ISOLATION AND SCREENING OF BACTERIA WHICH CAN DEGRADE LINGO-CELLULOSIC COMPOUND PRESENT IN PULP AND PAPER MILL EFFLUENT

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1 CHAPTER 4 ISOLATION AND SCREENING OF BACTERIA WHICH CAN DEGRADE LINGO-CELLULOSIC COMPOUND PRESENT IN PULP AND PAPER MILL EFFLUENT 4.1. Introduction Water pollution is the sector of environmental concern. It is necessary to treat the wastewater properly before discharge and devise proper treatment technology so that properly treated wastewater is discharged in to the natural water bodies. Life in our planet is sustained in a fragile biological balance; microorganisms play an important role on nutritional chains that are an important part of this biological balance. Ability of microorganisms to transform and degrade many types of pollutants in different matrixes (soil, water, sediments and air) has been widely recognized during the last decades. Microorganisms can survive in contaminated habitat because they are metabolically capable of utilizing its resources and can occupy a suitable niche. Contaminants are often potential energy sources for microorganisms. Bioremediation, a process that exploits the catalytic abilities of living organisms to enhance the rate or extent of pollutant destruction, is an important tool in attempts to mitigate environmental contamination. The effectiveness of bioremediation is often a function of the extent to which a microbial population or consortium can be enriched and maintained in environment. The major problems in pulp and paper industry wastewater are the presence of color and COD, this is due to the presence of lignin, cellulose and hemi cellulose. Lignin is a non-repeating amorphous polymer of high-molecular weight that is highly cross-linked and optically inactive. The heterogeneous polymeric structure is composed of phenyl-propanoid units linked by an array of stable C C and hydrolysis-resistant ether linkages. These features, together with insolubility and lack of stereo-regularity, offer high resistance towards microbial degradation. But, some microorganisms having 84

2 lignolytic enzymes such as laccase, lignin peroxidise and manganese peroxidase can degrade lignin. Cellulose is a linear, repeating polymer composed of D glucose units linked by glycosidic bonds and its chemical properties are mostly determined by the degree of polymerization, which is highly dependent on the plant species (15, 000 in cotton compared to around 10, 000 in wood). Alignment of the polymers leads to the formation of crystalline sections in native cellulose, which is a semi-crystalline polymer. These crystalline sections are held together by strong hydrogen bonds and Vander Waals forces between the planes. Cellulose can be degraded by microorganisms having enzyme cellulose. Hemicellulose refers to a wide variety of hetero-polysaccharides including arabinoxylans and glucans, gluco and galacto-mannans, pectins and xyloglucans, all of which vary in their degree of polymerization, the composition of monosaccharides and glycosidic linkages, and the substitution pattern. Hemicellulose can be de degradated by microorganism which enzyme system for degradation of these compounds. Due to the complex nature of effluent of pulp and paper industry, its treatment is questionable till date. The aforesaid mentioned problem has been overcome by isolating the bacteria from different sources within the industrial premises. Isolated bacteria were screened on the basis of their degradation capability of lignocellulosic compounds. These bacteria were used for degradation of pollutant of pulp and paper mill effluent Materials and Methods Chemicals, Equipments and Glassware (i) Chemicals All chemicals used in the present study were of analytical grade. Nutrient broth, agar powder, D-glucose, Tween-80 and glycerol were obtained from Hi-Media, India. Dyes and different substrate (azure B, congo red, malachite green, methylene blue 85

3 and ramazole brilient blue) were procured from Sigma Aldrich. 2,2-azino-di-[3- ethylbenzo-thiazolin-sulphonate] (ABTS), guaiacol, vanillic acid (VA), vertryl alcholol, ferulic acid (FA), gallic acid (GA), p cresol (PC), xylose, tannic acid, kraft lignin (KL), carboxymethylcellulose (CMC) and birch wood xylan were obtained from Sigma Aldrich. The routine chemicals were procured from S.D. fine, Qualigens and Merck India Limited. Distilled water was used throughout the study. (ii) Equipments The equipment used during the present study includes laminar flow (Kartos International), electronic balance (Sartorius), ph meter (Lab India), autoclave (Yorco), incubator shaker (New Brunswick Innova 4300), centrifuge (Sorvall RC 5B Plus), spectrophotometer (Pharmaspec UV , Shimadzu), lyophilizer (Vertis), micropipettes (Eppendrof), vortex mixer and magnetic stirrer (matrix). (iii) Glassware/Plastic Ware Storage bottles, tips and petri-dishes etc., were of Tarsons make. Measuring cylinders, conical flasks, beakers, erlenmeyer flasks, Durham, bottle, test tube, petridish were procured from M/s. Borosil. All glassware and plastic ware were cleaned by washing with mild detergent followed by rinsing with tap water and finally with distilled water Preparation of Media, Reagents and Stock Solutions (i) Media (a) Nutrient Broth (NB) (ph- 7.4) Peptic digest of animal tissue Sodium chloride Yeast extract Beef extract 5.0 g/l 5.0 g/l 1.50 g/l 1.50 g/l All of the above contents were dissolved in 1000 ml of distilled water and autoclaved at 121 o C, (15 lbs pressure) for 30 min. 86

4 (b) Nutrient Agar (NA) Peptic digest of animal tissue Sodium chloride Yeast extract Beef extract Agar 5.0 g/l 5.0 g/l 1.50 g/l 1.50 g/l 20 g/l All of the above contents were dissolved in 1000 ml of distilled water and autoclaved at 121 C (15 lbs pressure) for 30 min. After autoclaving, nutrient agar was poured in petri plates and allowed to solidify for further use. (c) Minimal Salt Media (MSM) Component Working Concentration K 2 HPO M KH 2 O4 0.01M MgSO M EDTA 0.003M ZnSO 4 MnSO 4 CuSO mm 0.02 mm mm FeSO M NaMoO mm (NH 4 ) 2 SO M Dextrose 2.0% *(substrate might be changed according to the specific media designed) H 2 O 1000ml *note: different substrate used were (kraft lignin (0.3%), birch wood xylan (0.5%), carboxymethyl cellulose (1%)) 87

5 All of the above contents were dissolved in 1000 ml of distilled water and autoclaved at 115 o C, (10 lbs pressure) for 10 min. (d) Xylanse Production Media (ph 7.0) Birch wood Xylan Peptone Yeast Extract di-pottasium hydrogen phosphate (K 2 HPO 4 ) Magnesium sulphate hepta hydrate (MgSO 4.7H 2 O) Potassium chloride (KCl) Ferrous sulphate hepta hydrate (FeSO 4. 7H 2 O) Sodium Chloride (NaCl) Potassium nitrite (KNO 3 ) 10 g/l 5 g/l 5 g/l 4 g/l 1 g/l 0.2 g/l 0.02 g/l 0.5 g/l 5 g/l All of the above components were dissolved in 1000 ml of distilled water and autoclaved at 121 o C, (15 lbs pressure) for 30 min. (e) Cellulase production Media (ph 7.0) Carboxymethyl cellulose Peptone Yeast extract Di-pottasium hydrogen phosphate (K 2 HPO 4 ) Magnesium sulphate hepta hydrate (MgSO 4.7H 2 O) Ferrous sulphate hepta hydrate (FeSO 4. 7H 2 O) Sodium chloride (NaCl) 10 g/l 5 g/l 5 g/l 5 g/l 0.25 g/l 0.02 g/l 0.5 g/l All of the above components were dissolved in 1000 ml of distilled water and autoclaved at 121 o C, (15 lbs pressure) for 30 min. (f) Composition of Laccase Production Media (Broth) Dextrose Peptone 10 g/l 5 g/l 88

6 Sodium chloride (NaCl) Beef Extract Magnesium sulphate hepta hydrate (MgSO 4.7H 2 O) Calcium carbonate (CaCO 3 ) Ferrous sulphate hepta hydrate (FeSO 4.7H 2 O) Zinc sulphate hepta hydrate (ZnSO 4.7H 2 O) Manganese (II) Sulphate Monohydrate (MnSO 4.H 2 O) Cooper Suplahte (CuSO 4 ) 5 g/l 3 g/l 1 g/l 0.2 g/l 1 g/l 0.9 g/l 0.2 g/l g/l The components (except the salts) were mixed in distilled water and autoclaved for 30 min at 15lb/inch 2 pressure and 121 C temperature. The stock solutions of mineral salts were autoclaved for 10 minutes at 10lb/inch 2 pressure and 115 C temperature to prevent decomposition of salts at high pressure conditions. 2% of Agar was added in case of solidified agar media. The salts were added into the broth from their respective stock solutions after autoclaving inside the laminar hood. (g) Composition of Lignin Peroxidase Production Media (Broth) Dextrose Yeast Extract Peptone Calcium Carbonate Veratryl Alcohol 4 g/l 4 g/l 4 g/l 2 g/l 20 mm The components (except the salts) were mixed in distilled water and autoclaved for 30 min at 15lb/inch 2 pressure and 121 C temperature. The stock solutions of mineral salts were autoclaved for 10 minutes at 10lb/inch 2 pressure and 115 C temperature to prevent decomposition of salts at high pressure conditions. 2% of Agar was added in case of solidified agar media. The salts were added into the broth from their respective stock solutions after autoclaving inside the laminar hood. (h) Composition of Dye-Incorporated Nutrient Broth Nutrient broth Dye concentration 13 g/l 25 mg/l 89

7 The nutrient broth media was mixed in distilled water and autoclaved for 30 min at 15lb/inch 2 pressure and 121 C temperature. The stock solutions of dyes to be used were autoclaved for 10 minutes at 10lb/inch 2 pressure and 115 C. 2% of Agar was added in case of solidified dye incorporated agar media. The dyes were added into the broth from their respective stock solutions after autoclaving inside the laminar hood. (ii) Reagents (a) Composition of 3,5-Dinitrosalicylic acid (DNS) Solution Sodium Potassium Tartrate Sodium hydroxide (NaOH) di-nitro salycilic acid (DNS) Phenol Sodium Sulphite 182 g/l 10 g/l 2 g/l 0.5 g/l 0.5 g/l All the components were mixed in distilled water. The solution was filtered and stored in amber bottle for future use. (b) Preparation of Citrate Buffer (ph = 7) Citric Acid 2.101g of citric acid was dissolved in 100 ml of distilled water. Sodium Citrate 2.941g of sodium citrate was dissolved in 100 ml of distilled water. 46.5ml of the prepared citric acid was mixed with 3.5ml of the prepared sodium citrate solution and final volume was made up to 100 ml by adding distilled water. The resulting solution was 0.1M Citrate Buffer and the ph was adjusted accordingly. (c) Preparation of Sodium Acetate Buffer (ph = 4.6) Acetic Acid 1.5 ml of Glacial Acetic Acid was dissolved in 98.5 ml of distilled water. Sodium Acetate Solution 0.64 g of Sodium Acetate was dissolved in 100 ml of distilled water ml of Sodium Acetate solution was mixed with 14.8 ml of Glacial Acetic Acid solution and final volume was made up to 100 ml by adding distilled water. The resulting solution was 0.2M Sodium Acetate Buffer and the ph was adjusted accordingly. 90

8 (d) Preparation of Phosphate Buffer Saline (ph = 7) Sodium chloride (NaCl) Potassium chloride (KCl) Di-sodium hydrogen phosphate (Na 2 HPO 4 ) Potassium di-hydrogen phosphate (KH 2 PO 4 ) 8 g/l 0.2 g/l 1.44 g/l 0.25 g/l All the components were mixed in distilled water and the ph was adjusted to 7.0. The solution was filtered and stored at 4 o C in a reagent bottle for future use. (e) Preparation of Citrate Phosphate Buffer (ph = 7) Citric Acid Solution 1.921g of Citric Acid was dissolved in 100ml of distilled water. Dibasic Sodium Phosphate Solution 2.682g of Dibasic Sodium Phosphate was dissolved in 100ml of distilled water ml of Citric Acid solution was mixed with 5.4 ml of Dibasic Sodium Phosphate solution and final volume was made upto 100 ml by adding distilled water. The resulting solution was 0.2M Sodium Acetate Buffer and the ph was adjusted accordingly. (iii) Stock Solutions Hydrochloric Acid (HCl) 10%: 31.3 ml of 32% concentrated HCl was added in 68.7 ml of distilled water. The solution was mixed properly and kept at room temperature for future use. Sodium Hydroxide (NaOH) 10%: 10 g of sodium hydroxide was dissolved in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Carboxymethylcellulose (CMC) Solution 1%: 1 g of CMC was added to 100 ml of hot/warm distilled water. The solution was mixed properly and kept at 4 0 C for future use. Birchwood Xylan Solution 1%: 1g of birchwood xylan was added into 100ml of distilled water. The solution was mixed properly and kept at 4 C for future use. 91

9 Sodium Potassium Tartrate Solution 40%: 40 g of sodium potassium tartrate was dissolved in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Potassium Di-hydrogen Phosphate Solution (KH 2 PO 4 ) 10%: 10g of potassium di-hydrogen phosphate was dissolved in 80ml of distilled water and the final volume was made up to 100ml. The solution was mixed properly and kept at room temperature for future use. Di-Potassium Hydrogen Phosphate Solution (K 2 HPO 4 ) 10%: 10 g of Dipotassium hydrogen phosphate was dissolved in 80ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Magnesium Sulphate Solution (MgSO 4.7H 2 O) 10%: 10 g of magnesium sulphate was added in 80ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Sodium Chloride Solution (NaCl) 5%: 5 g of sodium chloride was dissolved in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Potassium Nitrate Solution (KNO 3 ) 10%: 10 g of potassium nitrate was added in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Calcium Carbonate Solution (CaCO 3 ) 2%: 2g of calcium carbonate was dissolved in 80 ml of distilled water and the final volume was made up to 100ml. The solution was mixed properly and kept at room temperature for future use. Ferrous Sulphate Solution (FeSO 4.7H 2 O) 2%: 2 g of ferrous sulphate was added in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Zinc Sulphate Solution (ZnSO 4.7H 2 O) 1%: 1 g of zinc sulphate was dissolved in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. 92

10 Manganese Sulphate Solution (MnSO 4.H 2 O) 1%: 1 g of manganese sulphate was dissolved in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Copper Sulphate Solution (CuSO 4.5H 2 O) 1%: 1 g of Copper sulphate was dissolved in 80 ml of distilled water and the final volume was made up to 100 ml. The solution was mixed properly and kept at room temperature for future use. Pyragallol with Hydrogen Peroxide (H 2 O 2 )1%: 0.5 g of pyragallol was dissolved in 50 ml of absolute ethanol and added 0.65 ml of H 2 O 2 in 50 ml of absolute ethanol and mixed both the solutions. α- Naphthol 0.5%: 0.25 g of α- Naphthol was dissolved in 25 ml of Acetic acid and mixed well. Guaiacol 20mM: µl of 9M Guaiacol was added in about 20 ml of distilled water and made up the volume to 25 ml. The solution was mixed properly and kept at room temperature for future use. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) 50mM: g of ABTS was dissolved in about 20 ml of distilled water. The solution was mixed properly and kept at room temperature for future use. Hydrogen Peroxide (H 2 O 2 ) 0.4%: 0.66 ml of 30% H 2 O 2 was mixed with 50ml of distilled water. Veratryl Alcohol 20mM: 0.11 ml of 9M veratryl alcohol was mixed with 40 ml distilled water to and finally the volume was make upto 50 ml. Dye Solution Stock: 1 g of the required dye was added to 25 ml of distilled water. The solution was mixed properly and kept at room temperature for future use Isolation Collection of Soil Sample Soil samples (3 in number) were collected from industrial premises. Sample 1 was collected from wood yard section (WYS). Sample 2 was collected from combined effluent discharged point (CEDP) and sample 3 was collected from near the final discharge point (FDP). All the samples were collected in plastic bags. After collection the bags were kept at 4 C till further use. 93

11 Preparation of Enrichment Media The soil samples were homogenized and suspended in different designed MSM media. For each soil sample three *enrichment media flasks were prepared containing different substrate i.e., lignin, carboxymethylcellulose (CMC) and birch wood xylan in addition to glucose. *For preparing the enrichment media sub culturing was done. After every 15 days 5% of the enrichment culture was transferred to fresh media and the concentration of glucose decreased subsequently. The flasks were incubated for 15 days. This process was repeated 3 times. Therefore, the total time of incubation was 45 days. In the last cycle the concentration of the glucose was kept to zero (Flow Chart 4.1). Flow Chart 4.1: The preparation of enrichment media 94

12 Isolation of Pure Cultures For the isolation of pure cultures, serial dilutions of the incubated broth medium (up to ) were prepared by adding 1ml of the incubated sample in 9 ml of sterilized normal saline (0.85% w/v sodium chloride in distilled water). 100 l inoculum from the dilutions 10-7, 10-8, 10-9 and was spread on MSM lignin, MSM CMC, MSM xylan and nutrient agar plates (in duplicate) separately. These plates were incubated at 35 2 C, for hrs. Single isolated colonies, which appeared on incubated plates were picked up with the help of sterile nichrome wire and streaked on fresh plates of the same medium. These plates were incubated at 35 2 C for hrs in incubator Maintenance of Isolated Pure Cultures In order to ensure the availability of pure microorganisms and their initial metabolic activities, the isolated cultures were sub-cultured periodically. Finally, the isolated pure cultures were stored. Generally, two storage methods viz., short term and long term storage methods have been used. (i) Short Term Storage Methods These methods are applied when the microorganisms are in continuous use. They include preservation techniques such as, storage of microorganisms on agar plates and slants. Preparation of Nutrient Agar Slants: Nutrient agar media prepared as mentioned above. To prepare slants, the autoclaved nutrient agar was poured in sterile glass vials when it was warm (~45 C) and the glass vials were tilted at C until solidification. The slants were incubated at 35 C (overnight) to check for contamination. (ii) Long Term Storage Methods Methods such as the use of 50% glycerol and lyophilization of bacteria have been used for the long term storage of bacteria. 95

13 (a) Glycerol: Double strength Nutrient broth media i.e. 13 g / 500 ml distilled water was autoclaved at 15 psi for 15 min. The cultures were inoculated individually and the flasks were incubated at 35 C for 16 hrs at 200 rpm. The cultures were streaked for purity check and the plates were incubated (overnight) in incubator. Finally 500 µl of the cultures was inoculated in autoclaved* cryovials (1.5 ml capacity) containing 500 µl of glycerol. * The cryovials containing glycerol were autoclaved at 10 psi for 10 min. (b) Lyophilization: The bacterial cultures were grown in double strength medium and centrifuged at 6000 rpm for 10 min. Pellet thus obtained was re-suspended in phosphate buffer ph 6.8 and the slurry was layered on to round bottom flask using chilled acetone ( _ 80 C). The layered suspension was freeze dried; bacterial powder was transferred to Duran bottle and stored at room temperature Screening of Isolated Bacteria Isolated bacterial strains were screened to check the presence of lignocellulosic compound degrading enzymes in them Lignin Degrading Enzyme Degradation of Low Molecular Weight Aromatic Compounds (LMWACs) Three bacteria were isolated from enrichment flask containing MSM and kraft lignin (KL) as sole carbon source. Further screening was carried out on MSM-agar plate containing various lignin-related low molecular weights aromatic compounds (LMWAC 50 mg/l) as sole carbon source. These LMWAC were vanillic acid (VA), ferulic acid (FA), gallic acid (FA), and tannic acid (TA). The plates were incubated at 35 C. Growth was observed after 3 days of incubation Degradation of Kraft Lignin Biodegradation experiment was carried out in 500 ml flask containing 100 ml of different media designed (table 4.1) of ph 7.0. The inoculum was prepared by inoculating one loopful of individual bacterial isolate (PNP 1 and PNP 3), separately 96

14 in 50 ml of sterilized nutrient broth having 0.01% Tween 80. The inoculated broths were incubated in an orbital shaker at 35 C for 16 hrs. so as to obtain actively growing mother cultures. These mother cultures were used for sub-culturing. 100µl of culture was inoculated in to 100 ml of NB and incubated at 35 C under shaking conditions for a period of 16-18hrs. Bacterial culture was harvested by centrifugation at 4 C and 7000rpm followed by washing twice with sodium phosphate buffer (ph ). Supernatant was discarded and pellets were stored for the further experiments. The 5% of pellets was inoculated in different media composition. Flasks were incubated in the orbital shaker at 35 C and 200 rpm for 50 hrs, uninoculated medium was used as control. Samples were withdrawn periodically at 10 hrs intervals and analysed for reduction of colour and residual KL content. Table 4.1: Different media designed for the biodegradation experiment S.No. Components Kraft liginin MSM Glucose Xylose Media 1 0.3% 100 ml 0.1% - Media 2 0.3% 100 ml - 0.1% Media 3 0.3% 100 ml Dye Decolorization Preparation of flasks for dye decolorization assay: Isolates PNP 1 and PNP 3 were inoculated in 25 ml of nutrient broth and incubated overnight at 35 C and 200 rpm. These mother cultures were checked by streaking on nutrient agar plates which were then incubated at 35 C for further experiments. In nutrient broth media different dyes (malachite green, congo red, methylene blue, azure blue and remazole brilliant blue) was present in the concentration of 25 mg/l. Nutrient broth media was prepared as mentioned in section Stock solution of dye was prepared as mentioned in section Media and dye were autoclaved at 15 psi for 20 min. After autoclaving media & dye were mixed and was poured in sterile flasks of 100 ml. 97

15 Above mentioned mother cultures were used for testing. Overnight grown culture was inoculated in each flask. The flasks were incubated at 35 C for 50 hrs. Flasks were observed at 10 hrs of time intervals by using spectrophotometer. The maximum absorbance wavelength of the dyes was scanned with the help of spectrophotometer. The color of the pellet was also visually inspected to establish whether the dye had adsorbed to the cells rather than being degraded. The percentage of decolorization efficiency of bacterial isolate was calculated as; (%) = ( ) Laccase Activity Laccase activity was tested by performing different experiment based on various substrates like, tannic acid, α- Naphthol, ABTS, and guaiacol. E coli was used as negative control and Phlebia ratiata was used a positive control for the experiments. (i) Tannic Acid Isolates PNP 1 and PNP 3 were inoculated in 25 ml of nutrient broth and incubated overnight at 35 C and 200 rpm. These mother cultures were checked by streaking on nutrient agar plates which were then incubated at 35 C for further experiments. The cultures were inoculated in laccase production media and incubate at 35 C and 200rpm for 48 hrs. 1 ml of the grown culture was transfer to test tube containing 0.3% of tannic acid. The test tubes were incubated at 35 C for 1hr at 200 rpm. Presence of laccase activity is denoted as brownish color change in the media (Pointing, 1999). (ii) α- Naphthol For the α- Naphthol test, 1ml of 0.5% α- Naphthol was added to 1ml of 24 hrs old culture of two isolates PNP 1 and PNP 3 from laccase production broth media (Pointing, 1999). The blank test tube contained media only which is not inoculated by the isolates. The tubes were kept in incubator shaker at 35 o C for 48 hrs at 200 rpm. Presence of laccase activity is denoted as purple-blue color change in the media (Pointing, 1999). 98

16 (iii) ABTS and Guaiacol To test the laccase enzyme the qualitative assay was performed. For this PNP 1 and PNP 3 were inoculated in laccase production media and incubated for 48 hr at 55 C and 200rpm. Crude enzyme was prepared by centrifugation of culture at 7000 rpm for 10min. Pellet was discarded and supernatant was stored for further use. 100 µl of supernatant was mixed with 800µl of sodium acetate buffer (ph 7) and 100 µl substrate i.e., 50 mm ABTS / 50mM guaiacol. The tubes were incubated at 35 C for 30 min and 200 rpm. The test tubes shows the appearance of brown color in case of guaiacol and green color in case of ABTS were considered as positive (Niku et al., 1999) Peroxidases Activity Test by Using Pyragallol For the Pyragallol test, 1 ml of 1% pyragallol mixed with 0.4% H 2 O 2 this was then added to 1 ml of 48hrs old cultures of two isolates PNP 1 and PNP 3 from lignin peroxidase production media. The tubes were kept in incubator shaker at 35 C for 1 hr at 200 rpm. Peroxidase activity if present will be denoted as brownish-yellow color change in the media. In order to check the activity of isolates one blank test tube was placed under same conditions in which bacteria were not inoculated (Rayner et al., 1988) Cellulose Degrading Enzyme Cellulase Enzyme Production For the production of cellulase enzyme, 100 ml of the carboxy methyl cellulose (CMC) broth media was inoculated with 5% bacterial culture and separate flasks were used for each of the bacterial isolate (PNP 4, PNP 5 and PNP 6). Inoculated flasks were incubated at 35 C and 150 rpm for 2 days, one flask with uninoculated broth was used as control. After 2 days, cultures of the respective bacterial isolates were transferred to 50 ml centrifuge tubes and centrifuged at 7,500 x g for 15 min at 10 C. Supernatant was transferred to separate tubes and stored at 4 C for future use and the pellets were discarded. This supernatant was then used for cellulose activity by the carboxymethyl cellulose and filter paper assay for reducing sugars (Miller, 1959). 99

17 Standard Curve for Glucose: A glucose standard curve was calibrated by using different concentrations of glucose, and their respective absorbance was checked at 540 nm. Absorbance was plotted against glucose concentrations; the plotted graph is glucose standard curve. Calibrated standard curve was used to determine glucose concentration. Through this glucose concentrations and standard curve, cellulase activity was determined CMCase Assay To test the cellulase enzyme from enzyme assay, 1ml crude enzyme was mixed with 1ml 0.1M citrate buffer (ph= 7.0) and 1ml substrate i.e. 1% carboxy methyl cellulose solution. Separate test tubes were used for enzyme extract from the different bacterial isolates. Blank tube was made by adding 1ml of distilled water instead of crude enzyme. The crude enzyme will react with the substrate added producing simple sugar. The reaction between enzyme and substrate was carried out by placing the test tubes in water-bath heated at 55 C for 30 min. After that, a total of 3 ml DNS is added into the solution. The solution is heated at 100 C for 15 min in water bath. After heating, 1ml of sodium potassium tartrate was added to the tubes and the solution was brought into ice bath to cool. After that the analysis was made by using spectrophotometer. The optical density was measured at 540 nm (Miller, 1959). Derivation of the CMC Unit: The absorbance values of the sample tubes were translated into their respective glucose concentrations using the Glucose standard curve. The unit of CMC is based on the International Unit (IU). 1 = h = 0.18 h h The critical amount of glucose in the CMC assay is 0.5mg; 0.5 =

18 This amount of glucose was produced by 0.5ml in 30 min, i.e., in the CMC reaction 0.5 = / = ( ) Therefore, the estimated amount of enzyme (= critical enzyme concentration. ml.ml - 1 ) which releases 0.5mg glucose in the CMC reaction contains IU, and; = / Filter Paper Assay To test the total cellulase activity from enzyme assay, the substrate used was a Whatman No 1. Filter Paper strip (1cm x 6 cm). A rolled filter paper strip was put into each test tube containing 0.5 ml crude enzyme which was mixed with 1ml of 0.1M citrate buffer (ph= 7.0). Separate test tubes were used for enzyme extract from the three different bacterial isolates (PNP 4, PNP 5 and PNP6). Blank tube was made by placing the filter paper strip in 0.5 ml of distilled water instead of crude enzyme. The crude enzyme will react with the substrate added producing simple sugar. The reaction between enzyme and substrate was carried out by placing the test tubes in water-bath heated at 50 o C for 1 h. After that, a total of 3 ml DNS was added into the solution. The solution was heated at 100 C for min in boiling water bath. After heating, 1ml of sodium potassium tartrate was added to the tubes and the solution was brought into ice bath to cool down. The absorbance was measured at 540 nm and blank was used for auto zero and the absorbance of controls was subtracted from that of the samples (Mandels et al., 1976). Derivation of the FPU Unit: The absorbance values of the sample tubes were translated into their respective glucose concentrations using the Glucose standard curve. 101

19 The unit of FPU is based on the International Unit (IU). 1 = 1 = 1 h h = 0.18 h h The absolute amount of glucose released in the FPU assay at the critical dilution is 2.0 mg; 2 = This amount of glucose was produced by 0.5ml in 60 min, i.e., in the FPU reaction 2 = / = 0.37 ( ) Therefore, the estimated amount of enzyme (=critical enzyme concentration. ml.ml -1 ) which releases 2 mg glucose in the FPU reaction contains 0.37 IU, and; = 0.37 / Xylan Degrading Enzyme Xylanase Enzyme Production For production of xylanase enzyme, 100 ml of the xylanase activity (XC) broth media was inoculated with 5% bacterial culture and separate flasks were used for each of the bacterial isolate (PNP 7, PNP 8, PNP 9 and PNP 10). Inoculated flasks were incubated at 35 C and 150 rpm for 2 days. Uninoculated broth was used as controls. After 2 days the cultures of the respective bacterial isolates were transferred to 50 ml Falcon tubes and centrifuged at 7,500 x g for 15 min at 10 C. The supernatant was transferred to separate tubes and stored at 4 C for future use and the pellets were discarded. This supernatant was then used for the xylanase activity for reducing sugars. 102

20 Standard Curve for Xylose: A xylose standard curve was calibrated by using different concentrations of xylose, and their respective absorbance was checked at 540 nm. Absorbance was plotted against xylose concentrations; plotted graph is the xylose standard curve. The calibrated standard curve was used to determine xylose concentration at various absorbance. Through this xylose concentrations and standard curve, xylanase activity was determined Xylanase Enzyme Activity To test the xylanase enzyme from enzyme assay, 1ml crude enzyme was mixed with 1ml 0.1M citrate buffer (ph= 7.0) and 1ml substrate i.e. 1% birchwood xylan solution. Separate test tubes were used for enzyme extract from the different bacterial isolates. The crude enzyme will react with the substrate added producing simple sugar. The reaction between enzyme and substrate was carried out by placing the test tubes in water-bath heated at 55 C for 30 min. After that, a total of 3 ml DNS is added into the solution. The solution is heated at 100 C for 15 min in water bath. After heating, 1ml of sodium potassium tartrate was added to the tubes and the solution was brought into ice bath to cool. After that the analysis was made by using spectrophotometer. The optical density was measured at 540 nm (Ghose and Bisaria, 1987). The Xylanase Activity was calculated by using the formula; Where = h 540 = Results Isolation of Bacteria Ten bacteria were isolated from 3 soil samples (table 4.2). The bacteria were characterized on the basis of its colony morphology. The morphological characteristics studies were color (yellow, white and cream), size (1mm, 2mm, 3mm, 4mm etc.), and elevation (concave, convex, flat), shape (irregular, smooth etc.) (table 4.3 and figure 4.1). 103

21 Table 4.2: List of bacteria isolated from three different sites S.No. Enrichment media Laboratory name Isolates from wood yard section 1 MSM + KL PNP 1 2 MSM + CMC PNP 4 3 MSM + BWX PNP 7 4 MSM + BWX PNP 8 Isolates from combined effluent discharged point 5 MSM + KL PNP 2 6 MSM + CMC PNP 5 7 MSM + BWX PNP 9 Isolates from soil collected from near the final discharge point 8 MSM + KL PNP 3 9 MSM + CMC PNP 6 10 MSM + BWX PNP 10 Note : KL : kraft lignin, CMC: craboxy methyl celulose, BWX: birch wood xylan Table 4.3: Morphological characteristic of isolated bacterial colony S.No.o. Bacteria Configuration Margin Elevation Color Size mm Isolated bacteria from enrichment media containing 0.3 % lignin as substrate 1 PNP1 Round Smooth Convex Cream 1mm 2 PNP2 Round Smooth Raised Milky white 2-3mm 3 PNP3 Round Smooth Flat Cream 2-3mm Isolated bacteria from enrichment media containing 1.0 % carboxymethyl cellulose as substrate 4 PNP4 Round Smooth Flat White 2-4mm 5 PNP5 Round Smooth Convex Cream 2-3mm 6 PNP6 Round Smooth Convex Yellowish 2mm Isolated bacteria from enrichment media containing 0.5 % xylan as substrate 7 PNP7 Round Smooth Flat Cream 2-3mm 8 PNP8 Punchi form Smooth Flat Cream 3-5mm 9 PNP9 Round Smooth Concave Orange 3-4mm 10 PNP10 Round Smooth Convex white 1mm 104

22 PNP 1 PNP 2 PNP3 PNP 4 PNP 5 PNP6 PNP 7 PNP 8 PNP 9 PNP 10 Figure 4.1: Morphological characteristics of isolated bacteria on agar plate 105

23 Screening of Isolated Bacterial Degradation of Low Molecular Weight Aromatic Compounds (LMWACs) Since strain PNP 1 and PNP 3 uses 4 LMWACs as sole source of carbon and energy for its growth, they were selected as lignin degrader because these LMWACs are the basic components of lignin moieties. Results are summarized in table 4.4. Results revealed that the bacteria PNP 3 showed the maximum activity in the presence of gallic acid and tannic acid whereas, medium activity was observed in the presence of ferulic acid and vanillic. PNP 1 showed medium activity in the presence of gallic acid, ferulic acid and tannic acid. In case of PNP 2no activity was observed in case of ferulic acid and tannic acid. Both isolate in the presence of vanillic acid showed minimum activity (Table 4.4). Table 4.4: Screening of bacterial isolates on various lignin related low molecular weight aromatic compounds Bacterial isolates Growth of bacterial isolates on MSM agar plates containing lignin related LMWACs VA FA GA TA PNP PNP PNP Note: vanillic acid (VA), ferulic acid (FA), gallic acid (FA),, tannic acid (TA). Growth visibility of bacteria on agar plate: +++ (maximum), ++ (medium), + (minimum), _ (no growth) Degradation of Kraft Lignin The results of degradation of lignin were depicted in the figure 4.2 mentioned below. It was observed from the figure that maximum reduction was achieved by PNP 3. The results were measured spectrophotometerically. In case if first experiment where lignin was used as a substrate the more decolorization was observed in the test tube inoculated with PNP 3 after 50hrs of incubation. Whereas in case of second experiment where lignin and glucose was used as a substrate the decolorization was compared and it was observed that the test tube inoculated with PNP 3 was showing more decolorization. In third experiment where with lignin xylose was used as a substrate the test tube inoculated with PNP 3 shows maximum decolorization. 106

24 (a) MSM + lignin (0.3%) (b) MSM + lignin (0.3%) (c) MSM + lignin (0.3%) + glucose (0.1%) + xylose (0.1%) Figure 4.2: Decolorization assay for (a) MSM + lignin (0.3%), (b) MSM + lignin (0.3%) + glucose (0.1%) and (c) MSM + lignin (0.3%) + xylose (0.1%) by using PNP 1 and PNP 3 after 50 hrs of incubation Lignin degradation was observed by placing three experiments (i) cultures PNP 1 and PNP 3 were inoculated in the media containing MSM and lignin with the concentration 0.3% (ii) cultures were inoculated in the media containing MSM, lignin 0.3% and glucose with the concentration of 0.1% (iii) in the last experiment glucose was replaced by xylose concentration is 0.1%. All the test tubes were incubated in incubator shaker at 200 rpm and 35 C. The samples were collected after 10 hrs of intervals i.e., the first sample was collected after 10 hrs of incubation after that 20hrs, 30hrs and 40 hrs and 50 hrs respectively. It was observed from the figure 4.3 in case of the flask containing lignin as a substrate the bacteria PNP 3 shows reduction in lignin was 25.8% whereas, the flask inoculated with PNP 1 shows the reduction value of 20.9% after 50hrs of incubation. The control value for lignin was mg/l. For the same experimental sample colour was also calculated the control value of colour was 1590 PCU for PNP 1 and 1592 for PNP 3. The achieved reduction in case of these two isolates was 32.5% and 37.4% respectively. In second experiment the flask containing lignin and glucose as a substrate. The lignin reduction achieved by the bacteria PNP 3 and PNP 1 was 49.4% and 44.2% after 50 hrs of incubation whereas; the color reduction was calculated as 49.3% with PNP 1 and 58.3% with PNP 3. The control values for the colour and lignin was 1600 PCU and mg/l for both the isolate. The In case of third experiment in which lignin and xylose was used as a substrate the achieved percentage reduction in lignin was 34% in case of PNP 3 and 29.1% in case of PNP 1. While observing the color value it was about 36.5% by PNP1 and 42.6% by PNP 3. The control value for lignin and color was mg/l and 1590 PCU for PNP 1 and 1596 for PNP 3. For every experiment blank was also placed. Negligible reduction was observed in case of blank (figure 4.3). 107

25 (a) Lignin (i) MSM + Lignin (0.3%) (b) Colour (a) Lignin (ii) MSM + Lignin (0.3%) + Glucose (0.1%) (b) Colour (a) Lignin (b) Colour (iii) MSM + Lignin (0.3%) + Xylose (0.1%) Figure 4.3: Biodegradation study for (i) MSM + lignin (0.3%), (ii) MSM + lignin (0.3%) + glucose (0.1%) and (iii) MSM + lignin (0.3%) + xylose (0.1%) by using PNP 1 and PNP 3 after 50 hrs of incubation. Blue and red line denotes the PNP 1 and PNP 3 isolates. 108

26 Dye Decolorization In order to study ligninolytic potential independently from lignin utilization, the decolourization of synthetic lignin-like dyes was monitored. This approach was followed for the bacteria PNP 1 and PNP 3, employing a range of lignin-mimicking dyes (malachite green, methylene blue, azure B, congo red and remazole brilliant blue). Dye decolourization was assessed in liquid assays with growing cultures. (i) Malachite Green According to the results the maximum percentage reduction after 50 hours is observed to be 83.26% and 94.68% in isolates PNP 1 and PNP 3 respectively. The bacterial pellet was not colored, which shows that biosorption has not occurred. The high rate of percentage degradation suggests that malachite green was significantly decolorized by the bacterial isolates figure 4.4 and figure 4.9 (a). The optical density was recorded at 615 nm Table below depicts the percentage reduction at different time intervals for blank, PNP 1 and PNP 3. After 0hr the observed reduction was up to 0.7% by using PNP 1 and 0.46% by using PNP 3. This reduction value was measured after 10 hrs, 20 hrs, 30 hrs, 40 hrs and finally 50 hrs of incubation. The results showed that after 10 hrs the test tube inoculated with PNP 1 showed reduction up to 29.6%, whereas PNP 3 shows reduction up to 20.4%. After 20hrs the reduction value was up to 34.9% for PNP 1 and 86.2% for PNP 3. When the test tubes were analyzed after 30 hrs the reduction value was up to 51.3% for PNP 1 and 91.3% for PNP 3. The reduction value after 40 hrs for PNP 1 and PNP 3 was 69.5% and 94.5%. Finally after 50hrs of incubation PNP 1, PNP 3 in two separate test tube with malachite green showed reduction up to 83.2% and 94.6% respectively. Figure 4.4: Decolorization of malachite green by using PNP 1 and PNP 3. Blue and red line denotes the PNP 1 and PNP 3 isolates 109

27 (ii) Methylene Blue According to the results the maximum decolorization efficiency after 50 hours is observed to be 55.72% and 67.75% in isolates PNP 1 and PNP 3 respectively. The bacterial pellet was not colored, which shows that biosorption. The optical density was recorded at 665 nm. The bacterium PNP 1 and PNP 3 was inoculated in flask containing methylene blue separately. The flasks were incubated in incubator shaker at 200rpm and 35 C. The samples were collected at different time interval and reduction was observed. After 10hrs of incubation the observed reduction was % for PNP 1 and % for PNP3 whereas, this reduction value increased to % and % after 20hrs of incubation. Samples were collected after 30 hrs and 40 hrs of incubation and it was analyzed that the reduction value after 30hrs for PNP 1 was % and for PNP 3 it was %. After 40hrs the reduction value achieved by PNP 1 was % and by using PNP 3 the reduction value was %. After 50hrs the maximum reduction was achieved by PNP 3 than by using PNP 1. The reduction value for the two bacteria was % (PNP 1) and % (PNP 3) (figure 4.5 and figure 4.9 (b)). Figure 4.5: Decolorization of methylene by using PNP 1 and PNP 3, Blue and red line denotes the PNP 1 and PNP 3 isolates (iii) Azure B According to the results the maximum decolorization efficiency after 50 hours is observed to be 42.01% and 48.75% in isolates PNP 1 and PNP 3 respectively. The 110

28 bacterial pellet was not colored, which shows that biosorption not take place. The optical density was measured at 650.nm. It was observed from the table the achieved reduction for the flask inoculated with PNP 1 and PNP 3was 6.734% 3.151% after 10hrs, % 7.953% after 20hrs, %, 9.771% after 30hrs and %, % after 40hrs of incubation respectively (figure 4.6 and figure 4.9 (c)). Figure 4.6: Decolorization of azure B by using PNP 1 and PNP 3. Blue and red line denotes the PNP 1 and PNP 3 isolates (iv) Congo Red According to the results the maximum decolorization efficiency after 50 hours is observed to be 71.24% and 72.97% in isolates PNP 1 and PNP 3 respectively. The bacterial pellet was not colored, which shows that biosorption will not take place along with decolorization. The optical density was recorded at 470 nm. It was observed from the table that after 10 hrs of incubation the achieved reduction was % in case of flask incubated with PNP 1 and % in case of flask incubated with PNP 3. After 20 hrs the reduction was up to % and % in case of PNP 1 and PNP 3. The achieved reduction was % and % after 30hrs of incubation for PNP 1 and PNP 3. The samples were collected after 40hrs of incubation the results showed that the achieved reduction was % and % for PNP 1 and PNP 3 respectively (figure 4.7 and figure 4.9 (d)). 111

29 Figure 4.7: Decolorization of congo red by using PNP 1 and PNP 3. Blue and red line denotes the PNP 1 and PNP 3 isolates (v) Remazol Brilliant Blue R According to the results the maximum decolorization efficiency after 50 hours is observed to be % and % in isolates PNP 1 and PNP 3 respectively. The bacterial pellet was not colored, which shows that biosorption not occurred. The optical density was recorded at 595 nm. The table below showed the reduction value calculated from the measured absorbance. The flasks were incubated in incubator shaker at 200rpm and 35C. The bacteria (PNP 1 and PNP 3) were inoculated in separate flask containing remazole brilliant blue R. It was observed that after 10hrs of incubation the calculated reduction was 3.289% for PNP 1 and 4.561% for PNP 3. After 20hrs of incubation the reduction value was 5.985% for PNP 1 and % for PNP 3. The reduction value was % for PNP 1 and % for PNP after 30hrs of incubation. It was observed that after 40 hrs of incubation the reduction value was % for PNP 1 and % PNP 3 (figure 4.8 and figure 4.9 (e)). Figure 4.8: Decolorization of remazole brilliant blue by using PNP 1 and PNP 3. Blue and red line denotes the PNP 1 and PNP 3 isolates 112

30 (a) Malachite Green (b) Methylene Blue (c) Azure B (e) Congo Red (f) Remazole Brilliant Blue Figure 4.9: Dye decolorization assay for (a) malachite green, (b) methylene blue, (c) azure B, (d) congo red and (e) remazole brilliant bule by using PNP 1 and PNP 3 after 50 hrs of incubation Laccase Activity (i) α- Naphthol and Tannic Acid Two tests were performed to test the enzymatic activity by using (i) alpha naphthol (ii) tannic acid as substrate. These tests were qualitative test also known as conventional method to check the laccase. For these two tests overnight grown cultures of PNP 1 and PNP 3 were centrifuged, supernatant was discarded and the pellet was added to the individual test tube. The PNP 1 and PNP 3 gave positive results for the two experiments using different substrates. In case of alpha naphthol violet color appears in the test tube.. Similarly in case of tannic acid brown color appears in the test tube containing PNP 1 and PNP 3. Both the bacteria were giving positive results for laccase by using above mentioned substrate (figure 4.10). 113

31 (a) Alpha-naphthol (b) Tannic acid Figure 4.10: Enzymatic assay for laccase in the presence of (a) alpha naphthol and (b) tannic acid by using PNP 1, PNP 3 (ii) Guaiacol and ABTS Two tests were performed to test the enzymatic activity by using (i) guaiacol (ii) ABTS as substrate. For these two tests overnight grown cultures of PNP 1 and PNP 3 were centrifuged, supernatant was discarded and the pellet was added to the individual test tube. The PNP 3 gave positive results for the two experiments using different substrates whereas; PNP 1 shows negative results for the both. In case of guaiacol brown color appears in the test tube. Similarly in case of ABTS green color appears in the test tube containing PNP 3 (figure 4.11). (a) Guaiacol (b) ABTS Figure 4.11: Enzymatic assay for laccase in the presence of (a) Guaiacol and (b) ABTS by using PNP 1, PNP Peroxidase Test by using Pyragallol The tests were performed by using pyragallol as a substrate for peroxidase activity. Both the isolates PNP 1 and PNP 3 show positive results with the appearance of 114

32 yellowish brown color in the presence of the substrate. But the intensity of the color in case of PNP 3 is more than PNP 1 (figure 4.12). Figure 4.12: Peroxidase activity in the presence of pyragallol by using PNP 1, PNP Cellulose Degrading Enzyme Glucose standard curve was plotted in order to quantify the enzyme release. Different glucose concentration (2mg/l 12 mg/l) was used to plot the graph. The trend line was plotted to check the linearity of the line plotted. The R 2 value was about which shows that the plotted line was linear figure Figure 4.13: Glucose Standard Curve 115

33 CMCase Assay and Filter Paper Assay Cellulase activity of the three given isolates PNP 4, PNP 5 and PNPP 6 were calculated by performing the filter paper (FPase) activity and carboxymethyl cellulase (CMCase) activity assays. The results obtained were depicted in figure 4.14 and figure (a) CMCase (b) FPase Figure 4.14: Cellulase enzyme assay (a) carboxymethyl cellulose (CMCase) and (b) filter paper assay (FPase) by using PNP 4, PNP 5 and PNP 6 It was observed from the table that the PNP 6 shows maximum cellulase activity. The quantity calculated was about 0.96 IU/ml in comparison to PNP 4 with 0.69 IU/ml and PNP 5 with 0.84 IU / ml. Figure 4.15: Cellulase Activity from CMCase Assays 116

34 Similarly the experiment was performed by using filter paper to check the cellulase activity among these three bacteria results revealed that PNP 6 showed maximum activity in comparison to PNP 4 and PNP 5. The quantity of enzyme released was estimated. PNP 6 shows the value of 0.40 IU/ml whereas, the calculated values for PNP 4 and PNP 5 was 0.17 IU/ml and 0.32 IU/ml respectively (4.16). Figure 4.16: Cellulase Activity from FPase Assays In both the experiment PNP 6 was showing the better results in comparison to PNP 4 and PNP 5. The isolates in the decreasing order of activity PNP 6 > PNP 5 > PNP 4 in both the experiments Xylanase Activity Assays Xylose standard curve was plotted in order to quantify the enzymee release. Different xylose concentration (0.5 mg/l 5 mg/l) was used to plot the graph. The trend line was plotted to check the linearity of the line plotted. The R 2 value was about which shows that the plotted line was linear (figure 4.17). 117

35 Figure 4.17: Xylose Standard Curve Xylanase Enzyme Assay Xylanase activity of the three given isolates PNP 7, PNP 8, PNP 9 and PNP 10 were calculated by performing enzyme activity assay using 1% birchwood xylan as substrate. The results obtained were depicted in figure 4.18 and figure Figure 4.18: Xylanase enzyme assay by using PNP7, PNP8, PNP9 and PNP10 118

36 Figure 4.19: Xylanase Activity The experiment was performed by using birchwood xylan as a substrate to check the xylanase activity among PNP 7, PNP 8, PNP 9 and PNP 10. Results revealed that PNP 8 showed maximum activity in comparison to PNP 7, PNP 9 and PNP 10. The quantity of enzyme released was estimated. PNP 8 shows the value of IU/ml whereas, the calculated values for PNP 7, PNP 9 and PNP 10 was IU/ml, IU/ml and IU/ml respectively. The isolates in the decreasing order of activity PNP 8 > PNP 9 > PNP 7 > PNP 10 in the experiments Discussion The nature of pulpp and paper wastewater is complex therefore; specific microorganisms were required to bioremediate the effluent in a holistic manner. The goal in bioremediation is to stimulate microorganisms with nutrients and other chemicals that will enable them to destroy the contaminants. Microorganisms gain energy by catalyzing energy producing chemical reactions thatt involve breaking chemical bonds and transferring electrons away from the contaminant. In order to meet the variation in wastewater characteristics, one has to be specific in choosing the biological component therefore; the isolation of specific bacteria is necessary. In case of pulp and paper industry wastewater is rich in lingo-cellulosic compounds. So, the isolation was made in such a manner that the bacteria were able to degrade lingo- wastewater were cellulosic compounds. The components present in the pulp and paper broadly come under the category of lignocellulose. 119