Isolation of fungi and optimization of process parameters for decolorization of distillery mill effluent

Five different fungi isolated from distillery mill site in which two isolates (DF3 and DF4) had higher capabilities to remove color were identified as Emericella nidulans var. lata and Neurospora intermedia, respectively. Optimization of process parameter for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, %carbon, %nitrogen, duration, pH, temperature, stirring and inoculum size, at two levels applying L-8 orthogonal array were taken for both fungi. Maximum color was removed at pH 3, temperature 30°C, stirring 125 rpm, dextrose (0.05%) and sodium nitrate (0.025%) by both fungi. After optimization, there was two-fold increase in color removal from 38 to 62% (DF3) and 31 to 64% (DF4) indicating significance of Taguchi approach in decolorization of distillery mill effluent. The mechanism for decolorization was determined by enzyme analysis, laccase and glucose oxidase, and indicated significant activity in DF3 as compared to DF4.     

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.     

Bacterial decolorization of black liquor in axenic and mixed condition and characterization of metabolites

The pulping byproducts (black liquor) cause serious environmental problem due to its high pollution load. In order to search the degradability of black liquor, the potential bacterial strains Citrobacter freundii (FJ581026) and Citrobacter sp. (FJ581023) were applied in axenic and mixed condition. Results revealed that the mixed bacterial culture are more effective than axenic condition and can reduce 82% COD, 79% AOX, 79% color and 60% lignin after 144 h of incubation period. Additionally, the optimum activity of lignin degrading enzyme was noted at 96 h and characterized as manganese peroxidase (MnP) by SDS–PAGE analysis. Further, the HPLC analysis of control and bacterial degraded sample has shown the reduction as well as shifting of peaks compared to control indicating the degradation as well as transformation of compounds of black liquor. The comparative GC–MS analysis of control and degraded black liquor revealed that along with lignin fragment some chlorophenolic compounds 2,4,6-trichlorophenol, 2,3,4,5-tetrachlorophenol and pentachlorophenol were detected in black liquor degraded by axenic culture whereas these chlorophenolic compounds were completely absent in black liquor degraded by mixed bacterial culture. These chlorophenol inhibit the oxidative degradation which seems a major reason behind the low degradability of axenic degradation compared to mixed culture. The innovation of this aerobic treatment of alkaline black liquor opens additional possibilities for the better treatment of black liquor along with its metabolic product.     

Isolation and characterization of distillery spent wash color reducing bacteria and process optimization by Taguchi approach

The ability of five different types of bacterial strains isolated from a distillery mill site was analyzed for decolorization of distillery spent wash. 16S rDNA based denaturing gradient gel electrophoresis (DGGE) and amplified random DNA restriction analysis (ARDRA) were used to characterize the bacterial strains. One of the isolates had higher capability to reduce color (21%) and chemical oxygen demand (COD) (30%) was finally identified by 16S rDNA sequence analysis as Bacillus sp. Different parameters such as pH, temperature, aeration,% carbon,% nitrogen, inoculum size and incubation time were optimized by the Taguchi approach to achieve maximum decolorization of distillery spent wash by the Bacillus sp. Reduction in color (85%) and COD (90%) was observed within 12 h after optimization by the Taguchi method. The significant factor in the optimization process was duration followed by inoculum size to attain maximum color reduction. The Taguchi approach proved to be a reliable tool in optimizing culture conditions and analyzing interaction effects of process parameters in achieving the best possible combination for maximum decolorization of the distillery spent wash.     

白腐真菌处理灰法造纸黑液废水的研究

研究了不同白腐真菌菌株对灰法造黑液废水的处理,考察了黑液废水浓度和碳氮源添加量对黑液脱色及COD去除率的影响。研究表明,变色栓菌（Trametes verscolor)对黑液废水的处理效果最好,其COD去除率为64．25％,脱色率为47．31％,用自选的白腐真菌AH28－2菌株处理未经稀释的黑液废水,分别添加0．2％纤维二维糖和0．02％天冬酰胺,COD去除率达62．45％和68．60％,研究发现锰过氧化物酶（MnP)和木素过氧化物酶（LiP)对COD去除率有直接影响,MnP/LiP酶活力值越高,处理效果越好。     

Decolorization of practical textile industry effluents by white rot fungus Coriolus versicolor IBL-04

Textile industry discharges a vast amount of unused synthetic dyes in effluents. The discharge of these effluents into rivers and lakes leads to a reduction in sunlight penetration in natural water bodies, which, in turn, decreases both photosynthetic activity and dissolved oxygen concentration rendering it toxic to living beings. This paper describes the decolorization potential of a local white rot fungus, Coriolus versicolor IBL-04 for practical industrial effluents collected from five different textile industries of Faisalabad, Pakistan. Screening of C. versicolor IBL-04 on five effluents showed best decolorization results (36.3%) for Arzoo Textile Industry (ART) effluent in 6 days followed by Crescent Textile Industry (CRT), Itmad Textile Industry (ITT), Megna Textile Industry (MGT) and Ayesha Textile Industry (AST) effluents. Optimization of different process parameters for ART effluent decolorization by C. versicolor IBL-04 showed that manganese peroxidase (MnP) (486 U/mL) was the lignolytic enzyme present in the culture filtrates with undetectable lignin peroxidase (LiP) and laccase. The MnP synthesis and effluent decolorization could be enhanced to 725 U/mL and 84.4%, respectively, with a significant time reduction to 3 days by optimizing pH and temperature and using 1% starch as a supplementary carbon source.     

Repeated batch process for biological treatment of black liquor using brown-rot basidiomycete <Emphasis Type="Italic">Fomitopsis</Emphasis> sp. IMER2

A repeated batch operation is developed for the treatment of alkaline pulp black liquor, through a process of biological acidification precipitation of lignin using brown rot fungus Fomitopsis sp. IMER2. The results showed that COD and color removal of black liquor was dependent on the biomass concentration, pH decrease and initial COD. Based on these results, the repeated batch process was successfully carried out 12 times over 36 days in an air bubble column bioreactor. The average reduction of COD and color was approximately 40% and 70%, respectively.     

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.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

Decolorization of Direct Red 28 by mixed bacterial culture in an up-flow immobilized bioreactor

Aerobic mixed bacterial culture comprised of five isolates (Bacillus vallismortis, B. pumilus, B. cereus, B. subtilis and B. megaterium) identified by 16srDNA analysis was developed from wastewater samples from the aeration tank of an effluent treatment plant of a textile and dyeing industry and evaluated for its ability to decolorize azo dye Direct Red 28 in an up-flow immobilized packed bed bioreactor using marble chips as support matrix. The bioreactor was operated under two parameters: an aeration rate of 0.4 and 0.6 mmol/min at a flow rate of 60, 90 and 120 ml/h, respectively. At a constant aeration rate of 0.4 mmol/min and with flow rates of 60, 90 and 120 ml/h, optimum decolorization of 91, 75 and 72% was observed, while at an aeration rate of 0.6 mmol/min and flow rates of 60, 90 and 120 ml/h, optimum decolorization of 93, 78 and 72% was observed over 10 days. The study concluded that across the two aeration rates and the respective flow rates, the higher aeration rate of 0.6 mmol/min along with a flow rate of 60 ml/h was best suited to decolorize Direct Red 28 in the packed bed bioreactor. Spectral changes of the input and output of the bioreactor by UV–visible spectroscopy indicated decolorization of the dye solution by degradation in addition to the visual observation of the biosorption process.     

Exploring the antioxidant potential of lignin isolated from black liquor of oil palm waste

This study was conducted to evaluate the potential antioxidant activity of lignin obtained from black liquor, a hazardous waste product generated during the extraction of palm oil. Antioxidant potential of the extracted lignin was evaluated by dissolving the extracted samples in 2 different solvent systems, namely, 2-methoxy ethanol and DMSO. Results revealed high percent inhibition of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in the lignin sample dissolved in 2-methoxy ethanol over DMSO (concentration range of 1–100 μg/ml). Lignin extracted in 2-methoxy ethanol exhibited higher inhibition percentage (at 50 μg/ml, 84.2%), whereas a concentration of 100 μg/ml was found to be effective in the case of the DMSO solvent (69.8%). Fourier transform infrared (FTIR) spectrometry revealed that the functional groups from the extracted lignin and commercial lignin were highly similar, indicating the purity of the lignin extracted from black liquor. These results provide a strong basis for further applications of lignin in the food industry and also illustrate an eco-friendly approach to utilize oil palm black liquor. To cite this article: R. Bhat et al., C. R. Biologies 332 (2009).     

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。     

Effect ofStreptomyces viridosporus T7A on kraft lignin

Summary The ability of the lignino-cellulolytic actinomyceteStreptomyces viridosporus T7A to attack purified fractions of kraft lignin was examined. In the presence of 0.3% yeast extract, high-molecular weight kraft lignin (MW>3000, ether-insoluble fraction) does not affect growth of this microorganism significantly, whereas low-molecular weight kraft lignin (MW<3000, ether-soluble fraction) inhibits its development. Accordingly, average molecular weight of the ether-insoluble fraction after bacterial growth remained unaltered, as measured by Sephadex G-50 gel permeation chromatography. Slight modifications were detected by high performance liquid chromatography in the ether-soluble fraction after incubation with the microorganism.S. viridosporus T7A partially decolorized Remazol Brilliant Blue R during growth on wheat lignocellulose. However, decolorization of either fraction of kraft lignin was not observed. These results suggest that the filamentous bacteriumS. viridosporus T7A is not suitable for pulp mill effluent treatment.     

Depolymerization and decolorization of kraft lignin by bacterium Comamonas sp. B-9

There is no commercial or industrial-scale process for the remediation of black liquor using microorganisms to date. One of the most important causes is that most microorganisms are not able to use lignin as their principal metabolic carbon or energy source. The bacterial strain Comamonas sp. B-9 has shown remarkable ability to degrade kraft lignin and decolorize black liquor using lignin as its principal metabolic carbon and energy source. This report looks at the depolymerization and decolorization of kraft lignin by Comamonas sp. B-9. The degradation, decolorization, and total carbon removal reached 45, 54, and 47.3 %, respectively, after 7 days treatment. Comamonas sp. B-9 was capable of depolymerizing kraft lignin effectively as analyzed by gel permeation chromatography and decolorization via degrading benzene ring structures as shown using Fourier transform infrared spectroscopy analysis.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.     

A Constructed Alkaline Consortium and Its Dynamics in Treating Alkaline Black Liquor with Very High Pollution Load

Background

Paper pulp wastewater resulting from alkaline extraction of wheat straw, known as black liquor, is very difficult to be treated and causes serious environmental problems due to its high pH value and chemical oxygen demand (COD) pollution load. Lignin, semicellulose and cellulose are the main contributors to the high COD values in black liquor. Very few microorganisms can survive in such harsh environments of the alkaline wheat straw black liquor. A naturally developed microbial community was found accidentally in a black liquor storing pool in a paper pulp mill of China. The community was effective in pH decreasing, color and COD removing from the high alkaline and high COD black liquor.

Findings

Thirty-eight strains of bacteria were isolated from the black liquor storing pool, and were grouped as eleven operational taxonomy units (OTUs) using random amplified polymorphic DNA-PCR profiles (RAPD). Eleven representative strains of each OTU, which were identified as genera of Halomonas and Bacillus, were used to construct a consortium to treat black liquor with a high pH value of 11.0 and very high COD pollution load of 142,600 mg l⁻¹. After treatment by the constructed consortium, about 35.4% of color and 39,000 mg l⁻¹ (27.3%) COD_cr were removed and the pH decreased to 7.8. 16S rRNA gene polymerase chain reaction denaturant gradient gel electrophoresis (PCR-DGGE) and gas chromatography/mass spectrometry (GC/MS) analysis suggested a two-stage treatment mechanism to elucidate the interspecies collaboration: Halomonas isolates were important in the first stage to produce organic acids that contributed to the pH decline, while Bacillus isolates were involved in the degradation of lignin derivatives in the second stage under lower pH conditions.

Conclusions/Significance

Tolerance to the high alkaline environment and good controllability of the simple consortium suggested that the constructed consortium has good potential for black liquor treatment. Facilitating the treatment process by the constructed consortium would provide a promising opportunity to reduce the pollution, as well as to save forest resources and add value to a waste product.     

Dephenolization and decolorization of olive mill wastewater through sequential batch and co-culture applications

In a previous work it was reported adapted Trametes versicolor FPRL 28A INI culture was used to treat undiluted olive mill wastewater (OMW) without addition of any nutrients with significant amount of total phenolics were removed. However, decolorization was not so pronounced. Therefore, the aim of this study is to enhance the efficiency of dephenolization and decolorization of the primary treatment with adapted Trametes versicolor, incorporating a secondary biological treatment step using different microorganisms with sequential batch and co-culture applications. Through sequential batch applications Funalia trogii ATCC 20080 was found to have a higher potential in terms of total phenolics removal and decolorization amongst the tested organisms and better results were obtained from sequential batch applications as compared to co-culture experiments. In sequential batch applications, up to 91% total phenolics were removed and 64% decolorization was achieved after 24 days with 20% (v/v) inoculation rate of F. trogii when malt extract broth was used in inoculum preparation. In addition, significant accumulation of laccase (2019 ± 121.13Ul⁻¹) and manganese peroxidase (463 ± 33.89 Ul⁻¹) activities were attained. In co-culture applications highest total phenolics removal and decolorization were 78 and 39%, respectively, with non-adapted T. versicolor, whereas highest laccase and manganese peroxidase acitivities were obtained with F. trogii as 2219 Ul⁻¹  ± 176.14^. and 513 ± 4.12 Ul⁻¹, respectively.     

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.     

Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SU-EBT

The 16S rRNA sequence and biochemical characteristics revealed the isolated organism as Pseudomonas sp. SU-EBT. This strain showed 97 and 90% decolorization of a recalcitrant dye, Congo red (100 mg l⁻¹) and textile industry effluent with 50% reduction in COD within 12 and 60 h, respectively. The optimum pH and temperature for the decolorization was 8.0 and 40°C, respectively. Pseudomonas sp. SU-EBT was found to tolerate the dye concentration up to 1.0 g l⁻¹. Significant induction in the activity of intracellular laccase suggested its involvement in the decolorization of Congo red. The metabolites formed after decolorization of Congo red, such as p-dihydroxy biphenyl, 8-amino naphthol 3-sulfonic acid and 3-hydroperoxy 8-nitrosonaphthol were characterized using FTIR and GC–MS. Phytotoxicity study revealed nontoxic nature of the degradation metabolites to Sorghum bicolor, Vigna radiata, Lens culinaris and Oryza sativa plants as compared to Congo red and textile industry effluent. Pseudomonas sp. SU-EBT decolorized several individual textile dyes, dye mixtures and textile industry effluent, thus it is a useful strain for the development of effluent treatment methods in textile processing industries.     

Decolorization of synthetic melanoidins-containing wastewater by a bacterial consortium

The presence of melanoidins in molasses wastewater leads to water pollution both due to its dark brown color and its COD contents. In this study, a bacterial consortium isolated from waterfall sediment was tested for its decolorization. The identification of culturable bacteria by 16S rDNA based approach showed that the consortium composed of Klebsiella oxytoca, Serratia mercescens, Citrobacter sp. and unknown bacterium. In the context of academic study, prevention on the difficulties of providing effluent as well as its variations in compositions, several synthetic media prepared with respect to color and COD contents based on analysis of molasses wastewater, i.e., Viandox sauce (13.5% v/v), caramel (30% w/v), beet molasses wastewater (41.5% v/v) and sugarcane molasses wastewater (20% v/v) were used for decolorization using consortium with color removal 9.5, 1.13, 8.02 and 17.5%, respectively, within 2 days. However, Viandox sauce was retained for further study. The effect of initial pH and Viandox concentration on decolorization and growth of bacterial consortium were further determined. The highest decolorization of 18.3% was achieved at pH 4 after 2 day of incubation. Experiments on fresh or used medium and used or fresh bacterial cells, led to conclusion that the limitation of decolorization was due to nutritional deficiency. The effect of aeration on decolorization was also carried out in 2 L laboratory-scale suspended cell bioreactor. The maximum decolorization was 19.3% with aeration at KLa = 2.5836 h⁻¹ (0.1 vvm).