Use of laccase in pulp and paper industry

Laccase, through its versatile mode of action, has the potential to revolutionize the pulping and paper making industry. It not only plays a role in the delignification and brightening of the pulp but has also been described for the removal of the lipophilic extractives responsible for pitch deposition from both wood and nonwood paper pulps. Laccases are capable of improving physical, chemical, as well as mechanical properties of pulp either by forming reactive radicals with lignin or by functionalizing lignocellulosic fibers. Laccases can also target the colored and toxic compounds released as effluents from various industries and render them nontoxic through its polymerization and depolymerization reactions. This article reviews the use of both fungal and bacterial laccases in improving pulp properties and bioremediation of pulp and paper mill effluents.     

Colour and AOX removal from pulping effluents by algae

A mixed culture of algae was used to treat pulping mill effluent in terms of removing both colour and adsorbably organic halides (AOX). The removal of AOX from pulping effluent increased with increasing initial colour value of the effluent. However, for the total mill effluent (composed of both pulping and bleaching effluents), AOX removal was found to be independent of initial colour value, and was around 70%. Up to 80% removal of colour from pulping effluent was achieved within 30 days under continuous lighting conditions. It was found that algae reduced the colour of pulping effluent of relatively low initial colour more efficiently than that of high initial colour. Under simulated field lighting conditions, up to 60% colour removal from pulping effluent was observed after 60 days of exposure, whereas for the total mill effluent it was up to 64% after 45 days. Total organic carbon and lignin (UVA₂₈₀) were also removed to a significant extent, suggesting that the mechanism of colour removal might not be transformation of the coloured lignin molecules to non-coloured ones. Analysis of alkaline extraction of the algal biomass and material balance findings indicated that the main colour removal mechanism was metabolism rather than adsorption. The experimental results were also analysed using multiple regression techniques and a mathematical model was developed to express the removal of colour from pulping effluents in terms of initial colour value, exposure time and lighting periods as well as interactions between these variables. Received: 12 January 1999 / Revision received: 25 March 1999 / Accepted: 26 March 1999     

Screening of fungal isolates and properties of Ganoderma applanatum intended for olive mill wastewater decolourization and dephenolization

AIMS: To investigate different autochthonous isolates of wood-rotting fungi for the removal of both colour and phenolic compounds from olive mill wastewaters (OMW). METHODS AND RESULTS: The isolates Bjerkandera adusta Ba-100, Fomes fomentarius Ff-106, Ganoderma applanatum Ga-20, Irpex lacteus Il-3, Trametes versicolor Tv-101 and Tv-103 were preliminarily screened for their OMW-decolourizing potential on potato dextrose agar supplemented with different OMW concentrations. A further screening of batch cultures under different agitation speeds, to test the effect of shear stress, resulted in the selection of isolate G. applanatum Ga-20. Batch cultures grown in OMW-based medium exhibited strong laccase induction and significant decrease in the values of phenols, colour and chemical oxygen demand. Concomitant onset of laccase activity and colour removal was observed, and apart from laccase, neither lignin peroxidase nor manganese-dependent peroxidase activities were detected. Moreover, the depletion of aromatic compounds with high and low apparent molecular mass was observed by chromatographic analysis. CONCLUSIONS: Isolate G. applanatum Ga-20 exhibited interesting properties for its use in bioremediation of OMW, namely high removal of recalcitrant phenolic compounds and strong colour abatement. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the white-rot fungus G. applanatum proves to be effective for the decolourization and dephenolization of OMW.     

Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm

An investigation was conducted to explore the lignin-degrading capacity of attached-growth white-rot fungi. Five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22, grown on a porous plastic media, were individually used to treat black liquor from a pulp and paper mill. Over 71% of lignin and 48% of chemical oxygen demand (COD) were removed from the wastewater. Several factors, including pH, concentrations of carbon, nitrogen and trace elements in wastewater, all had significant effects on the degradation of lignin and the removal of COD. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity for lignin degradation at pH 9.0-11.0. The addition of 1 g l-1 glucose and 0.2 g l-1 ammonium tartrate was beneficial for the degradation of lignin by the white-rot fungi studied.     

Biodecolorization screening of synthetic dyes by four white-rot fungi in a solid medium: possible role of siderophores

AIMS: Four selected fungi were screened for their ability to decolourize a textile effluent and commercial reactive dyes in a solid medium. METHODS AND RESULTS: Ligninolytic enzymes activities (lignin peroxidase, manganese peroxidase and laccase) and siderophores presence were monitored in decolourized plates. RESULTS: The results showed low lignin peroxidase activity and no manganese peroxidase activity was detected for all fungi. Laccase activity was observed in Reactive Blue 19 decolourized plates by Trametes versicolor and Trametes villosa. Siderophores presence was observed in Trametes versicolor, Phanerochaete chrysosporium and Lentinus edodes decolourized plates. CONCLUSION: Lentinus edodes displayed the greatest decolourization ability both in terms of extent and rapidity of decolourization. SIGNIFICANCE AND IMPACT OF THE STUDY: The transformation observed for dyes open the possibility to study siderophores to treat dyes and textile effluents.     

Colour removal of anaerobically treated pulp and paper mill effluent by microorganisms in two steps bioreactor

In the present study sequential anaerobic and aerobic treatment in two steps bioreactor was performed for removal of colour in the pulp and paper mill effluent. In anaerobic treatment, colour (70%), lignin (25%), COD (42%), AOX (15%) and phenol (39%) were reduced in 15 days. The anaerobically treated effluent was separately applied in bioreactor in presence of fungal strain, Paecilomyces sp., and bacterial strain, Microbrevis luteum. Data of study indicated reduction in colour (95%), AOX (67%), lignin (86%), COD (88%) and phenol (63%) by Paecilomyces sp. where as M. luteum showed removal in colour (76%), lignin (69%), COD (75%) AOX (82%) and phenol (93%) by day third when 7 days anaerobically treated effluent was further treated by aerobic microorganisms. Change in pH of the effluent, and increase in biomass of microorganisms substantiated results of the study, which was concomitant to the treatment method.     

Biotreatment of pulp mill bleachery effluents with the coelomycetous fungusStagonospora gigaspora

Summary The coelomyceteStagonospora gigaspora degrades lignin derivatives within pulp mill bleachery effluents. Besides dechlorination, 90% of the color was removed from CEH bleachery effluents. Lignin derivatives in the effluents of the EOP bleaching stages revealed more persistent against fungal attack. Toxicity of both effluents was diminished significantly byS. gigaspora.     

Bioremediation of pulp and paper mill effluent by a novel fungal consortium isolated from polluted soil

Two basidiomycetous fungi (Merulius aureus syn. Phlebia sp. and an unidentified genus) and a deuteromycetous fungus (Fusarium sambucinum Fuckel MTCC 3788) were isolated from soils affected with effluents of a pulp and paper mill over several years. These isolates were immobilized on nylon mesh and the consortium was used for bioremediation of pulp and paper mill effluent in a continuously aerated bench-top bioreactor. The treatment resulted in the reduction of color, lignin and COD of the effluent in the order of 78.6%, 79.0% and 89.4% in 4 days. A major part of reductions in these parameters occurred within first 24h of the treatment, which was also characterized by a steep decline in the pH of the effluent. During this period, total dissolved solids, electrical conductivity and salinity of the effluent also registered marked decline. It is pertinent to note that this is the first report of bioremediation of pulp and paper mill effluent by an immobilized fungal consortium.     

Decolorization of phenolic effluents by soluble and immobilized phenol oxidases

Summary Colour removal from phenplic industrial effluents by phenol oxidase enzymes and white-rot fungi was compared. Soluble laccase and horseradish peroxidase (HRP) removed colour from pulp mill (E), cotton mill hydroxide (OH) and cotton mill sulphide (S) effluents, but rapid and irreversible enzyme inactivation took place. Entrapment of laccase in alginate beads improved decolorization by factors of 3.5 (OH) and 2 (E); entrapment of HRP improved decolorization by 36 (OH), 20 (E) and 9 (S). Beads were unsuitable for continuous use because the enzymes were rapidly released into solution. Co-polymerization of laccase or HRP with L-tyrosine gave insoluble polymers with enzyme activity. Entrapment of the co-polymers in gel beads further increased the efficiency of decolorization of E by 28 (laccase) and by 132 (HRP) compared with soluble enzymes. Maximum decolorization of all three effluents by batch cultures of Coriolus versicolor (70%–80% in 8 days) was greater than the maximum enzymic decolorization (48% of OH in 3 days by entrapped laccase). Soluble laccase (222 units ml^–1) precipitated 1.2 g l^–1 phenol from artificial coal conversion effluent at pH 6.0 and the rate of precipitation and enzyme inactivation was faster at pH 6.0 than at pH 8.5.Offprint requests to: R. G. Burns     

Biodegradation and Decolorization of Pulp and Paper Mill Effluent by Anaerobic and Aerobic Microorganisms in a Sequential Bioreactor

Summary Paecilomyces sp. and Pseudomonas syringae pv myricae (CSA105) were isolated from sediment core of drainage of the pulp and paper mill industry. Fungi and bacteria were applied for treatment of pulp and paper mill effluent in a two-step and three-step fixed film sequential bioreactor containing sand and gravel at the bottom of the reactor for immobilization of microbial cells. Degradation of chlorinated phenols and formation of their metabolites were determined by high performance liquid chromatography. The microbes exhibited significant reduction in colour (88.5%), lignin (79.5%), chemical oxygen demand (87.2%) and phenol (87.7%) in two-step aerobic sequential bioreactor, and colour (87.7%), lignin (76.5%), chemical oxygen demand (83.9%) and phenol (87.2%) in three-step anaerobic-aerobic sequential bioreactor.     

木质素生物降解与纸浆工业废水脱色

主要工业废水之一的纸浆工业废水中的木质素类有色物质的去除一直倍受关注。本文主要综述了木质素降解微生物、影响木质素降解的因素、木质素降解酶类及其基因工程研究和纸浆工业废水的固定化真菌法和酶法脱色。     

Decolorization of Pulp-Paper Mill Effluents by White-Rot Fungi

ABSTRACT:?

Phenolic effluents are waste products of pulp and paper, coal conversion, dying, textile, and olive oil industries. Such effluents impose coloration and toxicity problems in the receiving waters, causing serious environmental hazards. The pulp and paper mill effluent is highly colored, imparting black/brown color to the water body. The color is mainly due to lignin and its derivatives released during various stages in the paper-making process. The complex nature of such lignin compounds and their phenolic nature make them extremely resistant to microbial degradation. Conventional treatment methods such as aerated lagoons and activated sludge process are ineffective in removing color. However, physical and chemical treatment methods, including ultrafiltration, ion-exchange, and lime precipitation, are expensive and less efficient. Therefore, alternate low-cost biotreatment processes are now being considered, most of which are based on lignin-degrading fungi. Depending on the treatment process, the fungal inoculum for decolorization could be used in the form of mycelium, pellets, or in the immobilized state. The decomposition of lignin is an enzymatic process employing various ligninases being produced by the fungal species. Soluble and immobilized ligninolytic enzymes have also been employed for effluent decolorization. Therefore, the present review is an attempt to compile the scattered information on pulp-paper mill effluent decolorization employing microbes. The structure, distribution, physiology, and enzymology of lignin degradation is also briefly discussed.     

Influence of culture parameters on paper mill effluent decolourization by a white rot fungus Ganoderma lucidum

Efficacy of a white rot fungus G. lucidum for reduction of colour of paper mill effluent under various growth conditions was evaluated. G. lucidum cultured in IBME medium supported maximum colour reduction on 18th day of fungal growth. The optimization of growth parameters further improved colour reduction. The 18 day old culture at 4 g/l inoculum concentration resulted in maximum decolourization (89%) of the effluent with pH adjusted to 6.5 at 35 degrees C along with maximum reduction in biological oxygen demand and chemical oxygen demand. Relative contribution of lignin peroxidase and laccase to the decolourization of paper mill effluent by G. lucidum was also observed.     

Decolorization of Kraft effluent by free and immobilized lignin peroxidases and horseradish peroxidase

Color removal from Kraft effluent by lignin peroxidase and horseradish peroxidase was compared. Free lignin peroxidase and horseradish peroxidase removed color from kraft effluent. Immobilization of lignin peroxidase type III, lyophilized fungal culture and horseradish peroxidase on CNBr-Sepharose 4B improved the decolorization by factor of 2.9, 4.5 and 2.6, respectively in 48 h. Lignin peroxidase type I was effective only in the immobilized form in decolorization. In general, the immobilized form all the studied systems exhibited an average value around of 30% polymer consumption and very little of depolymerization. Lignin peroxidases and lyophilized fungal culture were shown to have considerable potential for treating Kraft effluents.     

Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach

Four different bacterial strains were isolated from pulp and paper mill sludge in which one alkalotolerant isolate (LP1) having higher capability to remove color and lignin, was identified as Bacillus sp. by 16S RNA sequencing. Optimization of process parameters for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, % carbon, % black liquor, duration, pH, temperature, stirring and inoculum size, at two levels, applying L-8 orthogonal array were taken. Maximum color was removed at pH 8, temperature 35°C, stirring 200 rpm, sucrose (2.5%), 48 h, 5% (w/v) inoculum size and 10% black liquor. After optimization 2-fold increase in color and lignin removal from 25–69% and 28–53%, respectively, indicated significance of Taguchi approach in decolorization and delignification of lignin in pulp and paper mill effluent. Enzymes involved in the process of decolorization of effluent were found to be xylanase (54 U/ml) and manganese peroxidase (28 U/ml). Treated effluent was also evaluated for toxicity by Comet assay using Saccharomyces cerevisiae MTCC 36 as model organism, which indicated 58% reduction after treatment by bacterium.     

Correlation of brightening with cumulative enzyme activity related to lignin biodegradation during biobleaching of kraft pulp by white rot fungi in the solid-state fermentation system.

Biobleaching of hardwood unbleached kraft pulp (UKP) by Phanerochaete chrysosporium and Trametes versicolor was studied in the solid-state fermentation system with different culture media. In this fermentation system with low-nitrogen and high-carbon culture medium, pulp brightness increased by 15 and 30 points after 5 days of treatment with T. versicolor and P. chrysosporium, respectively, and the pulp kappa number decreased with increasing brightness. A comparison of manganese peroxidase (MnP), lignin peroxidase (LiP), and laccase activities assayed by using fungus-treated pulp and the filtrate after homogenizing the fungus-treated pulp in buffer solution indicated that enzymes secreted from fungi were adsorbed onto the UKP and that assays of these enzyme activities should be carried out with the treated pulp. Time course studies of brightness increase and MnP activity during treatment with P. chrysosporium suggested that it was difficult to correlate them on the basis of data obtained on a certain day of incubation, because the MnP activity fluctuated dramatically during the treatment time. When brightness increase and cumulative MnP, LiP, and laccase activities were determined, a linear relationship between brightness increase and cumulative MnP activity was found in the solid-state fermentation system with both P. chrysosporium and T. versicolor. This result suggests that MnP is involved in brightening of UKP by white rot fungi.     

Involvement of lignin peroxidase in the decolourization of black olive mill wastewaters by Geotrichum candidum

AIM: Decolourization of black olive mill wastewaters (OMW) by depolymerization of phenolic compounds by Geotrichum candidum. METHODS AND RESULTS: Our results show that G. candidum is able to grow on black OMW supplemented with carbon source and nitrogen. The Geotrichum growth decreased the pH and induced a 49% of colour removal when the black OMW was supplemented with glycerol and diammonium tartrate (20 mm ammonium). An improvement of 10% of colour removal was observed when the culture was supplemented with veratryl alcohol. The decolourization was inhibited with glutamate as nitrogen source. CONCLUSION: Our results suggest the potential use of G. candidum in black OMW decolourization and support the concept that lignin peroxidase (LiP) of G. candidum is involved in the depolymerization of phenolic compounds. Significance and Impact of the Study: This is the first report of LiP production by G. candidum on OMW.     

Biogas production from water hyacinth and channel grass used for phytoremediation of industrial effluents

The paper reports on the biogas production from water hyacinth (Eichhornia crassipes) and channel grass (Vallisneria spiralis) employed separately for phytoremediation of lignin and metal-rich pulp and paper mill and highly acidic distillery effluents. These plants eventually grow well in diluted effluent up to 40% (i.e., 2.5-times dilution with deionized water) and often take up metals and toxic materials from wastewater for their metabolic use. Slurry of the two plants used for phytoremediation produced significantly more biogas than that produced by the plants grown in deionized water; the effect being more marked with plants used for phytoremediation of 20% pulp and paper mill effluent. Biogas production from channel grass was relatively greater and quicker (maximum in 6-9 days) than that from water hyacinth (in 9-12 days). Such variation in biogas production by the two macrophytes has been correlated with the changes in C, N and C/N ratio of their slurry brought by phytoremediation.     

Characterization of a lignin peroxidase gene from the white-rot fungus Trametes versicolor.

A genomic library of the white-rot fungus Trametes versicolor has been constructed and a gene coding for a lignin peroxidase has been isolated and sequenced. The gene, which contains 6 introns, encodes a protein of 346 amino acid residues, preceded by a tentative 26-residue signal peptide. The deduced amino-terminal sequence agrees with the amino-terminal end of a lignin peroxidase isozyme previously isolated from carbon-limited cultures of T versicolor.