Industrial Biotechnology Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan
Received November 28, 2011; Accepted January 20, 2012; Published January 22, 2012
Citation: Asgher M, Jamil F, IqbalT HMN (2012) Bioremediation Potential of Mixed White Rot Culture of Pleurotus Ostreatus IBL-02 and Coriolus Versicolor IBL-04 for Textile Industry Wastewater. J Bioremed Biodegrad S1:007 doi: 10.4172/2155-6199.S1-007
Copyright: © 2012 Asgher M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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In continuation of our studies on single fungal cultures, this study was aimed to investigate the potential of mixed culture of two indigenous white rot fungi Pleurotus ostreatus IBL-02 and Coriolus versicolor IBL-04 to decolorize and detoxify the dye based textile effluent collected from Sitara Textile Industry (SIT), Faisalabad, Pakistan. Different pHysical and nutritional factors were optimized to enhance the efficiency of mixed culture for SIT effluent color removal. Under optimum conditions, the mixed culture completely decolorized (100%) the effluent in 2 days using glucose and urea as carbon and nitrogen sources, respectively in 30:1 ratio. The mixed culture produced all the three major liginolytic enzymes like lignin peroxidase (LiP), manganase peroxidase (MnP) and lacaase. Mediators of ligninolytic enzymes including varatryl alcohol, ABTS and MnSO4 enhanced effluent decolorization showing variable effects on LiP, MnP and laccase activities. Analysis of the treated and untreated SIT effluent showed that fungal treatment significantly reduced the Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD) of the effluent.
Industrial wastewater; Fungal mixed culture; Ligninolytic enzymes; Decolorization; Detoxification
With increasing revolution in science and technology, there is bigger demand on opting for newer chemicals which could be used in various industrial processes . Growing environmental pollution resulting from rapid industrial developments is one of the major challenges confronting the modern world . The effluent discharged from textile industries is comprised mainly of residual dyes, auxiliary chemicals, surfactants, chlorinated compounds and salts . Such colored effluents affect pHotosynthetic processes of aquatic plants, reducing oxygen levels in water, cause damage to humans by mutagenic and carcinogenic effects and, in severe cases, results in the suffocation of aquatic flora and fauna .
Conventional methods for dealing with textile wastewater consist of various combinations of biological, pHysical and chemical methods [5-7]. Unfortunately, although the conventional chemical and pHysical methods are versatile and useful, but only transfer the pollutant from one form to another , and end up in producing secondary waste products. Innovative technologies, such as bioremediation, are needed as alternatives to conventional methods to find inexpensive ways of removing dyes from large volumes of effluents.
The use of biotechnology in the processing of fibers and textiles is rapidly gaining wider recognition because of its non-toxic and ecofriendly characteristics . Among the low-cost and viable alternatives available for effluent treatment, biological processes are recognized by their capacity to reduce biochemical oxygen demand (BOD) and chemical oxygen demand (COD) . Several microorganisms, including fungi, bacteria, yeasts and algae, can decolorize and even completely mineralize azo dyes under certain environmental conditions . In recent years, the possible utilization of the biodegradative abilities of white rot fungi has shown promise.
White rot fungi do not require preconditioning to particular pollutants and, produce non-specific extracellular free radical-based enzymatic systems. They can degrade to non detectable levels or even completely eliminate a variety of xenobiotics, including synthetic dyes [1,11]. This biodegradation capacity is assumed to result from the activities of non-specific ligninolytic enzymes secreted by these fungi, including lignin peroxidases, manganese peroxidases and laccases. Extracellular enzyme system also enables white rot fungi to tolerate high concentration of pollutants . WRF are also effective on mixtures of pollutants, making them useful for treatment of industrial effluents containing more than one residual dye .
Most of the studies on the biotreatment of dyes and effluents deal mainly with decolorization by single fungal cultures with few reports on mixed cultures  and reduction in toxicity [14,15]. In our previous studies [11,16,17] the single WRF cultures have been found to show variable potential on different dyes, thereby limiting their use for treatment of effluents that contain mixtures of different dyes. The present study was focused on bioremediation of dye based industrial effluents by a mixed culture of indigenous white rot fungi Pleurotus ostreatus IBL-02 and Coriolus versicolor IBL-04.
All the experimental flasks and all analysis were run in triplicate. The data values have been presented as mean ±S.E. The SE values have been displayed as Y-error bars in figures.
Practical textile industry effluent collected onsite from Sitara Textile (SIT) industries, Faisalabad, Pakistan was used for decolorization studies. The industry was using mixtures of different types of dyes to get fabrics of different colors and shades but they did not provide any information on the types of dyes or structures of dyes being used.
Pure cultures of indigenous white rot fungi Pleurotus ostreatus IBL-02 and Coriolus versicolor IBL-04 available in the Industrial Biotechnology Laboratory, Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad were used. The fungi were raised on slants of Potato Dextrose Agar (PDA) or sporulation medium at pH 4.5 at 28±2°C. After having sufficient population of spores, the slants were refrigerated (4°C) for subsequent use in decolorization studies.
Spore inocula preparation
The inoculum medium (100 mL) was the Kirk’s basal medium  prepared in two separate labeled Erlanmayer flasks (500 mL) and autoclaved (121°C) for sterilization. Glucose solution (1%) was added through millipore filter and the medium was brought to pH 4.5 with M NaOH/M succinic acid. Cultures of Pleurotus ostreatus IBL-02 and Coriolus versicolor IBL-04 were added to the respective inoculum media and the flasks were incubated (120 rpm) at 30°C for 5 days to get a homogenous suspensions of spores (1×106-108 spores/mL).
Three sets of triplicate flasks (each flask containing 90 mL of SIT effluent and 10 mL of Kirk’s nutrient medium) were prepared for screening of Pleurotus ostreatus IBL-02, Coriolus versicolor IBL-04 and their co-culture. All the decolorization flasks were maintained at pH 4.5 and sterilized (121°C) in an autoclave for 15 min. The flasks were inoculated with 5 mL inocula of individual and mixed cultures (1:1 ratio) of both strains in laminar air flow aseptically. The inoculated flasks were incubated for 10 days at 30°C in shaking cultures at 120 rpm. Triplicate flasks were harvested after every 48 h.
Optimization of culture parameters
Based on the results of screening experiment, SIT effluent was maximally decolorized by the mixed culture of Pleurotus ostreatus IBL- 02 and Coriolus versicolor IBL-04. SIT effluent was therefore, used for optimization of different parameters for decolorization by the mixed culture. For process optimization the Classical Method was adapted; varying one parameter in an experiment and maintaining the preoptimized in subsequent study.
Effect of inoculum size
For optimization of inoculum size, SIT effluent (90 mL) containing Kirk’s basal medium (10 mL) was inoculated with varying volumes (1-5 mL) of inoculum added in 1:1 ratio for the two fungi. After inoculation, all flasks were incubated in shaking culture conditions at 30°C and pH 4.5 for 6 days.
Effect of pH
The SIT effluent was adjusted at varying pH levels viz; pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0. All flasks were inoculated with 4 mL inoculum (optimum) of mixed culture of P. ostreatus IBL-02 and C. versicolor IBL- 04 (1:1 v/v ratio).
Effect of temperature
Triplicate flasks containing SIT effluent (optimum pH 3.5) were inoculated with 4mL inoculum and shaken (120rpm) at different incubation temperatures.
Effect of carbon and nitrogen sources
Glucose, fructose, lactose, molasses and starch were used as carbon sources in the presence of yeast extract, ammonium sulpHate, urea, peptone and beef extract as nitrogen sources under Completely Randomized Design (CRD) in the effluent decolorization media using optimum inoculum size, pH and temperature.
Effect of carbon/nitrogen ratio
After selection the best combination of carbon and nitrogen source, carbon/nitrogen ratio was varied (10:1, 15:1, 20:1, 25:1, 30:1, 35:1) using different concentrations of selected carbon and nitrogen sources under pre-optimized conditions.
Effect of mediators
The effluent flasks were supplemented with 1mM solutions of different mediators (1mL) such as ABTS, veratryl alcohol, MnSO4, oxalate and glyoxalate. The flasks were sterilized, inoculated and incubated under optimum conditions.
Determination of percent effluent decolorization
Triplicate flasks were harvested after every 24 h (48 h in screening experiment) by filtration through Whatman No.1 filter paper. These filtrates were centrifuged at 5000 g for 15 min at room temperature and clear supernatants were analyzed spectropHotometrically (T60, UV/Visible, PG Instruments, UK) at λmax of each effluent in order to determine the % age decolorization of textile effluents by P. ostreatus IBL-02, C. versicolor IBL-04 and their mixed culture. The λmax value of Sitara Textile, Crescent Textile, Nishat Textile and K&N Textile effluents were 590, 515, 615 and 667 nm respectively. Percent decolorization of effluents was calculated by:(1)
where, Aini = Initial absorbance of dye before incubation, Afin = Final absorbance of dye after incubation
Study of enzyme system
The culture filtrates of each experiment were centrifuged at 5000 g and supernatants were assayed for lignin peroxidase (LiP), Mnperoxidase (MnP) and laccase to study the enzymes secreted by the mixed culture of P. ostreatus IBL-02 and C. versicolor IBL-04 for decolorization of SIT effluent. Lignin peroxidase was assayed by the method of Tien and Kirk, . The oxidation rate of veratryl alcohol to veratraldehyde was determined at 310 nm in sodium acetate buffer (pH=3) in the presence of H2O2 (Є310 =9300). LiP activity is defined as amount of LiP required to produce a unit increase in absorbance at 310 nm per mL of reaction mixture. For the determination of MnP, the method of Wariishi et al.  was followed. Assay mixture (2.6 mL) contained 1 mL of 1 mM MnSO4 and 1 mL of 50 mM sodium malonate buffer of pH 4.5 and 100 μL of culture supernatant. 500 μL of 0.1 mM H2O2 was added as an oxidizing agent. Absorbance of all samples was read after 10 minutes interval at 270 nm (Є270 =11590 M-1cm-1). Laccase activity was measured by monitoring the oxidation of 2, 2 azinobis (3-ethylbenzthiazoline)-6 sulpHonate (ABTS) , by culture supernatants at 436 nm after 10 min interval. Blanks contained 100μL of distilled water instead of culture supernatants.
Decolorized effluent quality tests
The original and maximally decolorized effluents were subjected to the analysis for the determination of Biochemical Oxygen Demand (BOD) and Chemical Oxygen Demand (COD). The test samples along with specific micro-organisms were incubated at the specific temperature for 5 days. Dissolved Oxygen (DO) was measured initially and after incubation, and BOD was computed from the difference between initial and final DO . For COD determination, the test samples were oxidized by a boiling mixture of sulpHuric acid (H2SO4) and excess of potassium dichromate (K2Cr2O7). After digestion, the unreduced K2Cr2O7 was titrated against ferrous ammonium sulpHate to determine the amount of K2Cr2O7 consumed and the oxidizable matter was calculated in terms of oxygen equivalents.
Screening of single and mixed cultures
Single and mixed cultures of P. ostreatus IBL-02, C. versicolor IBL- 04 were screened for the decolorization of SIT effluent. The cultures showed variable decolorization potential for SIT effluent (Table 1). P. ostreatus IBL-02 and C. versicolor IBL-04 showed 51.7% and 53.4% decolorization of SIT effluent, respectively. However, mixed culture of P. ostreatus IBL-02 and C. versicolor IBL-04 caused significantly (P≤0.05) higher decolorization (60.8%) of SIT effluent in 6 days. All the three enzymes (LiP, MnP, laccase) were secreted by the mixed culture but MnP activity (507.0 U/mL) was significantly (P≤0.05) as compared to LiP and laccase. In single cultures, P. ostreatus produces high activiy of laccase, followed by lignin peroxidase (LiP) and Mn-peroxidase (MnP) . C. versicolor also produces the same three enzymes but MnP is the major enzyme produced by this fungus, followed by laccase and LiP . Mixed cultures are more efficient dye decolorizers than single cultures due to the combined action of the three enzymes produced by different fungi in different ratios .
|WRF cultures||Decolorization (%)|
Table 1: Screening of single and mixed culture of white rot fungi for decolorization of SIT effluent.
Optimization of SIT effluent decolourization process by mixed culture
Mixed culture of Pleurotus ostreatus IBL-02 and C. versicolor IBL- 04 maximally decolorized the SIT effluent. Different parameters were optimized for maximum bioremediation of the effluent by mixed culture and results have been discussed under the following subheadings.
Effect of inoculum size
SIT effluent (90 mL) containing Kirk’s basal medium (10 mL) was inoculated with varying volumes of inoculum i.e. 1-5 mL. The maximum decolorization (64.21%) of the effluent was observed after 5 days in the flaks receiving with 4 mL mixture of spore suspensions of the two fungi (1:1 ratio), followed by with 5 mL (59.90%) and with 3 mL (49.41%) inocula. The enzyme profile of the flasks (Figure 1) also showed maximum enzyme activities with that at 4 mL inoculum and MnP (705.5U/mL) was the major enzyme produced. Optimum inoculum density is vital for fermentation process since over crowding of spores can cause nutrient depletion and inhibit growth and development. Radha et al.  reported that optimum level of inoculum is necessary for best enzyme production and dye decolorization by WRF
Effect of pH
Since pH has a significant impact on fungal pHysiology and the efficiency of dye decolorization, the decolorization of SIT effluent by mixed culture of P. ostreatus IBL-02 and C. versicolor IBL-04 was carried out at varying pH of the medium. The maximum effluent color loss of 75.75% after 5 days was observed at pH 3.5, followed by 69.71% at pH 4, 57.85% at pH 4.5, and 43.25% at pH 5. MnP was the major enzyme produced in optimally decolorized pH 3.5 flasks having maximum MnP activity of 937.5 U/mL. The pH optima for a variety of WRF lies in the range of 4-6 depending upon the medium composition as well as on the nature of dye structures in the decolorization medium [26-28]. Dye decolorization by WRF increases in the beginning but decreases as pH exceeds from 4-4.5 . Pleurotus ostreatus has been found to maximally decolorize the textile dyes at pH 4 . Most constructive growth pH of ligninolytic fungi is around pH 3-5 and it has been shown previously that initial pH influences decoloration ability of WRF and pH adjustment of the medium would be obligatory  (Figure 2).
Effect of temperature
The triplicate effluent flasks were inoculated with 4 mL inoculum and incubated at 25, 30, 35, and 40°C for 5 days. The maximum decolorization (79.04%) was observed after 4 days in the medium incubated at 35°C followed by 30°C (72.55%) and 25°C (56.95%). The enzyme profiles of the experimental flasks revealed that at 35°C maximum MnP activity (1158 U/mL) was observed at 35°C after 4 days, indicating the role of temperature in enzyme action (Figure 3). As maximum decolorization and enzyme production was observed at 35°C after 4 days, the subsequent experiments were run at this temperature. An optimum temperature is a crucial factor for the growth of white rot fungi and activity of ligninases enzyme. For a variety of WRF cultures optimum temperatures were found to vary between 25-37°C [28,31]. Cetin and Donmez  reported that mixed cultures approximately removed 82-98% of Reactive Red RB dye incubated at 35°C. Srikanlayanukul et al.  found that C. versicolor RC3 decolorized Orange II at optimal temperature of 30-37°C. The optimum temperature for growth and dye decolorization studies with T. versicolor have been reported as 30°C .
Effect of carbon and nitrogen sources
To select the most effective combination of carbon and nitrogen source, different carbon (1%) and nitrogen (0.2%) sources were used in the effluent decolorization media, under optimum conditions of inoculum size, pH and temperature. Maximum effluent color removal (86.67%) was shown by the combination of glucose and urea as carbon and nitrogen sources, respectively in 4 days (Table 2). Maximum MnP synthesis (1288.74 U/mL) was also shown by the combination of glucose and urea. Additional carbon sources have been reported to enhance the fungal growth and enzyme activities to get maximum dye decolorization . Glucose plays multiple roles in dye decolorization mechanism which might be: the generation of H2O2 required for extracellular peroxidase activity and/or the generation of Mn+3 complexing agents necessary for MnP activity .
|Nitrogen sources (0.2%)||Carbon sources (1.0 %)|
|Yeast extract||Enzyme Activities
|Cell dry weight (g)||0.126||0.153||0.333||0.121||0.043|
|Ammonium Sulphate||Enzyme Activities
|Cell dry weight (g)||0.132||0.114||0.215||0.124||1.039|
|Cell dry weight (g)||0.133||0.187||0.355||0.143||0.526|
|Cell dry weight (g)||0.154||0.125||0.222||0.134||1.019|
|Beef extract||Enzyme Activities
|Cell dry weight (g)||0.098||0.157||0.379||0.133||1.026|
Table 2: Effects of different combinations of carbon and nitrogen sources on SIT effluent decolorization and ligninase synthesis by the mixed fungal culture.
Effect of carbon/nitrogen ratio
Microorganisms require an optimum carbon to nitrogen ratio (C:N ratio) for synthesis of different molecules including enzyme proteins. The experiment was conducted with varying concentrations of glucose and urea to get varying C: N ratios. The best decolorization (93.45%) enzymes production was achieved with 30:1 C: N ratio in 3 days (Figure 4). C: N ratio is a critical factor for LiP and MnP and laccase synthesis and dye removal  because some WRF grow better under carbon and nitrogen limitations and others give better performance under carbon and nitrogen sufficient conditions. At the low C:N ratio, the fungi are carbon starved and do not exhibit optimum growth and formation of enzymes while at a high C:N ratio (excess carbon and nitrogen limitation), fungal cultures produce large amounts of polysaccharides .
Effect of mediators
Different organic and inorgananic compounds perform the role of mediators of enzyme catalyzed reaction mechanisms involved in decolorization of xenobiotic compounds including textile dyes. To study the effect of different mediators on SIT effluent decolorization by mixed culture of Pleurotus ostreatus IBL-02 and Coriolus versicolor IBL- 04, the flasks were supplemented with 1 mL of 1 mM solutions of ABTS, veratryl alcohol, MnSO4, oxalate and glycoxylate and incubated for three days under optimum conditions. Dye decolorization was found to be enhanced to varying extents by the addition of all redox mediators except oxalate and glycoxylate. It was noted that there was a significant time reduction and maximum enzyme activities and effluent color loss (98.47%) was achieved in only 48 h by the addition of MnSO4 (Table 3), followed by ABTS and varatryl alcohol. The rates and extents of decolorization of dyes are significantly enhanced by the presence of different types of redox mediators . MnSO4 is mediator of MnP and increase in MnP production and activity by adding MnSO4 has been reported in a number of studies . Redox potential of laccases varies with the change in laccase source and this empHasizes the need/or nature of the redox mediator for the degradation of particular dye . The natural fungal secondary metabolites varatryl alcohol acts as mediator of LiP action on lignin and recalcitrant chemical pollutants .
|Mediators 1mM (1mL)||Decolorization (%) Mean±S.E||Enzyme activity (U/mL)||Cell dry weight (g)|
Table 3: Decolorization of SIT effluent by mixed culture of P. ostreatus IBL-02 and C. versicolor IBL-04 with different mediators.
Quality evaluation of decolorized effluent
Chemical oxygen demand (COD): Both treated and untreated samples of effluent were analyzed to determine the value of COD (mg/L). COD value was significantly reduced in fungal treated samples (28.4 mg/L) in comparison with that of untreated sample (82 mg/L). COD value of treated sample fell in permissible limits i.e. 30 mg/L (ISI: 2490; 1982). Chemical Oxygen Demand (COD) value useful measure of water quality that indicates the oxygen concentration required to oxidize all carbon compounds in a solution and is commonly used as an indirect measure of the amount of organic compounds present in water. Sanghi et al.  reported that C. versicolor removed 75% COD of reactive dye Remazol Brilliant Violet. Srikanlayanukul et al.  found that after treatment of Orange II dye by Coriolus versicolor RC3, 80% color removal was achieved with significant toxicity reduction.
Biochemical oxygen demand (BOD): Biochemical (or Biological) Oxygen Demand (BOD) is a measure of how rapidly biological organisms consume oxygen in a water body. Although BOD is not an accurate quantitative test, it can be considered as an indication of the quality of a water source. BOD value of treated sample (192 mg/L) was in the range of permissible limits (upto 250 mg/L; ISI: 2490 1982. Shin  found that BOD (330.8 ppm) and COD (370.1 ppm) of textile industry effluent were reduced by treatment with Irpex lacteus. In general, most of the previous reports on the biotreatment of dyes deal mainly with decolorization, but here are only a few reports on the reduction in toxicity or on the biodegradation products or intermediates of degradation. Driessel and Christov  reported that treatment with C. versicolor rendered the effluents essentially non-toxic. Vanhulle et al.  also reported 35% toxicity reduction in industrial effluents by fungal treatment.
This study was made to explore the prospective of mixed culture of two indigenous WRF Pleurotus ostreatus IBL-02 and Coriolus versicolor IBL-04 to decolorize and detoxify the dye based textile industry effluent. It was concluded that the potential of mixed culture was significantly higher as compared to single cultures for effluent decolorization. The treatment of SIT wastewater also led to reduction in Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD). The bioremediation potential of the co.culture could be further improved by careful optimization of pHysical and nutritional parameters.
Funds for this project study were provided by Higher Education Commission, Islamabad, Pakistan. The technical and analytical help extended by Environmental Biotechnology Division, NIBGE, Faisalabad and Central High Tech Laboratory, University of Agriculture, Faisalabad is thankfully acknowledged.
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