ISOLATION AND SCREENING OF BACTERIA WHICH CAN …shodhganga.inflibnet.ac.in/bitstream/10603/51025/13/13_chapter 4.pdf · were autoclaved for 10 minutes at 10lb/inch2 pressure and

Chapter 4

84

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

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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.

4.2. Materials and Methods

4.2.1. 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

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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 - 1700, 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.

4.2.2. Preparation of Media, Reagents and Stock Solutions

(i) Media

(a) Nutrient Broth (NB) (pH- 7.4)

Peptic digest of animal tissue 5.0 g/l

Sodium chloride 5.0 g/l

Yeast extract 1.50 g/l

Beef extract 1.50 g/l

All of the above contents were dissolved in 1000 ml of distilled water and autoclaved

at 121oC, (15 lbs pressure) for 30 min.

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87

(b) Nutrient Agar (NA)

Peptic digest of animal tissue 5.0 g/l

Sodium chloride 5.0 g/l

Yeast extract 1.50 g/l

Beef extract 1.50 g/l

Agar 20 g/l


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

K2HPO4 0.01M

KH2O4 0.01M

MgSO4 0.001M

EDTA 0.003M

ZnSO4 0.01 mM

MnSO4 0.02 mM

CuSO4 0.004 mM

FeSO4 0.01M

NaMoO4 0.004 mM

(NH4)2SO4 0.005M

Dextrose 2.0%

*(substrate might be changed according to the specific media designed)

H2O 1000ml

*note: different substrate used were (kraft lignin (0.3%), birch wood xylan (0.5%), carboxymethyl cellulose (1%))

Chapter 4

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at 115oC, (10 lbs pressure) for 10 min.

(d) Xylanse Production Media (pH 7.0)

Birch wood Xylan 10 g/l

Peptone 5 g/l

Yeast Extract 5 g/l

di-pottasium hydrogen phosphate (K2HPO4) 4 g/l

Magnesium sulphate hepta hydrate (MgSO4.7H2O) 1 g/l

Potassium chloride (KCl) 0.2 g/l

Ferrous sulphate hepta hydrate (FeSO4. 7H2O) 0.02 g/l

Sodium Chloride (NaCl) 0.5 g/l

Potassium nitrite (KNO3) 5 g/l

All of the above components were dissolved in 1000 ml of distilled water and

autoclaved at 121oC, (15 lbs pressure) for 30 min.

(e) Cellulase production Media (pH 7.0)

Carboxymethyl cellulose 10 g/l

Peptone 5 g/l

Yeast extract 5 g/l

Di-pottasium hydrogen phosphate (K2HPO4) 5 g/l

Magnesium sulphate hepta hydrate (MgSO4.7H2O) 0.25 g/l

Ferrous sulphate hepta hydrate (FeSO4. 7H2O) 0.02 g/l

Sodium chloride (NaCl) 0.5 g/l

All of the above components were dissolved in 1000 ml of distilled water and

autoclaved at 121oC, (15 lbs pressure) for 30 min.

(f) Composition of Laccase Production Media (Broth)

Dextrose 10 g/l

Peptone 5 g/l

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89

Sodium chloride (NaCl) 5 g/l

Beef Extract 3 g/l

Magnesium sulphate hepta hydrate (MgSO4.7H2O) 1 g/l

Calcium carbonate (CaCO3) 0.2 g/l

Ferrous sulphate hepta hydrate (FeSO4.7H2O) 1 g/l

Zinc sulphate hepta hydrate (ZnSO4.7H2O) 0.9 g/l

Manganese (II) Sulphate Monohydrate (MnSO4.H2O) 0.2 g/l

Cooper Suplahte (CuSO4) 0.159 g/l

The components (except the salts) were mixed in distilled water and autoclaved for 30

min at 15lb/inch2 pressure and 121° C temperature. The stock solutions of mineral

salts were autoclaved for 10 minutes at 10lb/inch2 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 4 g/l

Yeast Extract 4 g/l

Peptone 4 g/l

Calcium Carbonate 2 g/l

Veratryl Alcohol 20 mM

The components (except the salts) were mixed in distilled water and autoclaved for 30

min at 15lb/inch2 pressure and 121°C temperature. The stock solutions of mineral salts

were autoclaved for 10 minutes at 10lb/inch2 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 13 g/l

Dye concentration 25 mg/l

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The nutrient broth media was mixed in distilled water and autoclaved for 30 min at

15lb/inch2 pressure and 121° C temperature. The stock solutions of dyes to be used

were autoclaved for 10 minutes at 10lb/inch2 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 182 g/l

Sodium hydroxide (NaOH) 10 g/l

di-nitro salycilic acid (DNS) 2 g/l

Phenol 0.5 g/l

Sodium Sulphite 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.

36.2 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.

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91

(d) Preparation of Phosphate Buffer Saline (pH = 7)

Sodium chloride (NaCl) 8 g/l

Potassium chloride (KCl) 0.2 g/l

Di-sodium hydrogen phosphate (Na2HPO4) 1.44 g/l

Potassium di-hydrogen phosphate (KH2PO4) 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 4oC 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.

44.6 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 40C 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.

Chapter 4

92

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 (KH2PO4) 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 (K2HPO4) 10%: 10 g of Di-

potassium 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 (MgSO4.7H2O) 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 (KNO3) 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 (CaCO3) 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 (FeSO4.7H2O) 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


Zinc Sulphate Solution (ZnSO4.7H2O) 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


Chapter 4

93

Manganese Sulphate Solution (MnSO4.H2O) 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 (CuSO4.5H2O) 1%: 1 g of Copper sulphate was

dissolved in 80 ml of distilled water and the final volume was made up to 100 ml.


Pyragallol with Hydrogen Peroxide (H2O2)1%: 0.5 g of pyragallol was

dissolved in 50 ml of absolute ethanol and added 0.65 ml of H2O2 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: 55.55 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: 0.514

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 (H2O2) 0.4%: 0.66 ml of 30% H2O2 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.


4.2.3. Isolation

4.2.3.1. 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.

Chapter 4

94

4.2.3.2. 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

Chapter 4

95

4.2.3.3. Isolation of Pure Cultures

For the isolation of pure cultures, serial dilutions of the incubated broth medium (up

to 10-10) 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 10-10 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 24-48 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 24-48 hrs in incubator.

4.2.3.4. 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 30-35°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.

Chapter 4

96

(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.

4.2.4. Screening of Isolated Bacteria

Isolated bacterial strains were screened to check the presence of lignocellulosic

compound degrading enzymes in them.

4.2.4.1. Lignin Degrading Enzyme

4.2.4.1.1. 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.

4.2.4.1.2. 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

Chapter 4

97

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 6.8-7.0).

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 - -

4.2.4.1.3. 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 4.2.2. Stock solution of

dye was prepared as mentioned in section 4.2.2. 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.

Chapter 4

98

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;

�� (%) =(�� − �� )

�� ∗ 100

4.2.4.1.4. 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 35oC for 48 hrs at 200 rpm.

Presence of laccase activity is denoted as purple-blue color change in the media

(Pointing, 1999).

Chapter 4

99

(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).

4.2.4.1.5. Peroxidases Activity Test by Using Pyragallol

For the Pyragallol test, 1 ml of 1% pyragallol mixed with 0.4% H2O2 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).

4.2.4.2. Cellulose Degrading Enzyme

4.2.4.2.1. 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).

Chapter 4

100

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.

4.2.4.2.2. 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 �� =0.5

0.18 ��

Chapter 4

101

This amount of glucose was produced by 0.5ml in 30 min, i.e., in the CMC reaction

0.5 �� =0.5

0.18 ∗ 0.5 ∗ 30

��

��/��

= 0.185

��

��(

��

��)

Therefore, the estimated amount of enzyme (= critical enzyme concentration. ml.ml-

1) which releases 0.5mg glucose in the CMC reaction contains 0.185 IU, and;

�� =0.185

�� /��

4.2.4.2.3. 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 50oC for 1 h. After that, a total of 3 ml DNS was added into the

solution. The solution was heated at 100°C for 10-15 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.

Chapter 4

102

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 �� =2

0.18 ��

This amount of glucose was produced by 0.5ml in 60 min, i.e., in the FPU reaction

2 �� =2

0.18 ∗ 0.5 ∗ 60

��

��/��

=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

�� /��

4.2.4.3. Xylan Degrading Enzyme

4.2.4.3.1. 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.

Chapter 4

103

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.

4.2.4.3.2. 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;

��

�� = �� ℎ� �� 540 �� ∗ ��

Where

�� =��

��

�� ∗ 1000

�� 540 �� ∗ 30 ∗ 150

4.3. Results

4.3.1. 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).

Chapter 4

104

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

Chapter 4

105

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

Chapter 4

106

4.3.2. Screening of Isolated Bacterial

4.3.2.1. 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 1 + ++ ++ ++

PNP 2 + - + -

PNP 3 ++ ++ +++ +++

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)

4.3.2.2. 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.

Chapter 4

107

(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 296.2 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 296.2

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 296.2 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).

Chapter 4

108

(a) Lignin (b) Colour

(i) MSM + Lignin (0.3%)


(ii) MSM + Lignin (0.3%) + Glucose (0.1%)


(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.

Chapter 4

109

4.3.2.3. 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

Chapter 4

110

(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

18.158% for PNP 1 and 17.924% for PNP3 whereas, this reduction value increased to

33.826% and 31.549% 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 40.668% and for PNP 3 it was 47.276%. After 40hrs the reduction value

achieved by PNP 1 was 51.271% and by using PNP 3 the reduction value was

63.540%. After 50hrs the maximum reduction was achieved by PNP 3 than by using

PNP 1. The reduction value for the two bacteria was 55.724% (PNP 1) and 67.759%

(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



Chapter 4

111

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, 19.304% 7.953% after 20hrs, 30.188%, 9.771% after 30hrs and 37.117%,

19.136% 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



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

20.7481% in case of flask incubated with PNP 1 and 18.9024% in case of flask

incubated with PNP 3. After 20 hrs the reduction was up to 35.9096% and 42.8312%

in case of PNP 1 and PNP 3. The achieved reduction was 49.8846% and 54.6473%

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 54.5319% and

71.0778% for PNP 1 and PNP 3 respectively (figure 4.7 and figure 4.9 (d)).

Chapter 4

112

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



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 6.5421% for

PNP 3. The reduction value was 10.432% for PNP 1 and 13.263% for PNP after 30hrs

of incubation. It was observed that after 40 hrs of incubation the reduction value was

16.0321% for PNP 1 and 22.971% 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

Chapter 4

113

(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

4.3.2.4. 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).

Chapter 4

114

(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 3

4.3.2.5. 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

Chapter 4

115

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 3

4.3.2.6. 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 R2 value was about 0.990

which shows that the plotted line was linear figure 4.13.

Figure 4.13: Glucose Standard Curve

4.4.2.6.1. CMCase Assay

Cellulase activity of the three given isolates

by performing the filter paper (FPase) a

activity assays. The results obtained were

(a) CMCase

Figure 4.14: Cellulase enzyme assaypaper assay (FPase) by using PNP 4, PNP 5 and PNP 6

It was observed from the table that the PNP 6 shows maximum

quantity calculated was about 0.96

and PNP 5 with 0.84 IU / ml.

Figure

116

4.4.2.6.1. CMCase Assay and Filter Paper Assay

Cellulase activity of the three given isolates PNP 4, PNP 5 and PNP 6

filter paper (FPase) activity and carboxymethyl cellulase (CMCase)

The results obtained were depicted in figure 4.14 and figure

CMCase (b) FPase

Cellulase enzyme assay (a) carboxymethyl cellulose (CMCase) and (b) filter by using PNP 4, PNP 5 and PNP 6

It was observed from the table that the PNP 6 shows maximum cellulase activity. T

ty calculated was about 0.96 IU/ml in comparison to PNP 4 with 0.69 IU/ml

IU / ml.

Figure 4.15: Cellulase Activity from CMCase Assays

Chapter 4

PNP 6 were calculated

and carboxymethyl cellulase (CMCase)

and figure 4.15.

(b) FPase

carboxymethyl cellulose (CMCase) and (b) filter

cellulase activity. The

omparison to PNP 4 with 0.69 IU/ml

Cellulase Activity from CMCase Assays

Similarly the experiment was performed by using filter paper to check

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

PNP 4 and PNP 5 was 0.17 IU/ml and 0.32

Figure

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

both the experiments.

4.3.2.7. Xylanase Activity Assays

Xylose standard curve was plotted in order to quantify the enzyme release. Different

xylose concentration (

was plotted to check the linearity of the line plotted. The R

which shows that the plotted line was linear

117

Similarly the experiment was performed by using filter paper to check



6 shows the value of 0.40 IU/ml whereas, the calculated values

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


and PNP 5. The isolates in the decreasing order of activity PNP 6 > PNP 5 > PNP 4 in

Activity Assays


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 R2 value was about 0.996

which shows that the plotted line was linear (figure 4.17).

Chapter 4

Similarly the experiment was performed by using filter paper to check the cellulase



IU/ml whereas, the calculated values for

(4.16).


activity PNP 6 > PNP 5 > PNP 4 in


mg/l) was used to plot the graph. The trend line

value was about 0.996

Chapter 4

118

Figure 4.17: Xylose Standard Curve

4.3.2.7.1. 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 4.19.

Figure 4.18: Xylanase enzyme assay by using PNP7, PNP8, PNP9 and PNP10

The experiment was performed by using birchwood xylan as a

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 1.080939 IU/ml

whereas, the calculated values

0.898157 IU/ml and 0.582566 IU/ml respectively

of activity PNP 8 > PNP 9 > PNP 7 > PNP 10

4.4. Discussion

The nature of pulp 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 d

energy by catalyzing energy producing chemical reactions that involve breaking

chemical bonds and transferring electrons away from the contaminant. In order to

meet the variation in wastewater characteristics, o

biological component therefore; the isolation of specific bacteria is necessary. In case

of pulp and paper industry wastewater is rich in lingo

isolation was made in such a manner that the

cellulosic compounds. The components present in the pulp and paper wastewater were

broadly come under the category of lignocellulose.

119

Figure 4.19: Xylanase Activity

The experiment was performed by using birchwood xylan as a substrate




as, the calculated values for PNP 7, PNP 9 and PNP 10 was 0.672718 IU/ml,

0.898157 IU/ml and 0.582566 IU/ml respectively. The isolates in the decreasing order

of activity PNP 8 > PNP 9 > PNP 7 > PNP 10 in the experiments.

pulp and paper wastewater is complex therefore; specific



chemicals that will enable them to destroy the contaminants. Microorganisms gain



meet the variation in wastewater characteristics, one has to be specific in choosing the


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


broadly come under the category of lignocellulose.

Chapter 4

substrate to check the




7, PNP 9 and PNP 10 was 0.672718 IU/ml,

The isolates in the decreasing order

pulp and paper wastewater is complex therefore; specific



estroy the contaminants. Microorganisms gain



ne has to be specific in choosing the


cellulosic compounds. So, the

bacteria were able to degrade lingo-


Chapter 4

120

Lignocellulose is a polymer matrix composed of carbohydrate and lignin polymers.

The carbohydrate component consists of cellulose and hemicellulose polysaccharides

that contain hexose and pentose sugars. In plant cell walls the polysaccharides are

strongly bound to lignin, which fills the spaces between them. Chemical analyses

suggest that lignocellulose contains around 15 – 30 % lignin and 35 – 65 %

carbohydrate with varied composition of cellulose, hemicellulose in different types of

biomass. The lignin content is generally higher in wood than in grasses, of which

cellulose forms the primary component. 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.

Lignin modification and degradation have been most extensively studied in wood-

rotting basidiomycetes (Hatakka, 1994). White-rot fungi are known to secrete several

types of extracellular oxidative enzymes, namely laccases, lignin peroxidases (LiPs),

manganese peroxidases (MnPs), and versatile peroxidases, which have the capacity to

mineralize lignin (Bugg et al., 2011b; Hatakka, 1994). Owing to the fact that fungal

enzymes are often less robust in terms of thermal and pH stability, can be costly to

produce, and are difficult to optimize by protein engineering, the application of fungal

enzymes to lignin depolymerisation has not yet been commercialized (Bugg et al.,

2011a). Besides wood-rot fungi, there are reports of bacteria that have the ability to

break down lignin (Bugg et al., 2011a; Chen et al., 2012; Santhanam et al., 2011;

Vicuna, 1988; Zimmermann, 1990). These lignin-degrading bacteria have mainly

been isolated from soil and the guts of wood eating insects, and belong to three

classes, Actinomycetes, α-proteobacteria, and γ-proteobacteria (Bugg et al., 2011a).

By the mid-1980s, developments in the understanding of bacterial lignin degradation

mechanisms were obtained from work with gram positive actinomycetes and

Pseudomonas species. Reported lignin-degrading bacteria (Ohta et al., 1979; Kamaya

et al., 1981; Cui et al., 1995; Williamson et al., 1998 Faulds et al., 1994; Faulds et al.,

1995; Ferreira et al., 1999; Masai, 2002) include Actinomycetes such as Nocardia,

Chapter 4

121

Rhodococcus, Sphingomonas paucimobilis SYK-6 and Streptomyces viridosporus

T7A which, when grown on lignocellulose, produces extracellular peroxidases that

degrade both the lignin and carbohydrate components of lignocellulose (Saha et al.,

2010). In comparison to fungal lignin degrading enzymes, the bacterial enzymes

implicated in lignin breakdown are much less well studied. It has been suggested that

bacteria might use similar types of extracellular lignin degrading enzymes to

deconstruct lignin (McLeod et al., 2006; Ramachandra et al., 1988).

There are many reports of microorganisms such as bacteria and fungi which possess

lignocelluloses degradation activities. Nonetheless, none of these microorganisms are

efficient at cellulolytic, hemicellulolytic and ligninolytic activities simultaneously,

rendering the opportunity for discovery of better lignocellulase producing isolates.

Here, we define ‘lignocellulase producing’ as microorganisms which can produce

different enzymes such as cellulases, hemicellulases and lignases collectively, for the

decomposition of lignocellulosic biomass. We have focused on the isolation of

lignocellulolytic bacteria, i.e. bacteria which produce a greater variety of enzymes

including, in our case, cellulases, xylanases and lignases, in the hopes of finding

bacteria which can have greater decomposing impact on complex biomass which

could thus be potentially applied in industrial practices.

Several studies have focused time and again on isolation strategies for targeting

bacteria with more specific activities such as efficient cellulose producing bacteria,

yet some studies isolated bacteria with cellulase and xylanase activity, and very few

with lignase activities in addition to the previous (Maki et al., 2009, 2011; Sizoza et

al., 2011).

Lignocellulose compound degrading bacteria were isolated from the soil sample

collected from the premises of pulp and paper industry. For the soil sampling,

judgemental sampling was done. It is a non probability sampling technique where the

researcher selects units to be sampled based on their knowledge and professional

judgment. Bacteria present in a particular soil would be greatly influenced by

geographical location such as soil temperature, soil pH, soil type, organic matter

contents, cultivation and moisture content. Soil contains a variety of microorganisms

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included bacteria that can be found in any natural ecosystem. Microorganisms can be

used to determine the bioavailability of a given chemical compound in soil. The pulp

and paper industry premises soil supports a rich in lignocellulosic compounds. It is

thought that the microorganisms in these soils have developed enzymatic systems for

the rapid turnover of organic matter.

Isolation of bacteria was done by using enrichment culture and serial dilution method.

In the enrichment flask, initially MSM was supplemented with soil extract of bacterial

habitat lignin/CMC/birch wood xylan and glucose and later on only MSM and

lignin/CMC/birch wood xylan was used as only carbon source. Initially media was

supplemented with soil extract and glucose because sudden change in environment

may affect the growth of microorganism. For pure culture isolation agar plates were

used having same medium. Total ten bacteria were isolated, three having capability to

grow in the medium where lignin was only carbon source. Three bacteria were

isolated having capability to degrade CMC and four bacteria were isolated where

xylan was only carbon source.

Three lignin degrading bacteria were further screened to check their metabolic

activity to degrade lignin related low molecular weight aromatic compounds. In this

experiment, an aromatic hydrocarbon was the only organic molecule in the medium.

Three microbial soil inhabitants PNP1 and PNP3 were showed growth on low-

molecular weight lignin fractions, although growth of PNP2 was relatively poor. The

ability of the bacterial strains found here to grow on aromatic carbon sources and

consistent with a possible link between lignin degradation and aromatic compound

degradation. However, lignin-degrading activity has also been reported for certain

aromatic compound degraders such as Pseudomonas sp. and Burkholderia sp.

Furthermore, genera such as Burkholderia, Pseudomonas, Sphingomonas, Bacillus

and Pandoraea have been reported to degrade the structurally crucial biphenyl

component of lignin, which composes up to 10% of the structure, depending on the

lignin type (Khindaria et al., 1996; Kerem et al., 1998, 1999). Use of lignin-related

LMWACs by bacteria as a sole carbon source has been taken as criteria for selection

of ligninolytic bacterial strains (Gonzalez et al., 1986; Kato et al., 1998; Kumar et al.,

2001). The aerobic degradation of aromatic hydrocarbons by microorganisms has

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been investigated extensively (Hardwood and Gibson, 1988). In addition,

microorganisms, that can degrade aromatic compounds anaerobically by a reductive

enzyme system, have been reported (Hardwood and Gibson 1988; 1977). This study

provides the information on bacterial species living in soil of pulp and paper industry

premises and suggests that these bacteria have metabolic abilities to utilize aromatic

hydrocarbons derived from lignin biodegradation.

Following the steps, the bacterial isolates were further screened for degradation and

decolourization of Kraft lignin. Kraft lignin (KL), a waste polymer byproduct from

kraft pulping process of pulp and paper industry is disposed into the environment,

causing adverse impact on natural flora, fauna as well as aquatic bodies due to dark

colouration (Gaete et al., 2000). KL differs from natural lignin as it undergoes a

variety of reactions including aryl–alkyl cleavages, strong modification of side chains,

and various ill-defined condensation reactions causing the polymer to fragment into

smaller water/alkali-soluble fragments (Chakar and Ragauskas 2004). Inspite of this,

KL, though not identical to natural lignin, has been widely used as an experimental

lignin for microbial degradation studies (Forney and Reddy, 1979; Fiechter, 1982;

Perestelo et al., 1989; Morii et al., 1995). Several species of bacterial strains have

been reported to degrade the lignin monomeric substructure model compounds

(Vicuna, 1987; Zimmermann, 1990; Vicuna et al., 1993; Kumar et al., 2001), but only

a few strains are able to degrade kraft lignin from pulping industry (Perestelo et al.,

1989). Bacterial isolates from compost or soil, namely Azotobacter, Bacillus

megatarium and Serratia marcescens, were capable of decolourising or solublizing

industrial lignin (Perestelo et al., 1989; Morii et al., 1995). However, the ligninolytic

rates of reported bacterial species have been found to be much lower as compared to

fungi (Ulmer et al., 1983). For this reason there is still a need for continued search of

more efficient ligninolytic bacterial strains for bioremediation of lignin from pulp and

paper mill wastewater.

In the lignin degrading study by lignin degrading bacteria PNP1 and PNP3, kraft

lignin was added in the MSM as the sole carbon source. Glucose was added as

additional carbon source as co- substrate to promote lignin degrading bacterial growth

(Shi el al., 2012. A previous report showed that some lignin degrading bacteria have

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been able to use lignin as their sole source of carbon without depending on another

carbon source of energy (Chen et al., 2012). However Vicuna (1988) stated that

bacteria could not grow without glucose. Chandra el al., (2007) reported that lignin

degrading bacteria need glucose as co-substrate to aid in the degradation of lignin.

Therefore in lignin degrading experiment, glucose was added with in the medium as

co substrate to support bacterial growth to facilitate the lignin degradation. Similar

experiment for lignin degradation was performed where xylose was used instead of

glucose to check whether it support in lignin degradation. Initially it was observed

that very less reduction in lignin content and color, this can be explained by the low

growth rate of these strains due to lack of adoption to the lignin. It was also observed

that, when medium was supplemented with glucose more decolourization and

reduction in lignin content obtained. There was lesser decolourization in case of

xylose than glucose, but it was more than without glucose or xylose supplementation.

PNP 3 was found more efficient in kraft lignin degradation than PNP1. Other reports

also have been reported to degrade Kraft lignin (Zimmermann 1990; Leonowicz et al.,

2001; Hofrichter, 2002).

In order to study ligninolytic potential independently from lignin utilization, the

decolourization of synthetic lignin was monitored. This approach was followed for the

three isolates, employing a range of lignin mimicking dyes. Dyes usually have a

complex aromatic molecular structure, which makes them stable and resistant to

biodegradation. The chromophore groups in dyes are generally organic compounds

with conjugated double bonds. The largest class of commercially produced dyes are

azo dyes in which the chromophores are azo groups. In dye decolourization, azo

linkage is split either by reduction, the reaction used by some bacterial consortia

under anaerobic conditions, or by oxidation, the reaction used by ligninolytic white

rot fungi. Since reduction of azo dyes can result in accumulation of toxic aromatic

amines, oxidative decoloration is attracting much more interest. The white rot fungus

Phanerochaete chrysosporium was already reported to decolourise azo dyes in 1990.

Since then a number of reports studying the ability of different white rot fungi for

decoloration of various dyes have been increasing (Heinfling et al., 1997, 1998;

Swamy and Ramsay, 1999; Zhang et al., 1999; Novotny et al., 2001 and Kasinath et

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al., 2003) Researchers found that lignolytic enzymes such as lignin peroxidase,

manganese peroxidase and laccase were responsible for dye decolourization. (Moldes

et al., 2003; Martins et al., 2003; Heinfling-Weidtmann et al., 2001; Robinson et al.,

2001; Abadulla et al., 2000 and Kirby et al., 2000). Laccases from fungi have been

predominantly studied; where they are believed to have a major role in lignin

degradation. Their ability to use compounds, with irregular structures such as lignin,

as a substrate means laccases have a broad spectrum of substrates. The broad substrate

specificity means these enzymes have a wide range of applications, ranging from

effluent decolorization and detoxification (Eduardo et al., 2003) to pulp bleaching

(Widsten and Kandelbauer 2008), and removal of phenolics from wines (Minussi et

al., 2002). The ligninolytic system of these microorganisms enables them to degrade

different xenobiotic compounds, including polycyclic aromatic hydrocarbons (PAHs),

polychlorinated biphenyls (PCBs), and synthetic dyes. (Pointing, 2001).

Some actinomycetes and anaerobic bacteria have also dye degrading activity, being

involved lignin degrading enzymes (McMullan et al., 2001). Efforts to isolate

bacterial cultures capable of degrading azo dyes started in the 1970s with reports of

Bacillus subtilis (Chen et al., 2003), then Aeromonas hydrophila (Idaka and Ogawa,

1978) followed by Bacillus cereus (Wuhrmann et al., 1980). Numerous bacteria

capable of dye decolorization, either in pure cultures or in consortia, have been

reported (Verma and Madamwar, 2003; Bunat et al., 1996; Coughlin et al., 2002;

Pearce et al., 2003; Yatome et al., 1991; Rajaguru et al., 2000). Kalme et al., 2007

and Telke et al., 2010), reported bacterial dye decolorization by oxidoreductive

enzymes, including laccase, azoreductases and NADH-DCIP reductase It has been

reported that laccase containing spores of B. subtilis SF and the corresponding

immobilized spores could decolorize textile dyes such as Mordant Black

(Kandelbauer and Guebitz 2005). Laccase positive bacterium Stenotrophomonas

maltophilia AAP56 was also proven to be capable of decolorizing several synthetic

dyes (Galai et al., 2009).

Although laccases have been found in plants, fungi, and bacteria, the best

characterized laccases so far are mainly from fungi, especially from white rot

basidiomycetes that are efficient lignin degraders. The first observation of bacterial

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laccase activity was performed in Azospirillum lipoferum based on observed phenol

oxidase activity (Givaudan et al., 1993). Subsequently, laccases and laccase like

activity have been demonstrated in other bacteria such as Bacillus halodurans

(Ruijssenaars and Hartmans, 2004), Bacillus licheniformis (Koschorreck et al., 2008),

Bacillus subtilis (Martins et al., 2002), E coli (Roberts et al., 2002), Nitrosomonas

europea (Lawton et al., 2009), Streptomyces coelicolor (Machczynski et al., 2004),

Streptomyces griseus (Endo et al., 2003), and Thermus thermophilus (Miyazaki,

2005). A CotA-type laccase from B. pumilus was cloned and characterized; this has

potential as a biocatalyst (Reiss et al., 2011).

The production of lignolytic enzymes by using bacterial isolates PNP1 and PNP3 was

done. E coli was used as a negative control and Phlebia radiata was used as a positive

control in this experiment. The experiments were conducted to ensure that the

lignolytic enzymes were produced by these bacterial isolates. The substrate alpha

naphthol and tannic acid was studied previously as a substrate for laccase (Pointing,

1999). Therefore the alpha naphthol and tannic acid was used for the qualitative test

which was performed for the testing the presence of laccase activity. In presence of

alpha naphthol and tannic acid both the isolates i.e., PNP1 and PNP 3 showed positive

results. In literature it was mentioned that ABTS and guiacol was used as a substrate

in laccase enzymatic assay. These substrates were used to test the activity of the

strains PNP 1 and PNP 3. Only PNP 3 was able to produce color which shows the

positive result for the isolate. Both the isolates PNP 1 and PNP 3 showed positive

result for peroxidase when pyragallol was used as a substrate. Other researchers also

reported the presence of peroxidase and laccase in lignolytic bacteria (Shi et al., 2013

and Chen et al., 2012; Ahmed et al., (2010)). Some other reports are also on bacterial

lignin degrading enzymes isolated lignin degrading bacteria from palm oil plantation

soil by using a selective nutrient media medium. Bacteria were found to produce all

three lignin degrading enzymes peroxidase and laccase (Zhu et al., 2013; Oliveria et

al., 2009, Bibi and Bhatti 2012. Rahman et al., 2013). Jha et al., (2002a; 2002b

reported the strain Klebsiella aerogenes NCIM 2098 (K. aerogenes) of family

Enterobacteriaceae and find effective in lignin removal. In another study, Jha and

Patil (2013) isolated laccase from the bacterium K. aerogenes.

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Both fungi and bacteria have been exploited for their abilities to produce a wide

variety of cellulases and hemicellulases. Most emphasis has been placed on the use of

fungi because of their capability to produce copious amounts of cellulolytic enzymes

and often less complex than bacterial cellulase and easy for extraction and

purification. However, the isolation and characterization of novel cellulase from

bacteria are now becoming widely exploited. There are several reasons for these shifts

viz. i) bacteria often have a higher growth rate than fungi allowing for higher

recombinant production of enzymes, ii) bacterial cellulases are often more complex

and are in multi-enzyme complexes providing increased function and synergy iii)

bacteria inhabit a wide variety of environmental and industrial niches like

thermophilic or psychrophilic, alkaliphilic or acidiophilic and halophilic strains,

which produce cellulolytic strains that are extremely resistant to environmental

stresses. These strains can survive and produce cellulolytic enzymes in the harsh

conditions which are found to stable under extreme conditions and which may be used

in the bioconversion process (Miranda et al., 2009). Researchers are now focusing on

utilizing, and improving these enzymes for use in various industry such as pulp and

paper, biofuel and bioproduct industries.

Many bacteria can grow on cellulose and many produce enzymes that catalyze the

degradation of soluble derivatives of cellulose or the amorphous regions of crystalline

cellulose. However few bacteria synthesize the complete enzyme system that can result

in extensive hydrolysis of the crystalline material found in nature. These few bacteria

should be called “true cellulolytic “bacteria and those bacteria that produce some

endoglucanases and ß-glucosidases, but not the complete system, are called

“pseudocellulolytic” (Coughlan and Mayer 2006). There are different types of bacteria

isolated from different environment produced cellulase. Thomas et al., (2014), showed

the presence of cellulose enzyme in Clostridium sp. Kazemi et al., (2014), produced

cellulose enzyme from Bacillus sp. BCCS A3. Pandey et al., (2013), isolated bacteria

from different extreme environment samples and showed the presence of cellulose

enzyme. Bacteria were further identified as Bacillus, Paenibacillus, Lysinibacillus

Arthobacter, and Rhodococcus sp., Bacillus licheniformis isolated from soil samples

and reported cellulose activity Ghani et al., (2013). A Exiguobacterium sp. VSG-1 was

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isolated from the soil sample and characterized for the production of lingo cellulolytic

enzymes (Vijayalaxmi et al., 2013). Yang et al., (2013), isolated bacterial from pulp

mill wastewater storage pool and identified as Methanoculleus and Clostridium having

cellulose activity. Padaria et al., (2014) isolated cellulose degrading strain of Bacillus

pumilus from soils of Darjeeling hills of India and screened for the production of

extracellular cellulases. Some other reports are also available on bacterial cellulose

enzymes by various research groups (Ibrahim and Ahmed, 2007; Korpole et al., 2011;

Deka et al.,2011; Patel et al., 2005; Nizamudeen and Bajaj 2009; Acharya and

Chaudhary 2012; Mishra et al., 2008 and Sadhu et al., 2012).

In present study, isolate PNP 4, PNP 5 and PNP 6 were screened to test the activity of

cellulose by using CMC and filter paper assay. Results showed that the all the three

isolates gave positive results but on the basis of enzymatic assay PNP 5 and PNP 6

were selected for further studies. These strains were isolated from the soil sample

from paper industry premises rich in lignocelluloses. Therefore, the bacteria residing

in such habitats developed the capability to degrade cellulose.

The next targeting component in pulp and paper wastewater is hemicelluloses.

Hemicellulose is the second most abundant component in plant cell wall and xylan is

the major component of hemicelluloses, 15% - 25% of the total dry weight of plants

accounts for xylans. Xylan is a heterogeneous carbohydrate consisting of a backbone

of β-1, 4 linked D-xylopyranosyl units and short chain branches consisting of O-

acetyl, α-L-arabinofuranosyl and α-D-glucuronyl residues (Nair et al., 2008) which is

found in solid agricultural and agro industrial residues, as well as in effluents released

during wood processing (Collins et al., 2005; Anthony et al., 2005). Xylanase enzyme

is used by microorganisms for degradation of xylan. Xylanase is produced by many

bacteria and fungi (Marta et al., 2000; Polizeli et al., 2005). It has been exploited for a

range of industrial and environmental applications. Xylanase is commercially

important enzyme used in the pulp and paper industries to increase the brightness of

pulp without the use of bleach (Viikari et al., 1986; Garg et al., 2010). Xylanase

producing bacteria can be used for bioremediation of pulp and paper industry effluent.

Besides, xylanase is also used in number of other industries viz. food, beverage,

textile and animal feed industries (Polizeli et al., 2005; Sharma et al., 2013).

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In this study, bacteria isolated from soil sample from pulp and paper industry showed

the presence of xylanase enzyme in them. The isolate PNP 7, PNP 8, PNP 9 and PNP

10 were tested to check the activity of xylanase by using brich wood xylan. The

isolate PNP 8 and PNP 9 were selected on the basis of maximum enzymatic activity.

There are other reports also indicating secretion of xylanase by bacteria (Dheeran et

al., 2012; Shi et al., 2010; Mahatman et al., 2010; Vahino and Nakane, 1980; Blanco

et al., 1995; Mahatman et al., 2010; Nakamura et al., 1993; Araki et al., 1998; Lin et

al., 1999; Rattoet al., 1992; Saxena et al., 1991; Grabski et al., 1991).

Finally in the present study after isolation and screening for lingo cellulosic degrading

compounds strain PNP 1, PNP 3, PNP 5, PNP 6, PNP 8 and PNP 9 were selected for

further studies.

4.5. Conclusion

Soil samples were collected from industrial premises containing the micro flora which

were having the potential to degrade the lignocellulosic compounds. These bacteria also

have potential to degrade low molecular weight aromatic compounds. The

decolorization capability and degradation of kraft lignin was investigated in these

bacteria. It was seen that there degradation efficiency was increased when glucose and

xylose was added as a substrate. The bacteria screened to check the dye decolorization

activity. Followed by the experiment to check the lignin degrading enzyme in the two

bacteria and it was found that both the strains were showing the laccase and peroxidise

activity which is responsible for dye decolorization and kraft lignin degradation. After

the complete experiment the results showed that PNP 1 and PNP 3 were selected for

further studies. The next set of bacteria PNP 4, PNP 5 and PNP 6 were studied to

degrade cellulose. Results revealed that strain PNP 5 and PNP 6 were better than PNP 4

in cellulose production. The strains PNP 7, PNP 8, PNP 9 and PNP 10 were screened

for their xylan degradation capability. It was observed that PNP 8 and PNP 9 were

better than the PNP 7 and PNP 10 in xylanase activity. The strains selected for further

studies were PNP 1, PNP 3, PNP 5, PNP 6, PNP 8 and PNP 9. These bacteria were

isolated from the industrial premises and after several set of experiment it was found

that these strains have the lingo cellulose degrading capability. Therefore, they can be

use for the treatment of pulp and paper industrial effluent.

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