CHAPTER II. Physico-chemical Characterization of the Textile Dye Effluent and Isolation of Lignin Degrading Fungi

Transcription

1 CHAPTER II Physico-chemical Characterization of the Textile Dye Effluent and Isolation of Lignin Degrading Fungi Physico-Chemical Characterization of Textile Dye Effluent The world s ever increasing population and progressive adoption of an industrial based lifestyle has inevitably led to an increased anthropogenic impact on the biosphere. In textile production units, opportunities exist for the release into the ecosystem of potentially hazardous compounds at various stages of operation. Color is the first contaminant to be recognized in wastewater and has to be removed before discharging into water bodies or on land. Major pollutants in textile effluents are high suspended solids, chemical oxygen demand, heat, color, acidity and other soluble substances. Conventional biotreatment methods are not effective for most of the synthetic dyestuffs due to the complex polyaromatic structure and recalcitrant nature of dyes (McKay, 1979; Pagga and Brown 1986). Aromatic amines formed as the metabolites of reductive cleavage of the azo bond under anaerobic conditions are more toxic than intact dye molecules (Weber and Wolfe, 1987) while aerobic conditions are desirable as total mineralization can be achieved (Bannat et al., 1996). Wyene et al., 2001 noted that textile effluents are highly colored and saline, contain nonbiodegradable compounds and are high in Biochemical and Chemical Oxygen Demand (BOD, COD). The unplanned intrusion has negative effect on the environment. Efficiency of any physico-chemical or biological effluent treatment greatly depends on the nature and quality of the organic compounds. In addition, characterization of the effluent is important to determine its reuse as a 36

2 safe option due to its high water consumption (Pia et al., 2003). Based on these considerations, physico-chemical characterization of the textile dye effluent had been undertaken. Materials and Methods Collection of Sample Textile dye effluent of the first stage (direct effluent without any treatment) was collected from a textile industry at Jetpur, Gujarat utilizing azo and reactive dyes. The samples were brought to the laboratory in an ice box and analyzed within six hours of collection. Physico-chemical Analyses Standard methods (APHA, 2005) were used for analyses of various physicochemical parameters of the effluent. The physico-chemical parameters examined were temperature, ph color, BOD, COD, total solids, total dissolved solids, acidity and salinity. Temperature Temperature is a measure of heat in terms of a standardized unit. It is an important ecological factor and an environmental variable, not just seasonal- as it also fluctuates on daily or even hourly bases. It is one of the important parameter as it affects other properties of wastewater. It greatly influences vital activities like metabolism, behavior, reproduction and development of microorganisms (Saxena, 1990). Temperature was noted at the site prior to its collection with a thermometer. 37

3 ph ph is a measure of the acidity or basicity of a solution and measures the concentration of hydrogen ions in water (Saxena, 1990). ph of environment affects microorganisms and microbial enzymes directly and also influences the dissociation and solubility of molecules. The wastewaters with extreme concentrations of hydrogen ions are difficult to treat by any biological means as the concentration range suitable for the existence of most microorganisms is generally ph 5-9 ( If the effluent is typically acidic (<5) or alkaline (>7.5), it needs to be neutralized. ph was measured by a ph meter (ELICO-L1-612). Color Color in the wastewater is classified into two: true and apparent color. Apparent color is the total color due to both turbidity and the color of the wastewater. True color is the color after filtration of the wastewater. Color in the effluent was determined according to the Canadian Pulp and Paper Association (CPPA) standard method (CPPA, 1974). The effluent was adjusted to ph 7.6 by 2M NaOH and centrifuged at 10,000 rpm for 20 min. The absorbance of the clear supernatant was taken at 465 nm against distilled water as blank. Absorbance values were transformed in to color units (CU) according to the equation: CU = 500 A2 / A1 Where, A1 = Absorbance at 500 CU Platinum Cobalt Standard solution A2 = Absorbance of the effluent sample Absorbance was taken in a UV-Vis Spectrophotometer (Shimadzu 1800 UV-Vis). 38

4 Biochemical oxygen demand (BOD) and Chemical oxygen demand (COD) Oxygen demand is important because organic compounds are generally unstable and may be oxidized biologically and chemically to a stable inert end product. An indication of organic oxygen demand content of wastewater can be obtained by measuring the amount of oxygen required for its stabilization either as BOD or COD. BOD:COD ratio is an important factor in evaluating the extent of organic pollution. BOD:COD ratio reveals the treatability of waste water. If the ratio of BOD:COD is above 0.5, the waste water is considered to be highly biodegradable. If the ratio is less than 0.3, the waste water is deemed to undergo a chemical treatment before the routine biological treatment. Textile wastewaters exhibit low BOD:COD ratios (< 0.1) indicating non-biodegradable nature of dyes (Azbar, 2003). BOD determination is an empirical test used to determine the relative oxygen requirements of waste waters, effluents and polluted waters. The test measures the molecular oxygen utilized during specified incubation period for degradation of organic material and the oxygen used to oxidize inorganic material. COD measures the potential overall oxygen requirements of the wastewater sample including oxdizable components that are non-biodegradable and so not detected in BOD analysis. COD is used to measure the oxygen equivalent to organic material in the wastewater that can be oxidized chemically using dichromate in an acid solution. In textile dye effluent, the high molecular weight chromophores contribute little to BOD. However, these compounds are major contributors to effluent COD, color toxicity due to their inability to pass through the cell membranes. (Eriksson et al., 1985). BOD and COD were estimated as per the methods given in APHA,

5 Total Solids (TS) The most important characteristics of wastewater are its solids content. The total solid material can be characterized into non-filtrable and filtrable solids fraction. The non-filtrable fraction consists of settable and non-settable fraction and the filtrable fraction consists of total dissolved solids (TDS). TS is defined as the residue remaining after a wastewater sample has been evaporated and dried at ºC. Acidity Acidity of water is its quantitative capacity to react with a strong base to a designated ph. Acidity is a measure of an aggregate property of water and can be interpreted in terms of specific substances only when the chemical composition of the sample is known. If highly acidic effluents are released into the soil or natural water, it essentially decreases the ph, which can subsequently cause problems for the biota. Acidity was measured by potentiometric titration method as described in APHA, Salinity The content of dissolved salts in water and sediments is usually expressed as salinity ( ). Salinity is an important ecological determinant and is highly correlated with species and environmental composition. It also affects the amount of oxygen that can be dissolved. Salinity of effluent was measured by Salinity Refractometer (Atago Model S/Mill-E). 40

6 Results and Discussion Table 2.1 indicates the results of various physico-chemical characterization of the effluent sample. Table 2.1 Physico- chemical characterization of textile dye effluent Parameters Values CPCB Limits Temperature (ºC) 50 - ph Color (CU) 49,050 - BOD (ppm) COD (ppm) 80, TS (mg/l) 3753 TSS 200 TDS Acidity (mg/l) Salinity ( ) General standards by Central Pollution Control Board (CPCB) for discharge of environmental pollutants in marine coastal environments, Part A: effluents. There are numerous reports on physico-chemical characterization of textile dye effluents (Font et al., 2003; Oke et al., 2006) but the characterization of the first stage effluent is one of the rare studies that has been undertaken. Temperature of effluent was found to be 50 C. It is a very important ecological factor and an environmental variable parameter that limits the distribution of life. Generally the effluent characteristics need to be monitored properly for better environmental protection. The textile mills generally have their effluent 41

7 temperature between 35.7 C- 40 C. The result indicates that some reactions could be speeded up by the discharge of wastewater into the stream. It will also reduce the solubility of oxygen and amplify odor due to anaerobic reactions (Oke et al., 2006). The ph of effluent was found to be 3 due to the excessive use of azo dyes. The ph of the effluent being highly acidic needs careful attention during treatment. ph value of waste water has no health implication but many chemical reactions are controlled by the ph. Biological activities and some chemical treatment processes are usually restricted by ph. Waste water for biological process is narrowed in the ph range of 6 8, as highly acidic or alkaline wastewaters are undesirable because of hazardous effects and difficulties in treatment. Most of the reports state the effluent of textile dye industry to be highly alkaline, an observation contradictory to the results obtained (Azbar et al., 2003; Kdasi et al., 2004; Oke et al., 2006). Bhatt, 2008 have also reported ph of pulp and paper mill first stage effluents to be indicating its highly alkaline nature in contrast to the present observation of ph to be acidic (i.e. ph 3). Federal Environmental Protection Agency (FEPA) recommends ph value of range 6-9 for effluent to be discharged into stream, as either high or low ph will be harmful to man, aquatic animals and will disturb biological activity of stream if discharged untreated. The color units were found to be 49,050 CU. The effluent was highly colored indicating high content of different dyes. The color value of waste water is extremely ph-dependent and it invariably increases as the ph of the effluent is raised or lowered. Waste water was highly colored showing the presence of high concentrations of unused dyes. The above results are in accordance to Oke et al., 2006 showing high color of textile dye industry effluent. This high color may be 42

8 the combined results of ph, temperature and acidic conditions that do not allow the chromophore group of dye to degrade making effluent highly colored. BOD of the effluent was 3990 ppm, much higher as compared to CPCB limits indicating absence or less amount of oxygen for living organisms for utilizing organic matter. COD of the effluent was found to be 80,000 ppm, 320 times high as compared to limits set by CPCB. High levels of COD imply toxic conditions and the presence of biologically resistant organic substances. High COD concentration observed in the waste water might be due to the use of chemicals, which are organic, inorganic and diverse chemical structures that are highly oxygenic in nature. Font et al., 2003 have reported COD value of 1,80,000 ppm of effluents which are in agreement to the present results. The biodegradable COD fractions are used by organisms in the biological process with soluble material being used more rapidly than slowly biodegradable material. The values determined for all the above parameters are well above the limits set by the CPCB ( indicating that the effluent may need to be treated before their discharge into the receiving bodies. TS content of the effluent was found to be 3753 mg/l. Total dissolved solids being high may affect the operation of the treatment units. Nevertheless, the characteristics vary widely as with the raw material that is being used, especially type of dye and dyeing technique employed. Acidity of effluent was found to be 4800 mg/l. Increased acidity may be due to the use of highly acidic dyes in large amounts. The results are in contrast of Bhatt, 2008 who reported the effluent of pulp / paper mills to be highly alkaline. 43

9 Salinity of effluent was found to be 2. Salinity is an important ecological determinant as it affects the amount of oxygen that can be dissolved and is an important property of industrial waters. Salinity of effluent was very negligible as compared to the CPCB limits indicating negligible amount of salts. Isolation of Lignin Degrading Fungi The most important perceived environmental role of fungi are as decomposer organisms, plant pathogens and symbionts in the maintenance of soil structure due to their filamentous branching growth and exopolymer production. While the profound geochemical activities of bacteria receive considerable attention, especially in relation to carbon limited anaerobic environments, in aerobic environments fungi are of great importance (Kuhad et al., 1997). In relation to organic matter decomposition, most attention has probably been given to carbon and nitrogen cycles and the ability of fungi to utilize a wide spectrum of organic compounds is well known. The use of lignolytic fungi is one of the possible alternatives studied for the biodegradation of dyes. They can mineralize xenobiotics to CO2 and H2O through their highly oxidative and non-specific lignolytic system, which is also responsible for decolorization and degradation of a wide range of dyes (Fu and Viraraghavan, 2001). It is generally considered that white rots cannot degrade cellulose without prior removal of lignin. The threedimensional polyaromatic polymer lignin has a structural role in woody plants and also has a protective effect against fungal pathogens. It is the main barrier against enzymatic degradation of cellulose and it has been shown that degradation of lignocellulosic materials is slowed down by the presence of lignin, removal of which leads to accelerated cellulolysis. Fungi have evolved as chemical factories, secreting in their environment several classes of enzymes which break down polymeric constituents of dead organic matter into soluble 44

10 forms for absorption and utilization as sources of carbon and energy. (Maheshwari, 2006). The second part of this chapter relates to isolation of lignin degrading fungi, from various environmental sources and the effluent of textile dye industry. Materials and Methods Collection of samples The samples were collected from various environmental sources as forest soil (rich in wood biomass), humus, dead / decayed wood, textile dye effluent and effluent contaminated soils. The forest soil and samples of dead / decayed wood were collected from Victoria Reserve Forest, Bhavnagar. The soil and dead/decayed wood samples were collected in sterile aluminum foils kept in polythene bags with help of sterile spatula and the effluent samples in sterile glass bottles. The samples were brought to the laboratory in an icebox and inoculated within 6h of collection. Isolation of lignolytic fungi The following methods were used for isolation of lignin degrading fungi 1. The effluent samples were serially diluted from ml from each dilution was inoculated into Potato Dextrose Agar (PDA) (Anonymous, 1968) medium containing streptomycin (110 µg/ml) to inhibit bacterial growth and the plates were incubated at 30ºC. 3 4 days old cultures were purified by repeated sub culturing and stored at 4ºC until used. 2. Various dilutions of effluent samples (1:20, 1:50 and 1:100) were prepared in sterile Potato Dextrose Broth (PDB) containing streptomycin (110 µg/ml) 45

11 and incubated at 30ºC. At every fourth day, up to fifteen days, 1 ml of sample was withdrawn aseptically, from each dilution, diluted serially, plated on PDA plate and incubated at 30ºC 3. 5 g of soil and humus samples were taken in sterile distilled water (50 ml) and shaken on an environmental shaker (New Brunswick Excella R) at 200 rpm for 15 min at 30ºC. The samples were serially diluted upto Aliquots from each dilution were inoculated on PDA medium containing streptomycin (110 µg/ml). 4. To 1, 2 and 3 ml of sterilized effluent sample were added 99, 98 and 97 ml of sterilized PDB respectively in 250 ml Erlenmeyer flasks. 1 g of soil sample was added to each flask and incubated at 30ºC in static condition. This method allows selective isolation of soil fungi that would adapt to different concentrations of the effluent. Screening of fungi for lignolytic ability Plate assays The isolates obtained by above methods were screened for their lignolytic and decolorization abilities using plate assays as mentioned below. 1. Sundaman and Näse Plate Assay (Sundaman and Näse, 1972) Four days old cultures obtained as above were inoculated into media (Nagarathanama and Bajpai, 1999) containing (g/l) glucose 1, calcium chloride 1.5, magnesium sulphate 2, potassium dihydrogenphosphate 1.5, ammonium chloride 0.15, sodium lignosulphonate 0.25, agar 15, ph 4.5 and incubated at 30ºC. The mycelial mats after 4-8 days of growth were scrapped off and the plates were flooded with 10 ml freshly prepared 1% ferric chloride potassium ferricyanide reagent. The transformation of lignosulphonate is indicated by the formation of clear zones beneath and / or around the mycelia mats while, the 46

12 presence of untransformed lignosulphonate is marked with green coloration on the agar surface. 2. Bavendum Reaction (Kasinath, 2002) Four days old cultures obtained as above were inoculated into Sabaroud s Dextrose Agar (SDA) containing (g/l) peptone 10, dextrose 40, agar 15, o dianisidine 0.1, ph 5.6. Positive reaction is indicated by the formation of dark brown zone metabolizing dye o dianisidine, surrounding the fungal mycelia. 3. Solid State Decolorization Studies ( Novotny et al., 2001) Various concentrations of dye effluent such as 1:10, 1:20 and 1:50 were added to PDA and Yeast Extract Agar (YEA) media and inoculated with the isolates that showed positive reaction in Sundaman and Näse plate assay and Bavendum reaction. The plates were incubated at room temperature and observed for decolorization of effluent. Formation of clear halos, change in color from dark blue green to pink yellow indicates transformation of the dye and decolorization of the effluent. Results and Discussion A total of fifty fungi (designated as AJ 1 to AJ 50) had been isolated from various environmental samples, soils contaminated with effluents and effluents from textile dye industry (Table 2.2). From these, thirty fungi had been isolated from dead/decayed wood samples of the Victoria Reserve Forest. Among these, 11 belonged to Ascomycota (6 Aspergillus, 2 Fusarium, 2 Trichoderma, 1 Pencilium), 6 belonged to Zygomycota (4 Mucor, 2 Rhizopus), 1 belonged to Mitosporic fungi and the rest 12 remained unidentified. From soil and humus sample, total 10 fungi had been isolated. Among them, 2 belonged to Ascomycota (2 Aspergillus), 1 to Zygomycota (Mucor), 2 belonged to Mitosporic fungi and 5 remained unidentified. 47

13 From the soil samples contaminated with effluents, total 5 fungi were isolated from which 4 belonged to Ascomycota (2 Aspergillus, 2 Penicillium) and 1 belonged to Zygomycota (Rhizopus). From the textile dye effluent, a total 5 fungi had been isolated from which 3 belonged to Ascomycota (2 Aspergillus, 1 Penicillium) and 2 belonged to Zygomycota (1 Rhizopus, 1 Mucor). The isolates were identified based on their morphological characterization. As identification is complicated by the fact that fungal life cycles in the laboratory and in the environment are quite different, it can be one of the reasons why a majority of fungi remained unidentified (Domsch and Gams, 1972). Thus, maximum numbers of isolates were obtained from dead and decayed wood samples, suggesting forest ecosystem to be a rich source of lignolytic fungi. Lignin, next to cellulose is the second most abundant compound in plant biomass and is also known for microbial degradation because of its high molecular weight and presence of various biologically stable linkages. Fungi are found to be dominant in most forest soils because of low ph value, an observation in accordance to Yang et al., 2011 that supports flourished growth of fungi as main decay producers. Fungi belonging to Ascomycota were found to be dominant among all the environmental samples. The results obtained are in agreement to Moorthi, 2007 and Bhatt, 2008 suggesting the dominance of Ascomycota in forest ecosystem. The results are also in accordance to Yang et al., 2011 showing that fungi grow rapidly and have high antibacterial ability in natural environments. Fungi vary in their tolerance to temperature and humidity extremes. Certain fungi can survive in harsh conditions such as snow-covered soils of Antartica, refrigerators, highly acidic conditions, solvents and even petroleum products explaining their tolerance to extreme conditions, diversty and biodegradation efficacy (Yang et al., 2011). 48

14 A number of methods have been used to isolate lignolytic fungi. Fungi are nutritionally so diverse that no one medium can isolate all of them. The spatial distribution of microorganisms in soil and in nature needs to overcome the wide range of microbe - soil particle interactions that are the main limitations to representative and quantitative soil sampling for isolation of fungi (Satish et al., 2007). The results are also in agreement to Kuhad et al., 1997 who have reported the importance of Ascomycota, Zygomycota and Mitosporic fungi in lignolysis. Relative abundance of Ascomycota and Zygomycota (Table 2.2) is supported by the observations of Thorn et al., 1996 who showed that these groups overgrow on media for isolating lignolytic fungi, compared to slow growers as Basidiomycota. Table 2.2 Isolation of lignolytic fungi from various sources Sr No Sample source Ascomycota Zygomycota Mitosporic Fungi Unidentified Total Isolates 1 Dead / Decayed wood Forest soils / humus Soil contaminated with effluents Effluents from textile dye industry Total

15 The isolates were screened for their lignolytic ability and also for their decolorization ability by plate assay as Sundaman and Näse Plate assay, Bavendum reaction and Solid state decolorization ability. Among 50 isolates, 30 isolates as shown in (A) Sundaman and Näse Plate Assay (B) Bavendum Reaction (C) Solid State Decolorization Fig. 2.1 A, B and C Plate assays for lignolytic ability using representative fungi AJ 18 50

16 Table 2.3 showed clear zone in Sundaman and Näse Plate Assay within 48h indicating the depolymerization of lignosulphonate which had been added to the media as a synthetic lignin source (Fig.2.1 A). These thirty isolates also showed a positive Bavendum reaction by the formation of dark brown zone around the fungal colony on the plates containing o-dianisidine within 48h (Fig.2.1 B). The isolates were further examined for their decolorization ability in Solid state decolorization studies. The isolates showed pink- yellow to clear transparent halo indicating complete decolorization of the dye effluent (Fig. 2.1 C). Color of various shades had been observed on plate assays which might be due to varied efficiencies of the isolates to decolorize the effluent. Similar observations have been reported by Cripps et al., 1990 and Spardaro et al., 1992 using white rot fungi P. chrysosporium wherein rapid dye decolorization had been observed within 3 days with the pre grown culture, where as the present results indicate decolorization within 48h indicating the efficiency of the isolates in decolorization of textile dye effluent. 51

17 Table 2.3 Screening fungi for their lignolytic and decolorization abilities Sr No Isolates Sundaman and Näse Plate Assay Bavendum Reaction Solid State Decolorization 1 AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ AJ highly positive, + weakly positive, - negative 52