Indian Journal of Experimental Biology Vol. 41. November 2003. pp. 1239- 1248

Review Article

Bioremediation of paper and pulp mill effluents

K Murugesan*

Centre for Advanced Studies in Botany. University of Madras. Gu ind Campus, Chennai 600025 . India

Pulp and paper mill erfluents pol lute water, ai r and soil . causing a major threat to the envi ronment. Several methods have heen attempted by vari ous researchers throughout the world for the removal of colour from pulp and papcr mill cfflucnt s. The bio logica l colou r remova l process uses scveral classes of microorgan isms - bactcria, algae and fungi - to dcgrade thc pol ymcric li gnin derived chromophoric matcria l. Whitc rot fungi such as Plwnerociwere chrysosporilill/, Corills I'ersicolor. Trail/er e.\' versicolor etc .. arc crficicnt in dccolouri zing papcr and pulp mil l cfllucnts. Cliocladillrl1 I'irel/.\'. a saprophyti c soil fu ngus decolouriscd papcr and pulp mill ef'llucnts by 42 0/" due to thc production of hemiccllulase. lignin pcroxidasc. mangancse pcroxidase ;.lI1d laccasc.

Keywords: Paper mill crfluents. Pulp pollution. Microbes in bioremed iation. Water po llution

B ioremediation is a po llution contro l technology that uses biologica l sys tems to ca talyze the degradat ion or transformation of vari ous toxic chemica ls to less­harmful forms. Bioremediation could be employed for the treatment of va ri ous industria l effluents including sewage water, effl uents from tannery, distill ery, paper and pulp industry . The general approaches to bioremediati on arc to enhance natural biodegradati on by nati ve organisms ( intrinsic bioremed iat ion), to carry out env ironmental modification by applying nutrients or aerat ion (biostimulation ) or through addit ion of microorganisms (b ioaugmentation). Unlike conventional techno log ies. bioremediation can be carri ed out on-site. B ioremed iat ion is limited to a number of tox ic material s it can handle, but where appl icable, it is cos t-effect i ve I. This article is an overview of the attempts made by several research and development organi za ti ons around the world to usc bi o~eehno l og i ca l methods for remova l of the toxic compounds from the environment.

Major pollutants-Synthetic chemicals The major chemical pol lutants are synthetic

chemicals li ke dichlorodipheny l-tri chloroethane (DDT) and polyv iny l chlori de. The li st also includes many chcmica ls such as tri chloroeth y lene, polych lorinated bipheny ls, nitroaromatic compounds like nitrobenzene, nitrotoluenes and many others.

Polychlorobiphenyl s (PCBs) are a class of syntheti c. non-b iodegradabl e chemica ls , which were

* For corrcspondenee: E- Illai l: bOlall y@vsnl.co lll

w idely used all over the world before their harmful ef fects were reali zed. Polychlorobipheny ls were considered indestructibl e, super tox ic pollutants just as heavy metal s and diox ins. A lthough their manufacture in many countri es h:1s stopped for sometime, the PCBs continue to persist in the environment pos ing greater hea lth ri sks .

Ch lori nated organ ic compounds such as tetrachlorod ibenzod ioxin (TCDD) and tetrachlorodi­be llzo furall (TCDF) formed during bleaching operati ons were reported to cause cancer ill rats, but its effect on humans has been the center of much debate and is still being stud ied. Dibenzofuran (DBF) and dibenzodiox in (DBD) were found in defoamers and oi Is.

Apart from these synthetic chemicals there are vari ous other non-degradab le and degradable pollutants present in the environment such as lignin and its derivat ives. They offer resistance to degradat ion due to the presence or carbon -to-carbon linkage of bipheny l type and other linkages in the molecule.

Microbes in bioremediation The US army scient ists for instance, stumbled on

A llenllollos sp. , wh ich can neu trali ze a whole range of biologica l warfare agen ts, nerve gas li ke sari n. This bacterium has an enzyme, organophosphorous anhydrase, which breaks down the nerve gas agents. Since A llerl1l 01l0S sp., is osmophilic and alkali tolerant , sc ienti sts have suggested thi s organIsm for bioremed iat ion appli cations.

1240 INDI AN J EXP BIOL, NOVEM BER 2003

Similarly , Ultramicrobacteria (UMB), generally 0.3 .um in size, were isolated from deep oceans and they survi ve in a dormant state for more than 30 years. In thi s state, their adherence property is so modified that they move rapidl y through porous media li ke layers of soil. Scienti sts are probing into the members of UMB population wh ich can digest tox ic wastes. They are toy ing with the idea of pushing such UMBs through waste dumps for cleaning up the tox ic material s.

Recombinant DNA approaches have been used to construct desired bacteria to degrade the supertox ic pollutan t - PCB s. A t the university of M innesota, Professor Lawrence Wackctt had designed such a recombinant PCB rcducing bacteri a. They have combined seven genes taken from di f ferent bacteri a and which code for 2 types of oxygenase enzy mes ­the cy tochrome P-450 coenzyme and toluene deox ygenase and tran sferred to Pselldolllonas. The combination enabled the bacterium to dismantle pen tach loroethane, a complex non-b iodegradab le

organic chemical to CO2, a feat never observed so far, in nature. The recent methods used in bioremediati on are to put together different genes I"l"Om di fferent organisms into a single organi sm, so th at they act in tandem to reduce the pollutant chemical to simple, harmless ones.

A site poisoned by TCE near Sav nnah River in South Carolina, USA was cleared using the naturall y occurring M eth anotroph in 1992. With thi s method, TCE levels were reduced to 5 ppb (parts per billion) concentration in just half the time requ ired by the conventional remediation methods, hence bioremediation procedures are attracti v .

The release of genetica ll y engineered organisms (GMOs) in the environment could be r isky . Attempts were made to include indigenious mechan isms, which keep GMOs under control. They intend to include a " death gene" which will be sw itched on as soon as the microbes complete the task they are designed for.

The fungi are unique among microorganisms since they secrete a vari ety o f extracell ular enzymes. The

Table I - Economic bGncfit s of bioremed iation

Application

Pet ro leu m-h ydrocarbon ­contaminated soil - urban brown field site

Pet rolelJ m- hydroca rbon­contaminated soil - natura l gas proCl:ss ing plant

Petroleu m- hydrocarbon ­cOlllaminated groundwatcr­gaso li ne from unck rgrolJnd storage tank

TCE contaminated so il and ground wa ter - industrial site

Multiplc conta minant ­superfund site Multiple contami nant­Supcrfund site contai ning BTEX and arscnic

Chlori nated hydrocarbon­i ndu, [rial site

M arine nil spill

Ph ys ica l /Chemica l T reatmcnt Excavati on and ol"i"s it e di sposal: proj ected cost. $ 3 milli on

J::xcavat ion and olT sitc disposa l: proj ected cost. $3mi II ion

Pumping. treati ng with air stripping and skimm ing: projected cost. $ 2 milli on and time peri od greater than 12 years

Pump and trea t: projec ted cost. $ 20 mill ion

Phys ica l capp ing; proj ected cost es timatcd al. $ 25 milli on Pump and treat and capping: cos t es timated at $ 50 milli on

Excavat ion: cost est imated at $ 15 milli on

Ph ys ical washing; cost es timated at $ 1. 1 mill ion per km of oil ed shorcline

BTEX. benzene. toluene, ethy l benzene, xylene; TeE, tri chloroethene

Biorcmcd iation

Biove llting ollsite: proj ec ted cos io $ 0.2 llIil lion

Bioventing on site: proj cc ted cost. $ 0.2 mi l l ion

Soi l vapour ex trac ti on. bioventing; projected cost, $ 0.25 mil li on and time peri od less than I yea r

Bioven ting: projectcd cost, $ 2 mi lli on

It1 .villi biotreatment ; est imated cos t. $ 5 milli on 111 S illl biostimulati on. oxygen spa rging. bi oventing plus biological immobili zat ion or metals; es ti mated cost. $ 2 milli on

Bioventing; es timated cost, $ 2 III i II ion

Biostimuia tion through fert ili zer additi on; cost estimated at $0.005 III

Bendit

Project cost sal' i ngs. $ 2.X milli on

Project cost ,avi ngs. $ 2.8 milli on

Projec t cost savi ngs. $ 1.75 mi Ilion and over decade in time sav ing

Projec t cos t savi ngs. $ I X million

Project cost sav ings, $ 20 mill ion Proj ec t cos t sa vi ngs. $ 48 mill ion

Projec t cos t sa\ ings, $ 13 million

Proj ect cos t savings. of over 5; I mil li on per km of oiled shorel ine

i

MURUGESAN : BIOREM EDIATIO OF PAPER PULP MILL EFFLUE TS 124 1

Tab le 2 - Geneti c engineering solutions and benefi ts

Limitation Genetic engineering solution Benefit

Incomplete degradation I. Uncoupling metabolism from degradation 2. Deregulate genet ic contro ls

I . Support activity with inexpensive. nontoxic substrates

2. Eliminate the need of tox ic-inducing substrates

3. Achieve diflicult clean up

Degradation I . Se lect high-performance host organism 2. Remove degradati ve 'bottlenecks'

f . Usc smaller. less ex pensive bioreaClOr, 2. Decrease fermentatio n treatment cost

Reca lci trant target compou nd I. Add suhstituti on- speci fi c functi ons (e.g .. I . Increase range of treatable compounds dchalogenation activit y) 2. Increase substrate range of single

2. A lter enzyme speci fic it y organi sms

Formati on of toxic intermediates I . Red irec t metabol ites I. Ex tend treatment li fe 2. Add complementary ac ti vities/pathways 2. Ex tend range of treatable compound

Chemica l mi :>. tures (e.g. , PCB s, mixed I . Combine metabolic activities I . Decrease fermentation cost, (s ingle organic wastes ) 2. Broaden substrate spec i ficity organism)

decomposition of lignoce llulose is rated as the most important degradative event in the carbon cyc le of earth2

. Enormous literature ex ists on the rol e of fungi in the carbon and nitrogen cycles of nature3

.. ,. The role of fungi in the degradation of complex carbon compounds such as starch, cellulose, pectin , li gnin, li gnocellulose, inulin , xy lan, araban etc., is well known. Trichoderma reesei is known to po. sess the complete set of enzymes required to breakdown cellulose to glucose. Degradati on o f lignocellulose is the characteri stic of several bas idiomycetous fungi.

The ability of fungi to transform a wide variety of hazardous chemicals has aroused interest in using them in bioremedi ati on6 The white rot fun gi are unique among eukaryotes for hav ing evo lved nonspeci fic methods for the degradation o f li gnin ; curiously they do not use li gnin as carbon source for their grow th 7. Lignin degradation IS therefore, essentiall y a secondary metabo li c process, not required for the main growth process . Lamar et al. x

compared the ability o f 3 lignin-degrading fungi , P/wilerochal:'le chrysosporiulI1 , P. sordida and Trall1etes hirsute to degrade PCP (pentachlorophenyl ) and creosote in soil9. Inoculation of soil with 10% (wtlwt) Phaenerochaete sordida resulted in the significant decrease of PCP and creosote. P.sordida was also most useful in the degradation PAHs (polycyc lic aromati c hydrocarbons) from soil. Dav is el al.9 showed that P. sordida was capable of degrading the three ring PAHs efficiently. However, four nng PAHs were less efficientl y degraded.

2. Se lec t envi ronmentall y robust host.

Pulp and paper mill ef11uents-A major threat The pulp and paper industry is one of the largest

and most polluting industries in the world . It is the third most polluting industry in orth A meri ca. The world populati on used over 2 14 milli on tons of paper and board products in 1987 and all estimates showed that paper consumption wi ll increase in the foreseeable future to. There are about 500 kraft mill s, and many thousands of other types of pulp and paper mill s in the world . The Indian pulp and paper industry presently has an install ed capacity of about 3.0 million tons per annum ". Primary concerns include the use of chlorine-based bleaches and resultant tox ic emi ss ions to air, water and so il with global annua l growth forecast at 2-5 %, the industry and its negati ve impacts could double by 2025 .

Efl'ects of pulp pollution

Waler pollurion Pulp mill effluents can seri ously harm habitat nea r

mill s, reduce water levels necessary for fi sh and alter water temperature, a crit ica l environmental factor for fi sh. Mill wastes continue to wreak havoc on surrounding ecosystem. Lignin and its deri vati ves impart an 9ffensive colour to the water, which is not only aes thetica lly unacceptab le, but al so inhibit the natural process of photosynthesis in the stream due to absorbance of sunlight. Thi s leads to a chain of adverse effects on the aquatic ecosystem as the growth of primary consumers as well as secondary and tertiary consumers is adversely affec ted.

1242 INDIAN J EX P BIOL, NOVEMBER 2003

Di scharge of untreated or partially treated wastewaters from pulp and paper mill s results in the colour persisting in the receiving body for a long distance. Recent studies have also indicated that lignin and its derivatives are toxic . A study by Roald l 2

showed that the growth rate of young rainbow trout was affected when exposed to a concentration of> 160 mg/l ligno-sulphonate. Nazar and Rapson 13 in an assay of the mutagenicity of kraft pulp bleaching plants found that the component of pulp is mainly responsible for the mutagenicity produced by chlorination of lignin .

Studies on the impact of pulp and paper mill effluents on the river Kallada in Kerala (India) also indicated the harmful effects of kraft pulp bleaching effluents on the fi shes, cope pods and other aquatic forms l4

.

The major toxic compounds identified so far in chlorinated stage wastewaters in pulp mjlls are mixtures of chlorinated lignins. Das l5 isolated tetrachloro-o-benzoquinone, a compound of low tox icity in wastewaters. Other toxic compounds present in caustic extraction stage wastewaters from a pulp mill are trichloroguaiacol, tetrachloroguaiacol, mono- and dichlorodihydroabietic acid, epoxystearic and dichloro stearic acid 16. It becomes necessary to remove colour due to lignin before they can be accepted into sUlface waters . Each Canadian rrull produces an average of 40 oven-dry tonnes of sludge per day, which is de-watered and then either land filled or burned. Because of this disposal, sludge pollutes soil, air and water.

Air pollution Air pollution from pulp mills is not we ll studied.

Mill s are monitored for a range of air emiss ions, such as particulate matter, CO2, S02, H2S, vo latile organic compounds, chlorine, chloroform and ch lorine dioxide. Incomplete data from Briti sh Columbia's Environment Ministry indicates that in 1997, mill s in the Canadian province emitted 17000 tonnes of particulates and 2.7 million tonnes of CO2, plus other unreported emissions.

Air discharges from pulp mills contain hormone di srupting and carcinogenic chemicals such as chlorinated phenols, polycyclic aromatic hydrocarbons (PAHs) and VOCs. Briti sh Colombia ' s coastal pulp mills are the largest provincial source of airborne dioxins and furans, which are among the most toxic substances known .

Biological decolourization The problem of colour removal from pulp and

paper mill wastewater has been a subject of study in

the last few decades. The colour in these wastewaters is mainly due to lignin and lignin derivatives. Waste waters containing huge quantities of lignin from pulp and paper mill results main ly from processes such as pulping, bleaching and chemical recovery sections.

Several methods have been attempted for the removal of colour from pulp and paper mill effluents. Physical and chemical processes are quite expensive and removes high molecular weight chlorinated lignins, colour, tOXICity , suspended solids and chemical oxygen demand but biological oxygen demand (BOD) and low molecular weight compounds are not removed efficiently . The biological colour removal process is particularl y attractive since in addition to colour and chemical oxygen demand (COD) it also reduces BOD and low molecular weight chlorolignins.

Biolog ical deco louri zation methods use several classes of microorganisms - bacteria, algae and fun gi to degrade the pol ymeric li gnin derived chromophoric material. Among these, wood degrad ing white rot fungi have been shown to efficiently and completely degrade and metabolize lignin, res lting in rapid decolourization of the effluents 17-20.

Decolourization with bacteria Bacterial cultures of Pseudomonas aerugillosa are

capable of reducing Kraft mill e fflu ent colour by 26-54% or more under aerobic conditions2o

. Bourbonnais and Paice21 tested Bacillus cereus and 2 strains of P. aeruginosa for decolourization of bleach Kraft effluent. Colour was primarily removed by adsorption. Kawakami22 found that P. ovalis degraded alkali lignin more read ily than Kraft lignin su lphonate. A mixed population of bacteria and protozoa derived from lake bottom sediment near the effluent Kraft paper mill was shown to degrade lignin sulphonate source. Although numerous bacteri a can decompose monomeric lignin substructure models, only a few strains are able to a ttack lignin derivati ves obtained from different pulping processes.

The extracellular xylanase from Bacillus stearothermophiLus T-6 is a thermostable alkali tolerant enzyme that bleaches pulp optimally at pH-9 and 65°C and was successfully used in a large-scale bio-bleaching. Streptomycetes badius and S. viridosporous were able to use a commercial Kraft lignin as sole carbon source. The acid precipitable polymeric lignin derived from thi s deg radati on was characterized by Fourier transformed infra-red spectroscopy, arruno acid analysis, elemental analysis

MURUGESAN: BIOREMEDIATION OF PAPER PULP MILL EFFLUE TS 1243

fo r C, H, N and high performance size exc lusion I ') chromatograp lY- .

Two bac teri al strains Pseudomonas putida and Acielletobacter calcoacet icus were studied for degradation of black liquor from a Kraft pulp a.nd paper mill in a continuous reactor. They were a.b le to remove 70-80% of COD and lignin while the colour remova l efficiency was around 85 o/c in 8 day s2~.

The degradation of di sso l ved and co lloidal substances from thermo-mechanica l pulp (TMP) by bacteri a isolated from a paper mill was studi ed. Burkholderia cepacia strains hydrolysed tri glycerides to free fally acids and the liberated unsa turated fally acids were then degraded to some ex tent. Saturated fally acids were not degraded. However, the branched anteisoheptadecanoic fa tty acid was degraded almost like the un sa turated fatty acids. About 30% of the stary l es ters were degraded during II days , increasing the concentrat ion of free stero ls. A pprox imately 25% of the dehydroabiedic and 45 % of the abietic and isopimari c res in ac ids were degraded during II days. The degree of unsa turation seemed to be of greater importance for the degradation of fatty acids25.

The effec t of pH, nutrient and aerati on on the remova l of co lour and reducti on of BOD, COD and heavy metal s, nitrogen and phosphorous was studi ed. Active microbial consorti a effectively degraded recalc itrant compounds. The iso lated bacteria -Pseudolllonas I'lltida (S I ), CitrobaClCl" sp. (S4) and ElIl erobaCler sp. (S5), not onl y decolorized effluent upto 97 % but reduced BOD, COD, phenoli cs and sulfide upto 96.63, 96.80, 96.92 and 96.67% respectively w ithin 24 hrs of growth and the heavy metals were removed upto 82-99.80%. The TSS and TDS were sharpl y reduced due to degradation 16

.

A compari son of organochlorine remova l from bleached kraft pulp and paper mill effluents by deha logenati ng Pseudolllonas, Allcylobacter and Methylubacleriul1I strains were assessed. These bacteri a were tested for growth on chlorinated acetic acids alld alcohols for adsorbable organ ic halogen (AOX) reducti on in batch cultures of steril e bl eached kraft-mill effluents (BKM E) from three sources. A. aquatics (A 7) exhibited the broadest substrate range, but could only effec t significant AOX reduction in so ftwood effluents. Melhylobacterium CP J 3 exhibited a limited substrate range, but was capable of remov ing significant amounts of AOX from both hardwood and softwood effluents. By contrast, Pseudomonas sp.Pt exhibited a limited substrate range and poor to negligible reduction in AOX levels

from both effluent types. Mixed inocula of all three species combined and inocula of sludge from mill treatment systems removed as much AOX from softwood effluents as did pure populations of Methylobacterium CP 13. When BKME was hydro lysed pri or to AOX analys is, the subsequent es timates of recalcitrant or non-hydrolysab le, AOX levels were far less vari ab le th an their counterpart tota l AOX measures. It is suggested that this IS a relevant and useful measure o f AOX for b· d d ' d' n 10 egra atlon stu les- .

Resin acids, a group of diterpenoid carboxy lic acids present mainl y in softwood species are present in many pulp mill effluents and are tox ic to fish in recepient waters. They are considered to be readily bi odegradab le. However, their remova l across biologica l trea tment systems has been shown to vary. Recent studies indicated that natural res in acids and transformation products might accumulate in sediments and pose acute and chronic tox icity to fish. Several res in ac id biotransformation compounds have al so been shown to bioaccumulate and to be more resistant to bi odegradation than the original materi al. A lthough wood inhabiting fungi have been shown to decrease the level of resin present in wood, there is no conclusive ev idence that fungi can completely degrade these compounds. In contrast, a number of bacterial isolates have recentl y been described wh ich are able to utili ze dehydroabieti c or isopimari c acids as their sole carbon source2X

.

An alkalophi li c strain of Bacillus SAM3, producing high leve ls of cellulase-free xy lanase act i ve and stable at alkaline pH, was iso lated from a soda lake. The enzyme was tested as a means of bleaching sugarcane bagasse pulp from a paper mi II where the bagasse was subjected to hot alkali cook ing and washing with water to neutrality . Enzymatic treatment for 2 hI' at 60°C and pH 8 with an enzyme dose of 1.2 JU/g pulp led to a decrease of 4 units in the kappa number. Similar treatment of the pulp at pH 7 and p H 9 indicated that the Sam-3 xylanase was effective at lowering the kappa number of the pulp over a w ide

?9 pH range- . The optimal cond itions for the deco lori zation of a

paper mill effluent by several strains of Streptomyces, were investigated . Strains able to decolorize this effluent were identified from 50 test strain s isolated from li gnocellulosic substrates. The decolorized effluent was also parti all y characterized. Fractionation of the decolori zed effluent by gel permeati on chromatography suggested that the fractions of high,

1244 I DIAN J EX P SIOL. OVEMB ER 20tH

and medium-molecular weight compounds were mainl y responsible for the co lour of the effluent. The res idual co lour of the deco lori zed effluent was attributed to the low-molecular-weight compounds'o.

L ign i n is apparentl y not biodegraded anaerobi call y. either rapid nor ex tensive bacteri al degradati on

occurs even under aerobi c conditions. Lignin degradation has not been most ex tensi ve ly studi ed in SI rel'lOlIlyces viridosp0f"OlIS and SI replOlIlyces elollii' l.

Act i nomycetes iso lated from di rferent so i I samples were tes ted fo r their ability to utili ze spent sulfite bleach effluents from a papermill . Degradati on and dechlorinati on o f the chlorin ated compounds in the eflluents of the first two bleaching stages (ie) ch lor inati on stage Ired l and alka line ex trac ti on stage, ,,,ere monitored by determining total organic compou nds ,\J1d ac t i va ted carbon adsorbable organic­bound halogen (AOX). The iso lates showed increased degradati on rates after repeated incubati ons in the effluent-containing med ium. Separation of the culture supern atants by ultrafiltrati on into 3 frac ti ons of di fferent molecular we ights revea led substanti al AOX and TOC reducti on in the low molecular we ight fract ion. The AOX va lues o f the higher-molecul ar weight frac ti ons we re also reduced. Ex trace llular perox idase and ce ll wa ll -bound ca talase ac ti v iti es were produced during growth of the microorgani sms on bleach elll uentsJ2

.

Dccolollrizalion with algae It has been reported that some algae can

deco lourize diluted bleach Kraft mill efflu ents'-'. It was found th at pure and mi xed al ga l cultures removed upto 70% of co lour within 2 months of incubati on. All cultures ex hi bited a similar co lour reducti on pattern consisting of a phase with dec lining rate. Co lour remova l was most crrcc ti ve during the first 15 to 20 days o f incubati on, then graduall y dec lined. Complete remova l o f co lour did not occur. Co lour removal by algae is caused by metaboli c transformati on of co loured molecu les to nonco loured molecu les w ith limited ass imilation or degradati on of molecular entiti es. A dsorpti on is not a major colour remova l mechani sm.

Demlollrizalion with fungi The 'vvood degrad ing white-ro t fungi are capab le of

dcgrad i ng I igni n effi cientl y. Sclzi:oplzy ll UIII 17 7'" . l l . 18 PI I COJllllllllie 1I1C/opOr/ a ,)O l"'}OIlICa wCll eroc wele

I . 19 d 7' . I x h b C lIys()SpO rlUlil an rWl/ eles \Jer .\·ICO or ave een

found to degrade li gnin and metaboli ze it along w ith carbohydrates. Asp erg illus nige r and Trichoderlll a

sp., one of the fungi imperfec ti , are also capable of degrading li gnin and deco louri zing effluent of hardwood pulp bleaching'us.

Belsare and Prasad 17 reported that the effl uent from bagasse based pulp and paper mill s could be deco louri zed w ith the white rot fungus S. commulle.

However, thi s fungus could not degra e li gnin un less a more easily metaboli zable carbon source was made available simultaneously. The add ition of carbon and nitrogen not only improved the decolouri zing effi ciency o f the fungus but also resu lted in a reducti on o f BO D and COD of the effl uent. Sucrose was found to be the bes t co-substrate for the breakdown of li gnin. A 2 day incubat ion peri od was suff icient fo r li gn in degradation by S. C(}I l/ ll l/llIe. The effi ciency of trea tment of efflu ent wi th thi s fungus was highest at pH 4-5 and was fu rth I' improved by intermittent aerati on. Under opti mum conditions, S. COII/IIIUII (' removed the co lour of efrIu ent by 90% and also reduced BOD and COD by 70 an d 72% respecti ve ly in 2 days incubati on.

A white, rot f ungus, Till c loporia bor /Jo ll ica . has been reported to deco louri ze the Kraft wa.'; te li quor to a li ght ye llow co lour ls . A bout 99% co lour reducti on was achieved after 4 days of cul ti vat ion. M easurement of the culture filtratc by ultrav iolet­spec troscopy showed th at the ch luri ne - oxy l ign i n content also decreased with ti me and measu remen t of the culture filtrate plus mycelial ex tract after 14 days culti va ti on ~ how('d the total remova l of ch lori ne­oxy lignin con tent.

Addition o f carbon and nit rogen source was found to improve deco louri za tion of pu lp and paper Illill waste water by A. lIiger, leav ing 19% of the or igina l co lour and reduced about 43 % BOD and 4 1 % CO D

I· ') d f' . b ' 1~ a ter _ ays 0 Incu <1 tI OI1" . Prasad and Joyce}S used Tr icl/Odemw sp. \0

deco louri ze the hardwood effl uent. G lucose was found to be the most effec ti ve carbohydrate utili zed by the fungus as it stimulated substant ial co lour reduction withou t any increase in COD. A ddition of nitrogen did not stimul ate the deco louri za ti on process indica ting that it is not a rate- li miti ng fac tor. The optimum pH for deco louri za ti on and growth was 4.0. Under optimal conditions, total co lour an d COD decreased by almost 85 and 25 % respecti ve ly, after culti vation for 3 days.

Gliocladilllll l' irell s, a saprophytic soil fungus has been employed for the bioremed iati on of paper and

)I

MURUGESAN: BIOREMEDI AT ION OF PAPER PULP MILL EFFLUENTS 1245

Table 3 - Phys ica l and bioc hemi ca l parameters of the paper and pulp mill errIuents, after treatment with C. vi reJls

Parameters Cont ro l

p H 8.98 Colour unit s 1867.42 Lignin (mgll ) 455 Cellulose (mgll) 72.9 Protein (mgll ) COD (mgll) 3200 BOD (mgll) 290 CMC

CMC - Carboxy methyl ce llulose KNO.1 - Potassium nitrate COD - Chemical oxygen dema nd BOD - Bio log ica l ox ygen demand

No co- Glucose substratc 80 1 6.59

1257.57 1083 .33 264 545

29.7 155.7 56 55

2880 3360 100 280

0. 107 1 0.9636

pulp mill effluents. It grew efficiently in the eftluents and deco louri zed them by 42% and also decreased the level of li gnin (52%), cellulose (75%) and BOD (65%) in the eftluents36.

P/wenerochaete chrysosporiul1I has the ab ility to d d I· · d I· . d·· f'fi · I 1,) ? () 18-,P egra e Ignm an Ignm envatl ves e IClent y .- .- -. Lab scale studi es wi th P. cll rys()sporiul1I to paper mill el"fluent supp lemented with glucose and asparagines or urea, resulted in a significant reduct ion in co lour in 5-6 days37

The optimum conditions favouring fungal growth are qu ite different from those fa vouring decolouri zation. The pH range for optimum growth was 4.3-4.8 and decolourization is greatl y retarded below pH 4 or above 5 because of poor growth 19. However, i f the fungus was grown at an opti mum pH , decolourization occurs, even at a I)H as low as 3. In the pH range of 5-7, the situati on is less clear since fungal decolouri zation results in the formation of acids, which lowers the pH rapidl/'J. Thus it appears that clecolou ri zation is less sensiti ve to pH decrease th an fungal growth . The optimum temperature for the growth of fungus was 400 e , whereas decololll'i zation is not limited to the same narrow range of temperature bu t takes place with a little decrease in rate at temperature as low as 25°e 19 The funga l decolouri zation requ ires oxygen and a co-substrate but the addi tion of nitrogen source is not necessary.

A nother white ro t fungus, Coriolus versicolor, has shown good performance. It produces an ex tracellul ar laccase, which pl ays a ro le in li gnin biodegradation. It was found th at product ion of ex trace llular enzy me, such as laccase, did not follow any spec ific profi le. No correlati on could be deve loped between laccase producti on and the rate of decolouri za tion. Consequently, laccase production could not be considered as a safe indicator of li gnolytic ac iti vity in I

. 41 t lese experIments - .

Sucrose CMC KNO.1 Ammonium Peptonc tartrate

5.49 7.70 8. 17 8.02 8.45 1090.99 1306 1344 1409.09 1897 .72 4 17 360 260 320 470 155.25 178 30.6 18 57.6 54 47 62 73 56

3200 3200 2880 3040 3200 275 300 190 2 10 375

0.5740 0.4553 0.156 1 0.569

It requires a growth substrate such as ce llulose or glucose for the decompos ition of lignin44 . The culture conditions favouring lignin degradation are similar to those favouring funga l deco louri zation. Livernoche el al. ·H showed that C versicolor in liquid cul ture removed over 60% of the colour of the combined bleach kraft eftluent w ithin 6 days in the presence of sucrose. Deco louri zation of effluents was more effici ent when the concentrati on o f sucrose and inoculum was high.

Biologica l reac tors of the airlift type using calc ium alginate beads w ith immobili zed fungus C. versicolor have been used to study the continuous deco louri zati on or the kraft mill effluents45 Royer (' I al .46 described the use of the pellets of C.l'ersicolor to deco louri ze ullrafiltered kraft l iquour in non-steril e condition with a neg li gible loss o f ac ti vity.

Li gnin degradation by basidiomycetes fungi has been studied by Abbott and Wicklow47

. The gasteromycete Cyo lh us stercoreus , which is assoc iated with litter decompos ition, degrades l ignin as efricieml y as any other white rot fung i .

Bajpai et al.4K used pellets of Tr{//I/ eles versicolor strain B7 for deco louri zati on of E I effluent. The mycelial pellets ox idi zed the chromophores of the effluent in presence of either o f the carbohydrates, sucrose, glucose, starch, eLh anol, carboxy meLhyl ce llulose, microcrystalline ce llulose, pu lp and malt ex tract. The highest decolouri za ti on was obtained in the case of glucose. Optimum pH and temperature was 4.5-5.5 and 300 e respect i ve ly.

Deco louri zation of papermi ll effluent was studied in vilro , using glucose as a cosubstrate and urea as a nitrogen source. Sterili za ti on of substrate and adjusting the pH to lower va lues were unnecessary fo r the sedimentation of the chromophoric compounds. A max i mum decolouri zati on (34%) by T. versicolor was

1246 I DIA J EX P B IOL. OVEMBER 2003

observed on third day in the effluent supplemented with 1.0% (w/v) glucose co-substrate and 0.2% (w/v) urea . Further, co lour reducti on (24% ) was observed in Ihe effluenl supplemenled with glucose. It IS

conc luded for effluent deco louri za lion by T. I'ersicolor, the effluent should be supplemented with glucose as a cosubstrate and urea as the nitrogen sou rce~9 .

Decolouri zati ol1 of the effluent occurred rapidl y due to the growth of Glioc/adilllli virell s except in the ertl uents amended with peptone. Effluents supp lemented with ca rbon (glucose, sucrose) sources showed quicker and efficient colour reduction , when compared to that of unamended controls·'o.

The ability of 3 marine fungi (So rr/aria fimicola, Halosatpheia rat llogm ensis and an unknown basidiomycetes) to produce the lignin modify ing enzymes; laccase, manganese peroxidase (M NP) and lignin perox idase (UP) and to minerali ze 14 C ring labelled syntheti c li gnin was demonstrated. The abil ity of these marine fung i to decolorize paper mill bleach kraft efl'iuent was also demonstrated for the first lime50

Experiments were conducted to study the ability of A spergilllls ./oelidlls to remove colour, decrease COD (chemical oxygen demand) and metaboli ze li gnin from disso lved bagasse-based pulp and raw black and alka li -stage liquors in nutrient medium. Approx imately 90-95 0/£ of the total co lour was removed from growth media containing lignin at 0.05 or 0.1 % or diluted black liquor or alkali liquor. Decolorizati on and lignolyti c and COD removal processes occurred pri ncipall y duri ng the ex ponential growth phase of the fungus, with cOl1comitalll utili zat ion of the primary growth-supporting substrate. Strong correlations ex isted between the timin g of decolorizat ion and I ignol yt ic processes5 1

.

The degradation of ce llulose by Plell J'O llis sajor­cajll was rapid at the initial stages of growth . The ac ti vities of endogluc<l n<1se, exog lucanase and beta­glucos idase were maximum at 8, 12 and 26 d of growth , respecti ve ly . The acti vities of li gnin ­degrading enzymes were maximum at the later stages of growth. Such a delignifica ti on process is considered to have potential applications in the conversion of paper-mill sludge into food , animal feed and fibre products52

.

Decolourization with enzymes Some enzymes also seem to have the potential to

remove co lour from pulp and paper mill effluents.

Ligninase, cellulase, perox idase etc. , are the most important enzy mes, espec iall y perox idase. which is used for co lour remova l in bleaching effluents. It is also poss ible to mix enzy mes together w ith spec ial microbes, which normally do not have high enzyme activity. White rot fungi uses glucose as sub ·trate and produce peroxidase, an ex tracellular enzymes. It seems that thi s enzyme ox idi zes the chromophores and removes the co lour from bleaching was tewater)". The co lour removal from effluents at neutral pH by low leve ls of H20 2 was enhanced by the add i tion of perox idase.

Conclusion The compari son of deco lori za tion y different

organisms show that white rot fungi particular ly P. chrysosporiufIl and C. versicolo r are suitable for efficient degradation of the reca lcitrant chromophoric material in bleach plan t effluents. However, the requirements for high oxygen tension an d a growth substrate constrain the practi ca l implementation of funga l decolori zat ion. Further research is needed to develop fast biodegradation processes , which are likely to prov ide an economica ll y feasible co lou r remova l process.

Future outlook It may be poss ible to clone genes for the efficien t

degradat i ve enzyme into bacteria, which could be further transferred to suitable fungi. The high surface­to-cell rati o o f filamentous fungi makes them beller degraders under certain niches li ke contaminated soil s. Fungi have been shown to e\'en solubili ze partiall y coal, a highly polymeric sub ·tance more complex th an lignin . There is no doubt th at fungi can be harnessed more in environmental bioremediation work in future.

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