bonds mightaccount for the presence of some chloro-aromatics inHMM material. With HMM fractions (>30,000 Da),sequential depolymerisation does not necessarily leadto a complete degradation of aromatic compounds(Bergbauer et al, 1991). Once degradation of ligninderivatives by Trametes versicolor had reached acertain level, neither additional cosubstrates, norinoculation of the culture filtrate with fresh mycelium,could induce any further degradation. Thus, it seemsthat the end result of using this fungus could be theaccumulation of highly recalcitrant compounds in theecosystem.

Low Molecular Mass CompoundsCertain compounds (chlorinated phenols, quaiacols,

catechols, chlorosyringols and chloroaliphatics) in

446 INDIAN J BIOTECHNOL, JULY 2003

pulp and paper mill effluents (mole mass, < 1000g/mol) can be acutely toxic to aqueous organisms(Limura et al, 1996; Roy-Arcand & Archibald, 1991).Such compounds are divided into acidic, phenolic andneutral categories(Suntio et al, 1988). Chlorinatedphenols in bleach waste waters are typical chlorinatedlignin degradation products that contain methoxylgroups. The predominant compounds of the neutralfraction are chloroform, chlorodimethylsulfones andchloroacetones, while minor concentrations ofadditional chlorinated hydrocarbons, ethers, ketones,aldehydes, lactones and thiophenes have also beendetected (Smith et al, 1994). Chlorinated acidiccompounds in bleaching waste water are largelyaliphatic acids, mainly chloroacetic acid. Although avariety of low molecular substances do occur in thewaste water streams, most organic chlorine present isassociated with HMM material (Smith et al, 1994).

Cultivation Strategies for Decolourisation of WasteWaters

None of the traditional biological methods (aeratedlagoons, activated sludge systems and anaerobiccultivation) can degrade HMM chromophoriccompounds effectively (Boman & Frostell, 1988;Archibald et al, 1990). Aerated lagoons can reduceTOCl by about 25% at relatively high costs. Activatedsludge systems can reduce chlorinated compounds by40%, a substantial part of which is adsorbed onto thesurface of the biomass, which could lead to disposalproblems of excess sludge. Ultrafiltration of the E-stage waste water seems promising, but this will alsoresult in a disposal problem (Boman & Frostell,1988). Anaerobic treatment have gained popularity inthe pulp and paper industry because of low energyconsumption and the low production levels of wellstabilised excess sludge. However, bleach plant wastewaters are more diluted than desired for a lowretention time and the varying waste watercomposition can lead to toxic shock conditions,detrimental for the efficient functioning of theanaerobic systems. Boman & Frostell, (1988), bycombining ultrafiltration of the E-stage waste waterand incorporating permeate with the chlorinated wastewater, followed by anaerobic filter treatment,achieved an overall reduction of 62% inchloroorganics. However, they observed that white-rot fungi can degrade both high and low molecularweight compounds. One major drawback is the needfor the addition of an easily degradable, inexpensive

carbon source and the complicated physiologicaldemands of some of these fungi when degradinglignin. The requirements for high oxygen tension anda growth substrate can impede the practicalimplementation of fungal decolourisation (Eaton et al,1982). Various parameters and cultivation strategiesneed to be considered in an effort to develop asuccessful treatment process.

The Mycor ProcessIn the Mycor-process, called a FPLINCSU Mycor

method, the fungus. P. chrysosporium is immobilizedon the surface of rotating disks that can be enclosedfor additional oxygen supply (Eaton et al, 1982).Before decolourisation commences, a growth stage isnecessary so that nutrient nitrogen is depleted and thefungus becomes ligninolytic. The Mycor processreduced colour in an alkaline stage spent liquor by80% and furthermore converted 70% of theorganically bound chlorine into chloride at a hydraulicretention time of 2 days (Boman & Frostell, 1988).Although steam treatment initially might bebeneficial, further aseptic procedures would not berequired with fungi like P. chrysosporium. Wastewaters (temp, 28-40DC; pH, 4.5), in laboratory scalebench reactors, could be treated in over 60 days.Some limitations of the Mycor process seem to be thepoor surface to volume ratio since mycelia is inconstant contact with substrate to an extent of onlyabout 40%. Moreover, the mycelia is present in athick layer that could result in deficient oxygen andnutrient supply and a lower productivity in general.

Continuous-flow SystemsThree continuous-flow laboratory decolourisation

strategies were examined (Prasad & Joyce, 1991) asfollows:

Alternative I. It consisted of a set-up similar to aconventional oxidation basin with a surface area of510 ern' for fungal cultivation. Fungal mats ofTrichoderma sp, were transferred to the reactor anddecolourisation was carried out without aeration.

Alternative II. It utilized a vessel in which theheight was increased by decreasing the surface areaand four baffles were introduced to divide the vesselinto compartments. Trichoderma packed in syntheticwire bags were suspended in the middle of each zoneand continuous aeration supplied.

Alternative III. It was similar to the Mycor processin that fungal mats were clasped between circular

CHRISTOV & VAN DRlESSEL: WASTE WATER BIOREMEDIATION

wires, supported by outer central rings and securelyfixed in a metallic flame. The discs were rotatedcontinuously.

These systems could reduce colour by at least 50%for the first 6 days. However, Alternative III gave thebest results with a total delolourisation of extraction-stage effluent of more than 78% as well as a CODreduction of 25%. Colour removal targets in excess of57% were sustained for 18 days with Alternative III.

Fungal PelletsCoriolus (Trametes) versicolor as mycelial pellets

was used to decolourise lignin-containing kraft E-stage waste water (Royer et al, 1985). Decolourisationwas optimum (temp, 25-30°C; pH, 4 -5) andpractically non-existent at 4~°C. It corresponded tothe temperature of the liquor waste water at the E-stage. Magnesium ions improved the consumption ofchromophores, which might indicate that anenzymatic mechanism took place since magnesiumions serve as activators of many enzymes. At aninitial colour level of 7000 colour units (CU), themean colour removal proceeded at a rate of 300 CU/gmyceliumlhr. The authors suggested the use of airliftreactors employing the pelleted form of the funguswith the possibility of minimising operational costs.This strategy would facilitate recycling and the use oflarge amounts of fungal biomass. Mehna et al (1995)were able to achieve a colour reduction of 92% with aCOD elimination of 69% after 7 days using T.versicolor.

Flask CultivationsTrichoderma sp. was initially cultivated in shake

flasks and washed. Under optimal conditions (pH,4.0; carbon source, glucose), this fungus decreasedthe colour of kraft bleach plant waste water by 85%(68.6%without using additional carbon source) andreduced the COD by 25% after 3 days of incubation.Other carbon sources such as pulp and pith, which areabundant and inexpensive, increased thedecolourisation after 6 days, since they were notmetabolised immediately. Therefore, treatmentsshould be carried out strictly under defined conditions(Prasad & Joyce, 1991).

Fermentor CultivationsTrametes (Coriolus) versicolor was cultivated in a

laboratory fermentor with 0.8% glucose and 12 mMammonium sulphate at a controlled pH level of 5.0

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under optimal aeration. This resulted in 88%reduction in the colour units within 3 days, whereas80% reduction of waste water was observed in flaskcultures after 6 days (Bergbauer et al, 1991).

Immobilised Culture Treatment StrategiesThe application of immobilised filamentous fungi

in a continuous culture would alleviate the inherentproblems of biomass adherence, steady stateinstability and non-Newtonian behaviour (Anselmo &Novais, 1992). In liquid cultures, C. versicolorremoved over 60% colour of a combined bleach kraftwaste water within 6 days in presence of sucrose. Thiscontrasted drastically with the results obtained whenthe same waste water was treated with fungusimmobilised in beads of calcium alginate gel, where80% decolourisation was attained after 3 days in thepresence of sucrose. Recycled beads could removecolour efficiently in air repeatedly but not underanaerobic conditions (Livemoche et al, 1983). Afluidised bioreactor, containing polyurethaneimmobilised T. versicolor, operated at a residencetime of 9 hr and reduced colour of a bleach plantwaste water by 65%. A simultaneous reduction inAOX-levels of 57% was recorded. Biological activityand mechanical stability were maintained for 45 days.Up to 73% of colour and 55% of AOX were removedfrom bleach waste waters by mucoralean and white-rot fungi immobilised in a rotating biologicalcontactor reactor (van Driessel & Christov, 2001). Inthe Mycopor-process, white-rot fungi wereimmobilized in polyurethane foam and the systemwas operated continuously using a trickling filterreactor set-up. Using this process, waste water fromthe alkaline stage of a sulphate mill resulted in amaximum of 80% decoulorizing activity,accompanied by 50% COD reduction and 80%toxicity elimination after one passage through atrickling filter of I m length (Jaklin-Farcher et al,1992). The principle applicability of capillarymembrane technology using immobilised white-rotfungi in the bioremediation of industrial waste watersand aromatic pollutants has been demonstrated (Ryanet al, 2000).

Ligninolytic Enzymes and Their Possible Role inDecolourisation of Waste Waters

White-rot fungi produce a variety of keyligninolytic enzymes composed of lignin peroxidase(LiP), manganese dependent peroxidase (MnP) and

448 INDIAN J BIOTECHNOL, JULY 2003

multiple iso-enzyrnes of laccase. Confusion wascaused when mixtures of peroxidases and/or laccaseswere studied to demonstrate in vitro depolymerisationof high molecular mass lignin. Depolymerisation wasusually accompanied by repolymerisation of the lowmolecular weight fractions (Lamar, 1992). A mixtureof MnPs and laccases from Rigidoporus lignosuscould degrade Herea lignin (Lamar, 1992). Mutantsof P. chysosporium lacking the ability to produceMnP and LiP did not show significant decolourisationactivity when grown under nitrogen limitingconditions. Direct evidence linking MnP withdecolourisation was provided by the in vitrodepolymerisation of high molecular weightchlorolignin by MnP in the presence of Mn2+ andperoxide (Lamar, 1992). Laccase and MnP weredetected in bleach plant effluent treated by C.versicolor (Van Driessel & Christov, 2001). In vivostudies with purified MnP and LiP in thedecolourisation of kraft pulp bleach plant waste waterindicated that only MnP had a decolourising activity(Jaspers et al, 1994). Also MnP and not LiP activitywas detected when growing P. chrysosporium on thewaste water. Many aspects of the mechanisms andfunctions of the enzymes involved in ligninmineralisation are scarcely known (Manzanares et al,1995). More studies required to allow construction ofan enzyme mixture that prevents the repolymerisationreactions from taking place in a cell-free state(Eriksson, 1993). Therefore, processes based on thewhole fungus are still preferable (Ritter et al, 1990).

Adsorption Studies using Microbial BiomassBoman & Frostell (1988) reported that chlorinated

phenols (40-50%) and adsorbable chloroorganics(10%) were first adsorbed onto the fungal biomassbefore the actual breakdown started. The initial colourdecrease of Kraft pulp mill waste water (up to 30%after 3 days), using P. chrysosporium, could possiblybe attributed to adsorption of chromogeniccompounds during fungal growth (Feijoo et al, 1995).Jaspers & Penninckx (1996) reported that, dependingon the conditions of incubation, pellets of P.chysosporium strongly adsorbed colour and AOXfrom kraft bleach plant effluent. Royer et al (1985)regarded the decolourisation process by T. versicolorof bleach waste water to consist of two parts: i)Adsorption (completed after 24 hrs); and ii),Oxidation. About 10% of TOX removal, obtainedafter treatment of a mixture of first chlorination stage

and first alkaline stage waste water with Ganodermalacidum and C. versicolor, was estimated to be due toadsorption (Wang et al, 1992). The maximumpercentage removal of high-molecular weight organo-chlorine, using municipal waste water sludge assorbent, was 70%. After 24 hrs, only 8% of theorganochlorines desorbed from live biomass,indicating that the process was not readily reversible(Srinivasan & Unwin, 1995). Biosorption of colourfrom a sulphite mill bleach plant effluent by biomassof Rhizomucor pusillus, a mucoralean fungus,removed up to 90% of the adsorb able colour in thefirst few hours of treatment (Christov et al, 1999).Cell wall fractions (alkali-resistant, residual andchitosan fractions), extracted from the R. pusillusbiomass, appeared to be mainly responsible for theadsorption of chromophores from bleach plant wastewater. The residual and chitosan fractions (about 12%of the total dry biomass) removed more colour fromthe effluent (41%) than did the intact biomass (39%).These fractions, mainly composed of chitin and/orchitosan, seemed to be involved in the mechanism ofcolour removal from bleach plant effluent by R.pusillus (Van Driessel & Christov, 2002). Completeregeneration (by alkali treatment) and multiple reuseof this fungus were demonstrated in furtherdecolourisation treatments (Christov et al, 1999). Incontrast to R. pusillus, C. versicolor decolourisedeffluent by adsorption followed by biodegradation(van Driessel & Christov, 2001). Hernandez et al(1994), who used Streptomyces strains to decolourisepaper mill waste water obtained after semi-chemicalalkaline pulping of wheat straw, estimated that about20% of the initial colour was lost due to adsorption bythe mycelia. Furthermore, transformation of the majorchromophoric groups in effluent occurred only aftercolour adsorption by microbial biomass. Thus, bio-adsorption and transformation of coloured compoundswere linked and decolourisation required initialadsorption of the colour compounds to the mycelia.

ConclusionsHigher efficiencies of white-rot fungi during

biological treatment of bleach plant waste water arerequired to develop a practical biotreatment process.The high volumes of waste water from the pulp andpaper industry pose serious treatment problems.Biological treatments including lignolyticmicroorganisms, especially white-rot fungi, havepotential, but the necessity of including a co-substrate

CHRISTO V & VAN DRIESSEL: WASTE WATER BIOREMEDIATION

and the need for aeration would be costly affair.Fungal pretreatment of the bleach plant waste waterwas shown to be effective in colour reduction of thewaste water. Another alternative to the secondarybiotreatment of effluents would be their complexutilization in microbial fermentation processes toobtain valuable by-products. Biosorption ofchromophores from bleach waste water has severalpotential advantages. These include the fast rate of theadsorption process and the option of incineration ofthe spent biomass after repeated cycles ofadsorption/desorption and/or use as a supplement inthe paper-making process. Because of the complexityof treating paper mill waste water, combinations ofphysical, chemical and biological treatment strategiesmight lead to a synergistic, beneficial outcome thatwould facilitate the development of economic andefficient treatment procedures. The eventualimplementation of these biotechnologies may lead toconsiderable reductions of the AOX, chloride levels,toxicity and chemical load of the waste water.

AcknowledgementAuthors thank Sappi Management Services (Pty)

Ltd, Sappi Saiccor (Pty) Ltd, the Water ResearchCommission and the National Research Foundation(THRIP programme) for financial support, and SappiManagement Services (Pty) Ltd for giving permissionto publish this work.

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