OPTIMIZATION OF HYDROLYTIC ENZYMES PRODUCED

FROM THERMOPHILIC MICROORGANISMS FOR

DEGRADATION OF CELLULOSIC BIOMASS

Thesis
by

SHRUTI PATHANIA
Submitted in partial fulfilment of the requirements
for the degree of

MASTER OF SCIENCE
in
MICROBIOLOGY
(BASIC SCIENCES)

COLLEGE OF FORESTRY
Dr Yashwant Singh Parmar University
of Horticulture and Forestry, Nauni,
Solan - 173 230 (H.P.), INDIA
2011
Dr. (Mrs.) Nivedita Sharma Department of Basic Sciences
Associate Professor College of Forestry
Dr. Y.S. Parmar University of Horticulture
and Forestry, Nauni-173 230, Solan (H.P.)

CERTIFICATE-I

This is to certify that the thesis entitled, “Optimization of hydrolytic enzymes

produced from thermophilic microorganisms for degradation of cellulosic biomass”
submitted in partial fulfilment of the requirements for the award of degree of MASTER OF
SCIENCE in MICROBIOLOGY (Basic Sciences) to Dr. Yashwant Singh Parmar
University of Horticulture and Forestry, Nauni, Solan (H.P.) is a record of bonafide research
work carried out by Ms. Shruti Pathania (F-2009-09-M) under my guidance and
supervision. No part of this thesis has been submitted for any other degree or diploma.

The assistance and help received during the course of investigation has been fully
acknowledged.

Place : Nauni, Solan Dr. (Mrs.) Nivedita Sharma

Dated: , 2011 Chairperson
Advisory Committee
 CERTIFICATE-II

This is to certify that the thesis entitled, “Optimization of hydrolytic enzymes

produced from thermophilic microorganisms for degradation of cellulosic biomass”
submitted by Ms. Shruti Pathania (F-2009-09-M) to Dr. Yashwant Singh Parmar
University of Horticulture and Forestry, Nauni, Solan (H.P.) in partial fulfilment of the
requirements for the award of degree of MASTER OF SCIENCE in MICROBIOLOGY
(Basic Sciences) has been approved by the Student’s Advisory Committee after an oral
examination of the same in collaboration with the external examiner.

Dean’s Nominee External Examiner

Major Advisor
Dr. (Mrs.) Nivedita Sharma
(Associate Professor) Members of Advisory committee

Dr. (Mrs.) Mohinder Kaur

(Assistant Professor)

Dr. R. K. Gupta
(Associate professor)

Dr. (Mrs.) Neerja Rana

(Assistant Professor)
Professor and Head
Department of Basic Sciences

Dean
College of forestry
Dr. Y. S. P. University of Horticulture and Forestry,
Nauni, Solan
 CERTIFICATE-III
This is to certify that all the mistakes and errors pointed out by the external examiner
have been incorporated in the thesis entitled, “Optimization of hydrolytic enzymes
produced from thermophilic microorganisms for degradation of cellulosic biomass”
submitted to Dr Y.S. Parmar University of Horticulture and Forestry, Nauni-Solan (H.P.) by
Ms. Shruti Pathania (F-2009-09-M) in partial fulfilment of the requirements for
the award of degree of MASTER OF SCIENCE in MICROBOLOGY (Basic Sciences).

Major Advisor

Dr. (Mrs.) Nivedita Sharma

Associate Professor

Professor and Head

Department of Microbiology
Dr. Y.S. Parmar, UHF, Nauni-173 230, Solan (H.P.)
 ACKNOWLEDGEMENT

First of all I would like to offer my hearfelt salvation at the feet of the Supreme
Being for the unbroken health and vigour bestowed upon me, providing me everything that I
would hope for and in whose faith, I was able to complete this task.

I deem it to my proud privilege to express my deep sense of gratitude and profound

personal regards to my esteemed teacher and chairperson of my advisory committee, Dr. (Mrs.)
Nivedita Sharma (Associate Professor), for her unique way of guidance, critical analysis,
constructive criticism, valuable suggestions, constant encouragement and affection during the
course of present investigation are beyond the reach of my formal words.

I express my loyal and sincere thanks to Dr. A. K. Sharma (Professor and Head of
Department of Basic Sciences) for providing all the necessary facilities and means to carry out
the experiments successfully.

I express my venerable thanks to the members of my advisory committee, Dr. (Mrs.)

Mohinder Kaur, Dr. R. K. Gupta and Dr. (Mrs.) Neerja Rana for their guidance and
cooperation in the course of investigation.

Sincere thanks are also due to Dr. C. K. Shirkot and Mrs. Anjali Chauhan.

I take this precious moment to express my heartiest thanks to my papa, mumma and
Vikas whose association have always been a boost to me. I owe my success to them only, as
they rejuvenated my strength to fulfil every task of my life with perfection and clarity. It is
with personal touch of emotions that I express my gratitude to my nana ji and masi ji for
their care and affection and also to Baba ji and Rajinder Guleria for their heartfelt blessings.

Indeed the words at my command are not adequate to express heartiest vonoration
and heartful gratitude to my dignified seniors Sanjeev Sir, Neha ma’am, Hitender Sir and
Nisha, Richa and Divya ma’am for their knowledge of subject, able guidance, critical
appraisal, helpful suggestions and overwilling help which has guided my efforts to execute
the present investigations during crucial times.

Walking along sands of time, all my memories, be beautiful ones, ugly ones, joyous
ones having common quotient with Gaurav, Sakshi, Pallavi, Sushma, Rosy, Surbhi, Shweta,
Kitu and Madhu.

I would like to express my appreciation to laboratory and office staff of Department

of Basic Sciences, for their kind help and mean time support.

Words of appreciation also goes to Sohan lal, DPT Computers.

Needless to say errors and omissions are mine.

Nauni, Solan
Date: Nov, 2011 (Shruti Pathania)
 CONTENTS

CHAPTER TITLE PAGE(S)

1. INTRODUCTION 1-4

2. REVIEW OF LITERATURE 5-44

3. MATERIALS AND METHODS 45-74

4. RESULTS AND DISCUSSION 75-132

5. SUMMARY AND CONCLUSION 133-136

6. REFERENCES 137-158

ABSTRACT 159

APPENDICES I-V
 LIST OF TABLES

Table Title Page

REVIEW OF LITERATURE

1. Examples of sites serving as a source of microorganisms which 7

can provide thermotolerant enzymes

2. Source microorganisms and properties of thermostable 9

cellulases

3. Source microorganisms and properties of thermostable 9

xylanase

4. Showing the importance of thermostable enzyme in industries 12

RESULTS AND DISCUSSION

1. Physiochemical analysis of the water sample from hot springs 76

of Himachal Pradesh

2a. Isolation of cellulolytic and xylanolytic bacteria from hot springs 78

of Tattapani and their morphological and cultural characteristics

2b. Isolation of cellulolytic and xylanolytic bacteria from hot springs 79

of Manikaran and their morphological and cultural
characteristics

2c. Isolation of cellulolytic and xylanolytic bacteria from hot springs 80

of Vashisht and their morphological and cultural characteristics

2d. Percentage analysis of morphological and cultural 80

characteristics of bacterial isolates from hot springs of
Himachal Pradesh

2e. Isolation of cellulolytic and xylanolytic fungi isolated from the 85

hot springs of Himachal Pradesh and their morphological and
cultural characterization
Table Title Page

3a. Biochemical characteristics of cellulolytic and xylanolytic 82

bacterial isolates from Tattapani

3b. Biochemical characteristics of cellulolytic and xylanolytic 83

bacterial isolate from Manikaran

3c. Biochemical characteristics of cellulolytic and xylanolytic 84

bacterial isolate from Vashisht

4a. Screening of bacterial isolates from hot springs of Tattapani for 87

hypercellulase and hyperxylanase production

4b. Screening of bacterial isolates from the hot springs of 88

Manikaran for hypercellulase and hyperxylanase production

4c. Screening of bacterial isolates from the hot springs of Vashisht 89

for hypercellulase and hyperxylanase production

4d. Screening of fungal isolates from the hot springs of Himachal 93

Pradesh for hypercellulase and hyperxylanase production

5. Effect of media on xylanase production from Paenibacillus sp. 96

N1

6. Effect of incubation time on xylanase production from 96

Paenibacillus sp. N1

7. Effect of pH on xylanase production from Paenibacillus sp. N1 100

8. Effect of temperature on xylanase production from 100

Paenibacillus sp. N1

9. Effect of inoculum size on xylanase production from 104

Paenibacillus sp. N1

10. Effect of different amino acids on xylanase production from 104

Paenibacillus sp. N1

11. Effect of different carbon sources on xylanase production from 106

Paenibacillus sp. N1
Table Title Page

12. Effect of different nitrogen sources on xylanase production from 108

Paenibacillus sp. N1

13. Effect of additives on xylanase production from Paenibacillus 110

sp. N1

14. Cellulase production from Thermomyces sp. F1 using untreated 112

and microwave treated rice straw (Oryza sativa) under SSF

15 Cellulase production at different moisture level from 116

Thermomyces sp. F1 using untreated and microwave treated
rice straw (Oryza sativa) under SSF

16. Cellulase production at different temperature from 118

Thermomyces sp. F1 using untreated and microwave treated
rice straw (Oryza sativa) under SSF

17. Cellulase production at different incubation time from 120

Thermomyces sp. F1 using untreated and microwave treated
rice straw (Oryza sativa) under SSF

18. Xylanase production from Thermomyces sp. F1 using 123

untreated and microwave treated rice straw (Oryza sativa)
under SSF

19. Xylanase production at different moisture level from 126

Thermomyces sp. F1 using untreated and microwave treated
rice straw (Oryza sativa) under SSF

20. Xylanase production at different temperature from 128

Thermomyces sp. F1 using untreated and microwave treated
rice straw (Oryza sativa) under SSF

21. Xylanase production at different incubation time from 130

Thermomyces sp. F1 using untreated and microwave treated
rice straw (Oryza sativa) under SSF
 LIST OF PLATES

Plates Title Between

Page(s)

1. Molecular characterization of hyperxylanolytic bacterial 92-93

strain N1 using rrs (16S rRNA) technique

2. Hyperxylanolytic bacteria isolated from hot springs 92-93

3. Hypercellulolytic and Hyperxylanolytic fungus isolated from 92-93

hot springs

4. Production of cellulase enzyme from Thermomyces sp. F1 116-117

using untreated and treated rice straw in SSF

5. Effect of moisture level on cellulase production from 116-117

Thermomyces sp. F1

6. Effect of temperature on cellulase production from 119-120

Thermomyces sp. F1

7. Effect of incubation time on cellulase production from 119-120

Thermomyces sp. F1

8. Production of xylanase enzyme from Thermyces sp. F1 125-126

using untreated and treated rice straw in SSF

9. Effect of moisture on xylanase production from 125-126

Thermomyces sp. F1

10. Effect of temperature on xylanase production from 130-131

Thermomyces sp. F1

11. Effect of incubation time on xylanase production from 130-131

Thermomyces sp. F1
 LIST OF FIGURES
Figures Title Between
page(s)
REVIEW OF LITERATURE
1. Classification of microbial life 6
2. Representation of lignocellulosic structure showing 19
cellulose, hemicelluloses and lignin fractions
3. Structure of cellulose 21
4. Conformational structure of cellulose and modes of action 21
of various components of cellulase
5. Structure of xylan 22
6. Hydrolysis of xylan 24
7. Schematic representation of pretreatment effect on 24
lignocellulosic biomass
MATERIALS AND METHODS
1. Hot springs of district Mandi and Kullu 46-47
RESULTS AND DISCUSSION
1. Xylanase production from thermophilic bacterial isolates 88-89
from hot springs of Tattapani.
2. Xylanase production from thermophilic bacterial isolates 88-89
from hot springs of Manikaran.
3. Xylanase production from thermophilic bacterial isolates 90-91
from hot springs of Vashisht.
4. Effect of media on xylanase production from Paenibacillus 98-99
sp. N1 under SmF
5a. Effect of incubation time on xylanase production from 98-99
Paenibacillus sp. N1 under SmF
5b. Growth curve of Paenibacillus sp. N1 98-99
6. Effect of pH on xylanase production from Paenibacillus sp. 100-101
N1 under SmF
7. Effect of temperature on xylanase production from 100-101
Paenibacillus sp. N1 under SmF
8. Effect of inoculum size on xylanase production from 104-105
Paenibacillus sp. N1 under SmF
9. Effect of amino acids on xylanase production from 104-105
Paenibacillus sp. N1 under SmF
Figures Title Between
Page(s)
10. Effect of carbon sources on xylanase production from 108-109
Paenibacillus sp. N1 under SmF
11. Effect of nitrogen sources on xylanase production from 108-109
Paenibacillus sp. N1 under SmF
12. Effect of additives on xylanase production from 110-111
Paenibacillus sp. N1 under SmF
13. An overview of percent increase in xylanase activity from 110-111
Paenibacillus sp. N1 after optimization of different
parameters under SmF
14. Cellulase production from Thermomyces sp. F1 using 116-117
untreated and microwave treated rice straw under SSF
15. Comparison of cellulase activity at different moisture level 116-117
between untreated and microwave treated rice straw from
Thermomyces sp. F1 under SSF
16. Comparison of cellulase activity at different temperature 119-120
between untreated and microwave treated rice straw from
Thermomyces sp. F1 under SSF
17. Comparison of cellulase activity at different incubation time 119-120
between untreated and microwave treated rice straw from
Thermomyces sp. F1 under SSF
18. An overview of percent increase in cellulase activity from 119-120
Thermomyces sp. F1 after optimization of different
parameters under SSF
19. Xylanase production from Thermomyces sp. F1 using 125-126
untreated and microwave treated rice straw under SSF
20. Comparison of xylanase activity at different moisture level 125-126
between untreated and microwave treated rice straw from
Thermomyces sp. F1 under SSF
21. Comparison of xylanase activity at different temperature 130-131
between untreated and microwave treated rice straw from
Thermomyces sp. F1 under SSF
22 Comparison of xylanase activity at different incubation time 130-131
between untreated and microwave treated rice straw from
Thermomyces sp. F1 under SSF
23. An overview of percent increase in xylanase activity from 132-133
Thermomyces sp. F1 after optimization of different
parameters under SSF
 ABBREVIATIONS USED

% : Percent
& : And
°C : Degree Centigrade
µ mol/min : Micro mole per millilitre per minute
µg : Micro gram
µg/ml : Micro gram per ml
µl : Micro litre
µm : Micro meter
BSA : Bovine Serum Albumin
cfu : Colony forming units
cm : Centimeter
CMCase : Carboxy methyl cellulase
Distt. : District
DNA : Deoxyribonucleic acid
DNSA : Dinitrosalicylic acid
dNTPs : Deoxyribonucleotide triphosphate
et al : And others
etc : Et cetera
Fig : Figure
FPase : Filter Paperase
g : Gram
g/l : Gram per litre
Gt : Gigatonne
h : Hour
H.P. : Himachal Pradesh
Hrtz : Hertz
i.e. : That is
IU : International units
kDa : Kilodalton
kg/cm2 : kilogram per centimetre square
l : Litre
M : Molar
mg : Miligram
mg/ml : Miligram per millilitre
min : Minute
ml : Millilitre
mm : Millimetre
mM : Milimolar
MR/VP : Methyl Red/ Voges Prokaeur
N : Normal
nm : Nano meter
O.D. : Optical density
PCR : Polymerase Chain Reaction
ppm : Parts per million
RNase : Ribonuclease
rpm : Rotations per minute
rRNA : Ribosomal ribonucleic acid
SA : Specific Activity
SmF : Sumerged Fermentation
sp. : Species
SSF : Solid State Fermentation
U/gds : Unit per gram dry substrate
U/l/h : Unit per Litre per Hour
U/mg : Unit per milligram
V : Volts
v/v : Volume per volume
v/w : Volume per weight
viz. : Vizually
W : Watt
w/v: : Weight per volume
w/w : Weight by weight
α : Alpha
β : Beta
ρ : Para
 Chapter-1

INTRODUCTION
Hot spring is a spring that is produced by the emergence of geothermal-
heated water from the earth crust (Kauze et al., 2006). Terrestrial geothermal
areas are located in various regions of our planet, mostly around the border of
tectonic plates and in area where the earth crust is relatively thin, in these sites
thermal springs, fumaroles and geysers are the common places (Johnsson, 2003).
The study of extreme environment in the form of hot springs has considerable
biotechnological potential. Since ages, studies of diversity in such environment
are drawing attention to the microbiologist for two reasons:

• Some of the earlier microbial life on earth might have been thermophilic
and thermotolerant and thus research on microbial survivability and
adaptation in hot spring may help us to understand early life on earth or
life on other planetary bodies (Chapelle et al., 2002)
• Thermophiles in hot spring have lot of practical application, for example
some valuable enzymes (e.g. lipase, amylase, cellulase and DNA
polymerase) produced by thermophiles retrieved from hot spring have
high activity at high temperature and are being widely applied in industry,
medicines, agriculture and biotechnology (Tang et al., 2006).

Thermophilic microorganisms have gained a great deal of attention in the

last two decades (Beg et al., 2000). Enzymes produced from these
microorganisms are of special interest since these are not usually denatured by
high temperature and are even active at elevated temperature. Therefore interest
in these microorganisms has increased worldwide owing to their potential
commercial application namely production of bioactive compounds, including
antibiotics and enzymes (Malherbe and Cloete, 2002; Lynd et al., 2005). A
prominent example is the Taq polymerase, purified from hot spring bacterium
Thermus aquaticus which has found a number of applications including
molecular biology and biotechnology (Brock, 1978). One of the most attractive
attribute of thermophiles is that they produce enzymes capable of catalysing
biochemical reactions at temperature higher than those of mesophillic
microorganisms (Demirijian et al., 2001) and these are also resistant to chemical
reagents and extreme pH values in comparisons to mesophiles.

Among industrially important enzymes, cellulases and xylanases have

attracted the major attention globally because of their wide application in many
fields. These enzymes fetch extraordinary high prices in the market and main
factor responsible for that is the use of expensive substrates like pure cellulose
and xylan for production of enzymes

Alternatively use of inexpensive and easily available lignocellulosic

material as substrate can lead to a cost effective process for cellulase and
xylanase production. Plant cell wall is the most abundant renewable source of
fermentable sugars on earth (Saleem et al., 2002) and major reservoir of fixed
carbon in nature (Yang et al., 2006). Plant biomass comprises an average of 50%
cellulose, 30% hemicelluloses and 20% lignin by dry weight (Sa-Pereira et al.,
2003). Annually, 830 Gt of renewable plant biomass is formed consisting of
cellulose and hemicelluloses (Rauscher et al., 2006).

Cellulose and hemicelluloses present in plant biomass is recalcitrant in

nature. Therefore pretreatment is required to alter the biomass macroscopic and
microscopic size and structure as well as its submicroscopic structural and
chemical composition to facilitate rapid and efficient accessibility of
carbohydrates to enzyme producers.

Cellulose accounts for approximately 50% of the dry weight of the plant
biomass and 50% of the dry weight of secondary source of biomass such as
agricultural wastes (Haruta et al., 2003). Cellulose is an unbranched linear
homopolymer of glucose units linked by β-1,4-D-glucosidic bonds, that forms
insoluble, crystalline microfibrils which are highly resistant to enzymatic
hydrolysis, consisting of three different enzymes : endoglucanase,
cellobiohydrolase and β-glucosidase, which act together in synergism. An

2
important feature of the cellulose usually in the polysaccharide world i.e.
crystalline structure and insoluble nature and represents a formidable challenge
for cellulosic hydrolysis (Zhou et al., 2000). Cellulose degrading microorganisms
convert cellulose into soluble sugar by synthesizing cellulase thus causing an
enzymatic hydrolysis (Lynd et al., 2005).

Cellulases are industrially important enzymes that are sold in large

volumes for use in different industrial applications viz. in starch processing,
animal feed production, grain alcohol fermentation, malting and brewing,
extraction of fruit and vegetable juices, pulp and paper industry and textile
industry (Ogel et al., 2001). There is a growing merit for cellulase in the field of
detergents and saccharification of agriculture waste for bioethanol technology.

Among hemicelluloses, xylan is the main component which is present in

nature in large amount and major byproduct of farming industry. Xylan (20-30%
of total dry weight of plant biomass) is a heterogeneous polysaccharide consisting
of 1,4-glucosidically linked β-D-xylose with branches containing xylose and
other pentose hexose and uronic acids. Due to the structural complexity,
biodegradation of xylan require synergetic action of endo-1,4-β-xylanase
(E.C.3.2.1.8) and β-xylosidase (E.C.3.2.1.37) and several accessory enzymes to
hydrolyze the substituted xylan. The expanding area of xylanase application
requires a search for novel enzyme and new microbial producers with high
specific activities and higher productivity (Ustinov et al., 2008).

Xylanase represents one of the largest group of industrial enzymes with

increasing market demands and its applications in prebleaching of kraft pulps,
bioconversion of lignocelluloses into feed-stocks and fuels (Kim et al., 2000),
extraction of coffee and plant oils, improvement of the nutritional properties of
agricultural silages and degumming of plant fibres such as flax, sun hemp and
ramie (Kuhad et al., 1997; Beg et al., 2001; Subramaniyan and Prema, 2002). In
food industry, the enzyme treatments has favourable effects on dough handing,
bread volume, texture and stability (Bhat et al., 2001), in improving digestibility
of animal feeds (Wong et al., 1988). Xylanase randomly hydrolyze the β-1,4-

3
glucosidic bonds of xylan to produce xylo-oligomers of different length (Viikari,
2007).

Submerged and solid state fermentation are the two most popular modes
of enzyme production in industry. Submerged fermentation is the cultivation of
the microorganism in a liquid medium containing soluble carbon source and
nutrients maintained under agitation. The use of the submerged culture is
advantageous because of the ease of sterilization and process control rendering it
easier to engineer in these systems. Depending on the strain and the culture
conditions, the enzyme can be constitutive or inducible, showing different
production and is a preferable method for enzyme production from bacterial
fermentations (Vidhayalakshni et al., 2009).

On the other hand, solid state fermentation is an attractive model for

fungal enzyme production because it stimulates the natural growth of
microorganisms on moist insoluble substrates in the absence or near presence of
free liquid facilitating the penetration of mycelia dub into the substrates (Pandey,
2002). It can be performed on a variety of lignocellulosic materials such as rice
bran, wheat bran, ragi bran, corn bran, soya bran etc which are proved to be
highly efficient in the production of cellulase and xylanase (Sonia et al., 2005).
Solid state fermentation is an efficient process to produce enzyme as it is
economical due to its lower capital investment and lower operating expenses
(Singhania et al., 2009).

Keeping in the view the importance of thermophilic cellulase and

xylanase their production from novel isolates specially extremophiles and their
industrial importance, the proposed study was carried out with the following
objectives:

To isolate and screen the cellulolytic and xylanolytic thermophilic

microorganism from the hot springs of Himachal Pradesh

To optimize the production of hydrolytic enzymes-cellulase and xylanase.

4
 Chapter-2

REVIEW OF LITERATURE
Thermophiles are microorganisms that live and grow in the extremely hot
environment that would kill most other microorganisms. They can be isolated
from a number of marine and terrestrial geothermally heated habitats including
shallow terrestrial hot springs hyderothermal vents systems, sediment from
volcanic islands and deep sea hyderothermal vents (Bertoldo and Antranikian,
2002). They grow best at temperature that is between 50-70°C, they will not
grow if the temperature reaches 20°C (Reysenbach, 2005). A thermophilic
cyanobacteria Thermosynechococcus elongatus and Thermosynechococcus
vulcanus which were isolated from Japanese hot spring grow optimally at
temperature 57°C (Onani et al., 2004).

Thermophiles are separated into three categories based on their cardinal

growth temperature:

• Moderate thermophiles have adapted a very large optimum growth

temperature ranging from 35 to 70°C
• Extreme thermophiles having an optimum growth temperature between
55-85°C
• Hyperthermophiles having very high growth temperature between 75-
113°C.

On the other hand they can also be classified as:

• Obligate thermophiles having an optimal temperature between 65-75°C

do not grow below 40°C
• Facultative thermophiles have the optimum growth temperature between
50-60°C they are also able to grow at temperature around 37°C
• Thermotolerant thermophiles show the maximum growth at temperature
around 45-50°C and they are also grow below 30°C (Kristjansson and
Hreggvidsson, 1995).

2.1 THERMOPHILIC MICROORGANISMS

The classification scheme of organism recognises three major domains of

living organisms, archea, bacteria and eukaryotes (Allers et al. 2005). Living
organisms are usually divided into several classes based on the temperature at
which they grow well. Moderate thermophiles are primarily bacteria and display
optimum growth temperature between 60 and 800C, hyperthermophiles are
archea and grow optimally at 800C or above (Andrade et al., 1999).

Ecological studies have showed that both aerobic and anaerobic species
and many morphological and physiological types of microorganisms can exist in
thermophilic environments (Brock et al., 1978). Extreme thermophiles are mostly
distributed among the genera of Bacillus, Clostridium, Thermoanaerobacter,
Thermus, Thermotoga, Aquifex. Most hyperthermophiles, include the two
kingdoms of Archaea, Crenarchacota (Sulfolobus, Pyrodictium, Pyrolobus.),
Euryarchaeaota (Thermococcus, Pyrococcus ), methanogenes (Methanococcus,
Methanobacterium), sulfate reducers and halophiles.

Fig 1: Classification of microbial life (Woese, 1988)

6
 Microbes are rich source of enzymes and technologies for large scale
manufacture of several industrially useful enzymes. Extremophiles have studied
intensively the world over to discover enzymes stable to and active under
relatively harsh environment (Srinivasan et al., 1999).

Table 1. Examples of sites serving as a source of microorganisms which can

provide thermotolerant enzymes

Sources Microorganisms Enzyme References

Hot spring Thermus sp. αamylase Shaw et al.
(1995)
Hot spring Bacillus subtilis, B. Xylanase Singh et al.
Cereeus and (2010)
Paenibacillus
ehimensis
Decomposed plant Clostridium absonum Cellulase free Swaroopa and
samples froma CFR-702 xylanase Krishna (2000)
lake
Garbage dump Bacillus circulans Xylanase Ashita et al.
(2000)
Hot spring, yellow Thermus aquaticus, Cellulolytic Brock.(1978)
stone national Bacillus
park acidocalsarius
Hot spring Geobacillus spp. Xylanase Canakci et al.
(2007)
Hot spring Thermotoga sp. Xylanase and Saul et al.
cellobiohydrolase (1995)
Household Bacillus sp. AQ-1 Xylanase Wahyuntari et
aquarium al. (2009)
sediment
Hot spring Bacillus licheniformis Cellulase Acharya and
WBS1 and Bacillus Choudhary.
sp. WNS3 (2011)
Soil Actinomycetes Cellulase Aboul-Enein et
al. (2009)

7
2.1.1 Thermophilic bacteria

Hyperthermophiles that grow from 800C to above 1000C such as

Pyrococcus furiosus, Thermotoga neopoltana and Thermotoga maritime have
been isolated and these types of microbes are thought to be among the first form
of life to have evolved on earth. To date about 50 species, 20 genera and 11
orders of hyperthermophiles are known. Some of them belong to the eubacterium
by most of they are archea (Kristjansson and Hreggvidsson, 1995). Thermophiic
bacteria have received considerable attention as sources of highly active and
source of thermostable cellulolytic and xylanolytic enzymes. These are
thermostable cellulolytic enzymes characterized from thermophilic cellulolytic
bacteria such as Clostridium thermocellum, Caldocellum saccharolyticum and
Acidothermus cellulolyticus (Bergquist et al., 1999). Most of the enzyme isolated
from hyperthermophiles genus Thermotoga have temperature optima at 900C.
Thermotoga mariima and Aquifex pyrophilus exhibit the highest growth
temperature of 90 and 950C respectively (Herbert and Sharp, 1992). Among the
thermophilic aerobic bacteria only few actinomycetes are cellulolytic, notably
Thermonospora curvata.

Mawadza et al. (2000) purified and characterized the cellulase produced

by two Bacillus strains CH4 and CH8 isolated from hot springs in Zimbabwe,
showing 100% homology with endoglucanase from Bacillus subtilis. Similarly
Zuauya and Zvidzai (1995) reported the constitutive production of endoglucanase
by a Bacillus sp. isolated from a Zimbabwean hot spring. The sporulating and
aerobic Bacillus sp. produced endoglucanase when cultured on medium with
initial pH between 5.0 and 9.0 and at 30 and 600C.

Recently the xylanase from the strains of Cellulosomonas uda,

Microbacterium ulmi, M. xylanolyticum, B.pumilus, Streptomyces sp,
Microbacterium barkeri, Bacillus niabensis, B. funiculus, B. megaterium,
Pseudoxanthomonas suwonesis, Cupriavidus gildari and Rhodococcus have been
reported (Archana and Satyanarayan, 2003; Beg et al. 2001 & Duarte et al.
2000).

8
Table 2: Source microorganisms and properties of thermostable cellulases

Organisms Optimal Optimal pH Refrences

temperature(0C)
Anaerocellu 85-90 5.0-6.6. Zverlov et al.
thermophilum (1998)
Bacillus subtilis 65-70 5.0-6.5 Mawadza et al.
(2000)
Pyrococcus 102-105 - Kengen et al.
furiosus (1993)
Rhodothermus 95 6.5-8.0 Hreggvidsson et
marinus al. (1996)
Thermotoga 95 6.0 Bok et al. (1998)
nepoltana
Thermogota 106 6.0-6.6 Bok et al. (1998)
neapoltana

Table 3. Source microorganisms and properties of thermostable xylanase

Organisms Optimal Optimal pH References

temperature(0C)
Bacilllus 80 6.8-7.0 Breccia et al.
amyloliquefaciens (1997)
Bacillus circulans 80 6.0-7.0 Dhillon and
Khanna (2000)
Bacillus sp. 60-75 8.0-9.0 Gessessse (1998)
Bacillus subtilis 50 6.0 Paula et al. (2002)
Clostridium 75 8.5 Swaroopa and
abosum Krishna (2000)
Fusarium 55 5.0-5.5 Badal (2002)
proliferatum
Thermoascus 50 5.0 Kalegoris et al.
auranticus (1998)
Thermotoga 95 6.0 Gabelsberger et
neapolitana al. (1993)
Thermogota sp. 80 7.0 Ruthersmit and
strain FjSS3-B1 Daniel (1991)

9
2.1.2 Thermophilic Fungi

An aerobic thermophilic cellulolytic microorganism includes several

species of fungi and few species of filamentous bacteria belonging to the family
Actinomycetaceae. Among the fungi Mycelipothora thermophila is of
considerable interest as it grows well on cellulose in submerged culture at 500C
and pH 4.5 and synthesised the cellulolytic enzymes. Fungi Trichoderma sp. and
Aspergillus sp. are mostly exploited for cellulase production. Similarly
Aspergillus fumigates has been shown to grow well on cellulase in submerged
culture at 450C and pH 4.6 and to produce considerable amount of cellulase
(Shaker et al., 1984). Celluloytic and pectinolytic activities of five Capnodium
isolates (sooty mould) from Zimbabwe were investigated by Mwenje and Mguni
(2001). The five isolates showed the ability to produce endo-1-4-β -glucanase,
pectin lyase, polygalacturonase, pectin lyase.

2.1.3 Stability of thermophiles:

Thermophiles are reported to contain the proteins which are thermostable

and resist denaturation and proteolysis (Kumar and Nussinov, 2001). Specialized
proteins known as chaperonins are produced by these organisms, which help after
their denaturation to refold the proteins to their native form and restore their
functions (Everly and Alberto, 2000). The cell membrane of thermophiles is
made up of saturated fatty acids. The fatty acid provides a hydrophobic
environment for the cell and keeps the cell rigid enough to live at elevated
temperatures (Herbert and Sharp, 1992). Thermophiles also tolerate the high
temperature by using increased interactions, electrostatic, disulfidebridge and
hydrophobic interaction than non thermophilic microorganisms (Kumar and
Nussinov, 2001).

2.3 THERMOENZYMES AND THEIR APPLICATION

In 1976, Thomas D Brock of the University of Wisconsin reported for the

first time existence of microorganisms with optimal growth temperature higher
than 75°C growing in the hot spring, Yellow National Park (Brock, 1967). In
1980 the first hyperthermophilic enzyme were purified , which proved to be

10
extremely stable at high temperature and to exhibit no or low activity at
temperature below the growth temperature of the organism (Bouzas et al., 2006).

Natural terrestrial biotypes of hyperthermophiles include hot springs and

solfataric fields with a wide range of pH (0.5-9.0), low salinity (0.1-0.5%) and
deep geothermally heated oil containing stratification. Natural marine biotypes
are shallow hyderothermal system, abyssal hot vents (black smokers) and active
seamounts where the salinity levels are high (3%) and pH value ranges from the
5.0-5.8. Thermostable enzymes isolated from thermophilic microorganisms, has
number of commercial application because of their inherent stability (Demirijian
et al., 2001). Thermophilic microorganisms are of special interest to
enzymologists both at the fundamental and industrial level as a natural source of
enzymes that are active and stable at high temperature (Zeikus 1979), since most
of the process are carried out at high temperature, there is a need of thermophilic
enzymes (Haki and Rakshit, 2003).

2.3.1 Advantage of working with thermostable enzymes

Industries such as laundry, detergents, leather and paper require highly

stable enzymes that are able to remain stable at extreme pH and temperature. The
increasing stability at high temperature with respect to mesophiliic enzymes
make them more suitable for harsh industrial process, their stability is mostly
associated with the higher resistance to chemical denaturants commonly used in
industrial reaction, performing enzymatic reaction at high temperature, allows
higher reaction rates and process yield because of:

• a decrease in viscosity
• an increase in diffusion coefficient of substrates
• an increase in solubility of substrates and products
• an favourable equilibrium displacement in endothermal reactions (Haki
and Rakshit, 2003).
• the risk of contamination by the mesohiles are decreased

11
• Once expressssed in mesophilic hosts, thermophilic and
hyperthermophilic enzymes are easier to purify by the heat treatment
(Kumar and Swati, 2001).

2.4 THERMOSTABLE ENZYMES

2.4.1 Thermostable cellulases and xylanases:

Cellulolytic enzymes accounts for 20% of world enzyme market (Jaradat,

2008). More efficient hydrolysis of the lignocelluloses is obtained by using only
thermostable enzyme components (endoglucanase, exoglucanase, β-glucosidase
and xylanase (Li et al., 2006 & Gao et al., 2008). Cellulases are of important
interest because of their application in industries such as starch processing,
animal food production, grain alcohol fermentation, malting and brewing,
extraction of fruits and vegetable juices, pulp and paper industry and textile
industry (Adsul et al., 2007 & Kaur et al., 2004). In the current industrial
processes, cellulolytic enzymes are employed in the colour extractions of juices,
in detergents causing colour brightening, softening, in the bio stoning of jeans, in
the pre-treatment of biomass that contains cellulose to improve nutritional quality

Table 4. Showing the importance of thermostable enzyme in industries

Enzyme Temperature Bioconversions Application Refrences

range °C
α-amylase 90-100 Starch- dextrose Starch hydrolysis, Poonam and
syrups brewing, baking, Dalmel
detergents (1995)
α-amylase 50-60 Starch-dextrose Production of Haki and
fungal syrups maltose Rakshit (2003)
Xylanase* 45-65, 105 Craft-pulp- Pulp and paper Sharma et al.
xylan+ lignin industry (2009)
Cellulase** 45-55, 95 Cellulose- Cellulosic Ando et al.
glucose hydrolysis, (2002)
polymer
degradation in
detergents
Protease 65-85 Protein-amino Baking, brewing, Zuidzai and
acids and detergents, leather Zvauya (2001)
peptides industry

12
of forage (Buchert et al., 1997; Niehaus et al., 1999 & Bhat, 2000; Nakamura et
al., 2001; Van et al., 2001 & Zhou et al., 2001). In order to attack the native
crystalline cellulose, which is water insoluble and occurs as fibres of densely
packed structures, however, thermostable cellulases active at high temperature
and high pH are required. The biopolishing process of cotton in the textile
industry, for example, requires cellulase stable at high temperature close to 100°
C (Ando et al., 2002). Some filamentous fungi produce cellulases retain high
cellulose degrading activity at temperature of 50-70°C and is a good producer β-
glucosidase, show the high activity at 70°C of 23.5 h (Gomes et al., 2000),
optimum pH and temperature between 65 to 80°C and 4.5 h.

Thermostable xylanase were isolated from a number of bacterial and

fungal sources. Members of Bacillus sp., Streptomyces sp., Thermoascus
aurantiacus and Fusarium proliferatum have been reported to produce xylanases
which are active at temperature between 50-800C. Alkalophilic and cellulase free
xylanase with an optimum temperature of 650C from Thermoactinoyces
thalophilic (Kohilu et al., 2001) and cellulase free xylanase from Clostridium
abusonum (Swaroopa and Krishna, 2000) were reported recently. Thermostable
xylanases active at alkaline pH are of great importance for application in pulp and
paper industry to decrease the consumption of chlorine chemicals. Xylanases are
useful in bioconversion of lignocellulosics to fuel and chemicals, to improve
silage for better digestion by ruminants, to improve quality of detergents and also
for clarification of fruit juices in flour improvement of bakery products and in
controlling environmental hazards through biopulping.

Industries such as laundry, detergents, leather and paper require high

stable enzymes that are able to function and remain active at extreme pH and
temperature (Phitsuwan et al., 2010). Alkaline cellulase and xylanase are
extensively studied and successfully applied in detergents and pulp industries
(Horikoshi, 1999).

2.5 ISOLATION AND SCREENING OF CELLULOLYTIC

MICROORGANISMS
Samples of water from the hot spring of Uzon Caldera with temperature
from 68 to 87°C and pH of 4.1 to 7.0 supplemented with proteinacoeus (albumin,

13
casein, or α or β keratin) or carbohydrates (cellulose, carboxymethylcellulose,
chitin and agarose) biological polymer, filled with thermal water and incubated
at the same site with the contents of tube freely accessible to the hydrothermal
fluid, several enrichment culture growing in-situ on different polymeric gene
substrates were obtained Denaturating gradient gel electrophoresis (DGGE)
analysis of rrs (16S rRNA) gene fragment obtained after PCR with Bacterial
specific primer showed that the bacterial communities on carbohydrate rates
included the genera Cladicellulosiruptor and Dictyoglomus and that developing
on protein contained thermotogales order. DGGE analysis performed after PCR
with archea and crenarchaeota specific primes showed that Crenarchaeota
phylum were present in both carbohydrate and protein degrading communities
(Kublanov et al., 2009).

EHP1 (Anoxybacillus flavithermus), EHP2 (Geobacillus

thermodenitrificans) and EHP3 (Geobacter steriothermophiles) are the
thermophilic cellulase producing bacteria have been isolated from an Egyptian
hot spring by enrichment of the water and soil samples with cellulose for 3 weeks
at 70°C. Maximum cellulase production by Anoxybacillus flavithermus was
detected at the end of the stationary phase (36 h). Crude cell has the activity
towards the avicel, CMC, cellobiose and xylan but no activity towards the p-
nitrophenyl-β–D-glucopyranoside. The optimum temperature and pH for the
crude enzyme activity were 75°C and 7.5 respectively (Ibrahim and Ahmed,
2007).

Malkawi and Al-Omari. (2010) investigated the different microbial

diversity at five major hot spring in Jordan (Ashounah, Waggas, Zara, Zarqa
Ma’in and Afra springs) using microbiological culture based and molecular
culture independent approaches. 132 isolates were obtained from the different hot
springs. 125 bacterial isolates were gram positive rods while other were gram
positive coccobacilli, temperature for the bacterial growth was up to 750C, all
isolates will grow at the pH 7.0 and one single isolate isolated from the Zarqa
MA’spring, which was able to grow at pH 3.5. Culture independent approaches
using PCR identified it as green nonsulfur bacteria, heliobacteria and

14
methanogenic archea from methanogenic DNA extracted directly from water and
mat sample from each thermal spring. 9 water samples and 9 mat samples from
all springs revealed the bacteria yielding the amplicon size of (1500 bp). While 4
water sample from Zarqa Ma’in springs. 5 mat sample and wages well represent
the archea having amplicon size of 650 bp. PCR identification using primer pair
specific to the rrs (16S rDNA) gene sequencing of the genus Bacillus indicated
that 96.97% (128 out of 132) of bacterial isolates have the size of PCR amplicon
(320 bp) belong to genus Bacillus.

Chang et al. (2009) isolated the five strains of thermophiles from Brassica
wastes and studied there hydrolytic activity on the various cellulosic biomass
substrates. rrs (16S rRNA) sequencing identified these strains as
Thermoactinomycetes and Bacillus species. Thermoactionomycetes sp. strain
performs best among them producing a maximum cellulase activity of 2.3 IU/ml.
An increase in nitrogen contents from the 0.74 to 0.91 % and decreases in carbon
contents from 15.4 to 12.2 % resulted in higher efficiency and bioactivity during
compositing from Brassica waste..

Diversity of actinomycetes were isolated from soil samples obtained

from the five govermorates of Egypt (El-Behira, Tanta, Giza, Kafer El-Sheekh
and Alexindra), a total of 10 actinomycetes probably Streptomyces were obtained
from the different areas, the cellulolytic activity of these isolates was assayed,
one isolates from Giza gave the highest cellulolytic activity (Al-Enein et al.,
2009).

Geobaciliius pallius was isolated from the empty fruit bunch-palm oil
effluents (POME) compost at thermophilic stage, the isolation of microbes from
the compost heap was conducted at 7 days of compositing process, where the
mixture of compositing material consisted of 45.8% cellulose, 17% hemicellulose
and 28.3% lignin contents. Temperature, pH and the moisture contents were 58.3,
8.1 and 65.5 °C. The congo red test was conducted to test CMCase activity
(Baharuddin et al., 2010).

15
 46 thermophilic fungus were reported by Rajavaram et al. (2010) from the
different places of Andhra Pradesh using various substrates like ground coalmine
soil, bird nest material, vermicompost, cow dung, poultry litter, decompositing
pits prepared with agro waste, municipal waste, zoo dump material and industrial
waste etc. Thermophiles Humicola langinosus were present in all substrate and
Aspergillus fumigates found as a thermotolerant.

A circular, dark yellow, shiny and convex with entire edge, thermophilic
coccoid methanogen were isolated from the non thermal freshwater from the
Fritton lake, England. The minimum doubling time occurs at 57°C, upper and
lower limit for the growth were 26 to 62°C and pH was between 7.0-7.5. Electron
micrography showed a monolayer cell wall 20 nm thick, DNA ratio was 49.2
mol% guanine plus cytosine. A hydrogen plus carbondioxide and formate were
substrates for methanogenesis. Methane production was stimulated by the yeast
extract, casamino acid and trypsine (Harris et al., 1984).

To reduce the production cost of cellulase Gautam et al. (2011) isolated

two novel cellulase producing fungi (Aspergillus niger and Trichoderma sp.)
from municipal solid waste residue 4-5% (w/v). A combination of peptone and
yeast extract 1.0% (w/v) were found to be the best combination for the
production of cellulase by Aspergillus niger and Trichoderma sp. Optimum
temperature and pH of the medium for the cellulase production by A. niger were
40°C and 6-7, whereas those for the production of cellulase by Trichoderma sp.
were 45°C and 6.5. Cellulase production from Aspergillus niger and Trichoderma
sp. can be an advantage, as the enzyme production rate is normally higher as
compared to other fungi.

2.6 ISOLATION AND SCREENING OF XYLANOLYTIC

MICROORGANISMS:

Singh et al. (2010) reported the alkalothermotolerant bacteria from 17

samples collected from the Manikaran. Eleven isolates showed the growth and
production of xylanase at different temperature greater or equal to 50°C were
selected for quantitative estimation in modified reese mineral liquid medium
containing wheat bran. Maximum xylanase activity was produced by isolates H-7

16
followed by H-9 and R-9. The microscopic observation showed that the isolates
possessed the typical rod with endospore, characteristic of genus Bacillus. Using
BIOLOG MICOLOG 3 software the isolates H7, H9 and R9 were identified as
Paenibacillus ehemensis, Bacillus cereus/ B. thuringenesis and B. subtilis,
isolates were found to be oxidase and catalase positive.

A xylanolytic enzyme producing Bacillus subtilis strain was isolated from

hot spring. Optimal xylanase production of about 12 IU/ml was achieved at pH
6.0 and 500C, within 18 h of fermentation. Increasing temperature at 550C a
higher productivity was obtained in the batch reactor 45,000 U/l/h compared to
shake flask fermentation 12,000 U/l/h. Optimal xylanolytic activity was reached
at 600C on phosphate buffer (Sa-Pereira et al., 2002).

Tan et al. (2001) isolated a xylanolytic thermophilic bacterium (IT-08)

from the Gunung Pancar hot spring, enrichment of the sample was conducted in
modified thermus medium supplemented with 0.5 % oat spelts xylan, when
grown on xylan containing medium it produced the thermoactive xylanase, active
at temperature between 40-100°C, at pH values between 4.0 and 9.0, optimum
values obtained at 80°C and pH 6.0, rrs (16S rRNA) indicates that IT-08
resembles Bacillus thermolevorans.

Nakamura et al. (1995) isolated an extracellular xylanase producing

thermoalkalophilic Bacillus sp. strain TAR-1 from soil. The enzyme was most
active in the range of pH 5.0 to 10.0 at 500C. Optimum activities for activity were
750C at pH 7.0 and 700C at pH 9. Xylanase R had a Km of 0.82 mg/ml and Vmax
of 280 µmol/min /mg at 500C and pH 9.0.

A cellulase free and alkali-stable xylanase was isolated Bacillus pumilus

SV-85S from soil sample of Ambala Cantt, Haryana by Nagar et al. (2010), using
cheap and easily available agro-residue wheat bran. Optimization of fermentation
conditions enhanced the enzyme production to 2995.20±200.00 IU/ml, which
was 9.91 fold higher than the activity under unoptimized basal medium (302.20
IU/ml).

17
 An aerobic, thermophilic, xylanolytic bacterium was isolated from local
soil. The results of rrs (16S rRNA) sequence comparisons indicated that the
isolate was closely related to Bacillus caldoxylolyticus and Bacillus sp. strain
AK1. These organisms exhibited 94% levels of ribosomal DNA sequence
homology. Studies on the xylanase characterisation from liquid cultures grown on
beech wood xylan revealed that the enzyme retained 100% of activity for 2 h at
temperatures ranging from 30 to 500C, while at 60, 70 and 1000C, 10%, 11% and
29% of the original activities were lost, respectively. The optimum pH of the
enzyme was found to be between 6.5 and 7.0. After incubation of crude enzyme
solution for 24 h at 250C and at pH 5.5 to 8.0, a decrease of about 12% of its
original activity was observed by Cordeiro et al. (2002).

An aerobic thermophilic xylanolytic, spore forming bacterium has been

isolated by Touzel et al. (2000) from the farm soil situated underneath a manure
heap in northern France, stained negative in the gram stain, occurs as short rods
which sometimes form the chain, spores are ellipsoidal, central to sub terminal
and occur in swollen sporangia. It grow optimally at temperature up to 63°C and
in the pH ranges 6.5 – 8.5, CO2 act as the growth stimulator at the start of the
culture, utilizing xylose, maltose, arabinose, mannose, cellobiose, galactose,
sucrose and starch. The G+C contents of strain XE is 57.5 mol% 16S rDNA
indicated that the isolates represented a new genus and species Thermobacillus
xylanonilytic strain XEtp.

2.7 DIFFERENT SUBSTRATES USED FOR ENZYME

PRODUCTION

• Cellulose
• Xylose
• Lignocellulosic material are the different substrates that are being used for
the enzyme production. Cellulose and Xylan are conventional material for
the production of cellulase and xylanase enzyme, and are costly availably
material for the enzyme production. So, we proceed for the cost effective
material like lignocellulosic material for the enzyme production.

18
 Agricultural by products rich in hemicelluloses constitute one of the most
potential, economic and easily available substrate. Xylan of production medium
was replaced by various agricultural based crude substrate like wheat husk, wheat
bran, rice husk, sugarcane bagasse, wood husk and paper industry waste for
enzyme production (Sharma and Bajaj, 2005). Xylan is costly for large scale
production of xylanases, therefore lignocellulosic materials are used as cost
effective substrates for xylanase production (Haltrich et al., 1996 & Beg et al.,
2000).

2.7.1 Lignocellulosic biomass

Lignocellulosic biomass comprising forestry, agricultural and agro-

industrial wastes are abundant, renewable and inexpensive energy source,
including variety of materials such as sawdust, poplar tree, sugarcane bagasses,
waste paper, brewer spent grains, switch grass and straw, stems , stalk, leaves,
husks, shell and peels from cereals like rice, wheat, corn sorghum and barley.
Lignocellulosic waste accumulated every year in the large quantity causing larger
environmental problems. Due to their chemical composition based on sugars and
other compound of interest they could be utilized for the production of value
added products such as ethanol, food additives, enzymes, organic compounds and
other. Therefore beside the environmental problems caused by their accumulation
in the nature, the non use of these materials constitutes a loss of potentially
valuable source (Mussatto and Teixeira, 2010).

Fig 2: Representation of lignocellulosic structure showing cellulose,

hemicelluloses and lignin fractions (Mussatto and Teixeria, 2010)

In nature a variety of bacteria and fungi produces cellulase and xylanase

to hydrolyze the insoluble polysaccharides into soluble oligomers and

19
subsequently to oligomers. This conversion is quite difficult and owing to the
complex structure of plant cell wall degradation (Moiser et al., 2005).

2.7.2 Cellulose:

Biomass comprises on average of 50% cellulose, 30% hemicelluloses and

20% lignin by dry weight. Hemicellulose is an important biomass reservoir in the
plant cell wall (Hongpattarakre, 2002).

Celluloses are the most common carbohydrate occurring in plant

throughout the world. In fruits and vegetables cellulose comprising of almost
50% of carbohydrates while hemicelluloses comprise 15-34% (Das and Singh,
2004). Cellulose is an unbranched linear homopolymer of glucose units linked by
β-1,4-D-glucosidic bonds, forming an insoluble, crystalline microfibrils which
are highly resistant to enzymatic hydrolysis (Hongpattarakre, 2002).

Cellulases are inducible enzymes which are synthesized by

microorganisms during their growth on cellulosic materials. Cellulase enzyme ,
which can hydrolyze cellulose forming glucose and other commodity chemicals
can be divided into three type : endo-glucanase (endo-1,4-β-D-glucanse, EG, EC
3.2.1.4); cellobiohydrolase (exo-1,4-β–D-glucanase CBH, EC 3.2.1.91) and β-
glucosidase(1,4-β-D-glucosidase , BG, EC 3.2.1.21) (Da-Silva et al., 2005). The
endoglucanase randomly hydrolyse the β (1-,4) bonds in the cellulosic molecule
and exocellobiohydrolases in the most case release a cellobiose unit showing a
recurrent reaction from chain extremity. Cellulases are produced by the
microorganisms (molds, fungi and bacteria) during their growth on cellulosic
materials, major application of cellulose are in textile and detergent industries
(Bhat, 2000). Three main types of the enzyme involved in the cellulose
degradation i.e. exo-β-1,4-glucanase, endo-β-1,4-glucanase and β glucosidase , in
which the endoglucanase act internally on the chain of cellulose cleaving β linked
bonds liberating non reducing ends and exoglucanase act removing cellobiose
from this non reducing end of cellulose chain, finally β- glucosidase complete the
saccharification by splitting cellobiose and small cello-oligosaccharides into
glucose molecule (Raza and Shafiq-Ur-Rehman, 2009).

20
 Fig 3: Structure of cellulose (Simon and Scherage, 1988)

2.7.2.1 Cellulose hydrolysis

Chemical and enzymatic hydrolysis is the most common technique for the
hydrolyzing cellulose. Chemical method also called as concentrated acid
hydrolysis, is conducted with mineral acids such as H2SO4 or HCl at temperature
of about 160°C and pressure of about 10 atm (Sun and Cheng, 2002; Kumar et
al., 2009).

Fig 4: Conformational structure of cellulose and modes of action of various

components of cellulase (Karmakar and Ray, 2011)

21
 Enzymatic hydrolysis of cellulose is a reaction carried out by cellulase
enzyme, which is a mixture of several enzymes among which at least three major
groups are involved in hydrolysis of cellulose are:

• β-1,4 endoglucanase (EC 3.2.1.4) which attacks region of low crystallinity

in the cellulose fiber creating free chain ends
• β-1,4-exoglucanasae or cellobiohydrolase (EC 3.2.1.91) which degrades
the molecule further by removing cellobiose units from the free chain
ends
• β- glucosidase (EC 3.2.1.21) which hydrolyses cellobiose into glucose
(Prasad et al., 2007 & Cao and Tan, 2002).

2.7.3 Hemicellulose

Xylan are the heteropolysaccharides consisting of a β-1,4-xylopyranosyl

backbone with branches of acetyl arabinosyl and glucuronyl residues. Xylan is a
heteroploymer composed primarly of β-1,4-linked xylose with various amount of
arabinose, glucose, galactosides, uronic acid and other sugars as side group
depending on the plant source. In some higher plants and agricultural wastes,
xylan constitutes from 20-40% of the dry weight (Rifaat et al., 2005),
hemicelluloses has different classification, depending upon the sugar residue e.g.
xylans, mannans, glucans, glucouronoxylans, arabonoxylans, glucomannans,
galactomannnans, galactoglucomannans, β-glucans and xyloglucans. When
compared to cellulose, hemicelluloses differ by composition of sugar residue
units, by presence of shorter chain, a branching of main chain molecules and the
amorphous structure, which made its structure easier to hydrolyse than cellulose
(Butt et al., 2008).

Fig 5 : Structure of xylan (Roubroeks et al., 2001)

22
 Xylanases (EC 3.2.1.8) are ubiquitious in nature and play an important
role in the degradation by catalysing the endo-hydrolysis of the 1,4-β-D-xylosidic
linkage into short xylooligosaccharides (Beily, 1985). Xylanase are extracellular
enzyme produced by a variety of fungi, bacteria and yeast. Based on the primary
structure of the catalytic domain, most xylanase are confined to glycoside
hydrolase (GH) families 10 and 11 (Collins, 2005).

2.7.3.1 Xylan hydrolysis

Xylanases are the endoactive enzymes, are generally produced in the

medium containing xylan and also containing xylanase hydrolysates as the
carbon source, and attack the xylan chain in a random manner, causing a
decreases degree polymerization of the substrates and liberating shorter oligomer,
xylobiose and xylose. The mode of action of different xylanases and hydrolysis
products various accoding to the source of enzyme. Xylanase is a hydrolyse that
catalysis, complex sugar primarly xylan and related compounds to simple sugar
the primary product being xylose. β-3-1,4-xylan are heterogeneous
polysaccharides found in the cell wall of all land plants and almost in all plant
parts, the hydrolysis of their characteristic backbone, consisting of β-1,4-linked
D-xylosyl residues, involved β-1,4-xylanases (1,4-β-D-xylan xylanohydrolase;
EC 3.2.1.8 and β-xylosidase (1,4-β-D-xylan xylohydrolase, EC 3.2.1.3.7),
xylanase attack internal xylosidic linkage on the backbone and β–xylosidases
release xylosyl residue by endwise attack of xylooligosaccahrides (Rani and
Nand, 2001).

xylanase
Xylan + H2O -----------------------› xylose sugar

23
 Fig 6: Hydrolysis of xylan (Lemos et al., 2000)
Where A: Endoxylanase
B: Xylosidase

2.8 EFFECT OF PRETREATMENTS ON LIGNOCELLULOSIC

BIOMASS FOR ENZYME PRODUCTION

Waste from pulp and paper industries has long been recognized as
refractory effluents due to the presence of lignocellulosic (i.e. cell wall materials
of plants) including cellulose, hemicelluloses and lignin.

\ Hemicellulose

Fig. 7: Schematic representation of pretreatment effect on lignocellulosic

biomass (Mosier et al., 2005)

24
 The hydrolysis of lignocellulosic waste has been reported as a rate
limiting step in many anaerobic processes. Lignocelluloses are resistant to direct
enzymatic hydrolysis because of the two major hindrances related to the compact
cellulose structure and lignin barrier surrounding cellulose (Mansfield et al.,
1999).

Size reduction, alkali or acid treatment, steaming process and freeze

explosion have been suggested for the pretreatment of lignocellulosics to enhance
the efficiency of subsequent biological treatment of the wastewater (Schmidt and
Thomson, 1998) has been employed to modify the physical and chemical
characteristics, to reduce the polymerization of cellulose, creating a residue that
can be hydrolysed by enzymes. Heat treatment initially shatters the lignin seal by
altering the ligninocellulosic crystallinity. In addition mild acid loosen the
structure resulting in an improved overall rate of hydrolysis due to increased
accessibility of enzymes. Chemical pretreatment of the lignocellulosic material
resulted in better enzymatic saccharification as compared to the untreated
material because of the enhanced swelling which leads to the increase in internal
surface area and decrease in degree of polymerization causing separation of
structural linkage between lignin and carbohydrates apart from distruption of
lignin structure (Mussattos and Teixeria, 2010).

2.8.1 EFFECT OF DIFFERENT PRETREATMENTS ON CELLULASE

PRODUCTION

The production of cellulase by Trichoderma reesei F-418 cultivated on

alkali treated rice straw using solid state fermentation technique. Rice straw are
of the most abundant lignocellulosic waste by products worldwide, high cellulose
activity was obtained when the fungus was cultivated on substrate with 75%
(v/w) moisture, pH 4.8 for 5 days incubation at 28±2°C , giving 16.2 U/g
substrates cellulase of 1.2 IU/ml culture filtrate applied for saccharification (5%
w/v) of alkali treated rice straw, in 0.1M citrate buffer pH 4.8 in shaker water
bath of 100 rpm , sugary solution of 1.07% glucose was achieved by
Sacchararomyces cerevisiae SH-5 under static condition giving 5.1% (v/v)

25
ethanol after 24 h, fermented mash contained 3.6 g/L yeast cell can be utilized as
food yeast used for animal feeding (Fatama et al., 2010).

Thermomyces lanuginosus CAU44, a newly isolated thermophilic fungus

strain, used for the production of extracellular xylanase on various lignocellulosic
materials under shake flask conditions. High-level production of xylanase by the
strain achieved by optimizing the type of carbon sources, substrate concentration,
particle size and surfactants in the culture medium. The titre of xylanase activity
obtained was up to 4156 U ml−1 was reported by Jiang et al. (2005).

A thermophilic, cellulolytic, ethanologenic Clostridium thermocellum

CT2 isolated by Reddy et al. (2010) from the elephant droppings, the
optimization condition for cellulose fermentation were 60°C , pH 7.5 , inoculum
size 5% and incubation time 5 days, that was being used as the biodegradation of
cellulosic waste using siingle step fermentation. Clostridium
thermosaccharolyticum HG8 and Thermoanaerobacter ethanolicus ATCC 31937
were used in co-culture fermentation with CT2, coculture is more importance in
term of the ethanol production, cellulose degradation and reducing sugars
utilization. A maximum ethanol yield of 0.41 U/g obtained on co-culture with
CT2 with HG8 on alkali treated banana wastes, this was the first report on
anaerobic single step conversion of banana waste to ethanol by C. thermocellum.

Qi et al. (2009) optimized the enzymatic conversion of widely available

lignocellulosic biomass-wheat straw (WS), pretreatment facilitating the enzymatic
saccharification process was first performed by alkaline peroxide, resulting in a
substrate consisting of 60.17% cellulose, 29.53% hemicelluloses, and 4.59%
lignin. Using response surface methodology, the combined effects of enzyme
loading, substrate concentration, surfactant concentration, and reaction time on
hydrolysis yield from enzymatic saccharification of WS were further investigated.
The results showed that both enzyme loading and substrate concentration had
interactions with surfactant concentration. A quadratic polynomial equation for
predicting the hydrolysis yield was developed. The experimental results were in
good agreement with predicted values. Therefore, the model could be successfully

26
used to identify the effective combinations of the four factors for predicting
hydrolysis yield.

Effect of particle size on steam-explosion pretreatment of herbaceous

lignocellulosic biomass studied by Ballestros et al. (2002), effectiveness of
enzymatic hydrolysis of the cellulosic residue is presented for steam-explosion
pretreatment of an agriculture residue (Brassica carinata) using different particle
sizes. The parameters tested were: particle size (2/5, 5/8 and 8/12 mm),
temperature (190 and 210.8°C), and residence time (4 and 8 min). Larger steam-
exploded particle (8/12 mm) results in higher cellulose and enzymatic
digestibility. The use of very small particles in steam explosion would not be
desirable in optimising the effectiveness of the process improving economy.

The agricultural crop residues such as paddy straw, wheat straw and
sugarcane bagasse are abundantly available having rich source of sugars,
pretreatment process was developed for hydrolyzing paddy straw, wheat straw
and sugarcane bagasse into fermentable sugars. The powdered substrates were
delignified with 3% NaOH for microbiological and commercial cellulase enzyme
pre-treatment. Among the fungal cultures Trichoderma reesei has yielded
maximum reducing sugar of 22.30 mg/g in paddy straw, 25.56 mg/g in wheat
straw and 28.56 mg/g in sugarcane bagasse. In case of commercial cellulase
enzyme sugarcane bagasse has yielded maximum reducing sugar 136.86 mg/g
after hydrolysis with 2.5 per cent enzyme concentration at 45°C for 24 h of
incubation (Raghavendra et al., 2007).

Budarin et al. (2010) developed a microwave assisted low temperature

decomposition process for production of high quality fuels from biomass. 180°C
was identified as key in the process mechanism, as the amorphous region of
cellulose softens allowing a microwave induced rearrangement. Proton transfer is
then possible under the microwave field resulting in acid catalyzed
decomposition. This low temperature process has been shown to be suitable for
scale-up, producing a high quality char for use as a coal replacement and bio-oil
suitable for upgrading to liquid fuel. In one of the study, production of 5-
hydroxymethylfurfural (HMF) and furfural from lignocellulosic biomass was

27
studied in ionic liquid in the presence of CrCl3 under microwave irradiation. Corn
stalk, rice straw and pine wood treated under typical reaction conditions produced
HMF and furfural in yields of 45–52% and 23–31%, respectively, within 3 min.
This method should be valuable to facilitate energy-efficient and cost-effective
conversion of biomass into biofuels and platform chemicals (Zhang and Zhao,
2010).

Irfan et al. (2010) studied the pretreatment of corn cob with NaOH at
various concentrations and at different steaming time. These pretreated corn cobs
samples were tested for hydrolytic enzyme production from Aspergillus niger-
IR01. In 250 ml conical flask 25 ml of Vogel’s media with 2% pretreated
substrates were used for enzyme production with 2% inoculum size for 96 h of
fermentation period. Maximum CMCase activity (40.1 ± 1.3 IU/ml) was achieved
at 2.5% NaOH treated corn cob for 30 min of autoclaving time. Highest xylanase
and FPase activities of 140.3 ± 1.36 IU/ml and 10.1 ± 0.54 IU/ml was noted with
0.5% NaOH (for 15 min of steaming time) and 15 min steam hydrolyzed corn
cobs respectively

2.8.2 EFFECT OF DIFFERENT PRETREATMENTS ON XYLANASE

PRODUCTION

Keshwani (2010) investigated microwave- based alkali pretreatment of

switch grass. Pretreatment were conducted by immersing the biomass in dilute
alkali solutions and exposing the slurry to microwave radiation for 5-20 min.
Pretreatments were evaluated based on yields of total reducing sugars glucose
and xylose from enzymatic hydrolysis of pretreated biomass. Preliminary
experiments identified sodium hydroxide (NaOH) as the most effective alkali
reagent optimization of pretreatment conditions resulted in a glucose yield of
82% and xylose yield of 63%.

Jiang et al. (2010) first report on the purification, characterization and

application of thermophilic xylanase producing fungus, Chaetonium sp CQ31
noticed 131 IU/ml of xylanase activity when grown on a medium containing
corncob (3.5% w/v) at 37°C for 6 days, optimum pH and temperature was 7.5
and 65°C molecular mass was estimated to be 25.1 kDa, apparent km value for

28
the xylanase for birchwood, beechwood and oat spelt xylan was 1.3, 0.86 and 4.4
mg/ml on Chinese steamed bread was studied and found that the addition of
xylanase in the range of 2.5-5.0 ppm caused a 20-24.5% increase in specific
volume over the control and decrease (8.9-24.2%) in firmness was observed.

Sunflower stalks were analysed by Vaithanomsat et al. (2009) for

chemical compositions: pentosan 15.84%, holocellulose 70.69%, α cellulose
45.74%, glucose 27.10% and xylose 7.69% based on dry weight of 100g raw
material. The most optimum condition for steam explosion pretreatment was as
follows. Sunflower stalks were cut into small pieces and soaked in 0.02 M H2SO4
for overnight. After that, they were steam exploded at 207° C and 21 kg/cm2 for 3
minutes to fractionate cellulose, hemicellulose and lignin. The resulting
hydrolysate, containing hemicellulose, and cellulose pulp contained xylose sugar
at 2.53% and 7.00%, respectively. The pulp was further subjected to enzymatic
saccharification at 500C pH 4.8 citrate buffer) with pulp/buffer 6% (w/w) and
Celluclast 1.5 L/pulp 2.67% (w/w) to obtain single glucose with maximum yield
11.97%. After fixed-bed fermentation under optimum condition using
conventional yeast mixtures to produce bioethanol, it indicated maximum ethanol
yield of 0.028 g/100 sunflower stalk.

Horticultural waste in wood chips form collected from a landscape

company in Singapore was utilized as the substrate for the production of cellulase
and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-
C30. The effects of substrate pretreatment methods, substrate particle size,
incubation temperature and time, initial medium pH value, and moisture content
on cellulase and hemicellulase production were investigated. Enzyme complex
was obtained at the optimal conditions. This enzyme mixture contained FPase
(15.0 U/g substrate dry matter, SDM), CMCase (90.5 U/g SDM), β-glucosidase
(61.6 U/g SDM), xylanase (52.1 U/g SDM), and β-xylosidase (10.4 U/g SDM).
The soluble protein concentration in the enzyme complex was 26.1 mg/g SDM.
The potential of the crude enzyme complex produced was demonstrated by the
hydrolysis of wood chips, wood dust, palm oil fiber, and waste newspaper. The

29
performance of the crude enzyme complex was better than the commercial
enzyme blend (Xin and Geng, 2010).

Sun and Cheng (2005) reported the pretreatment of rye straw and
Bermuda grass for ethanol production by enzymatic hydrolysis at 121oC with
different sulphuric acid concentrations (0.6 0.9, 1.2, and 1.5% w/w) and residue
times (30, 60 and 90 min). It has been pretreated that Eucalyptus grandis
impregnated with 0.087 and 0.175% (w/w) H2SO4 at 200-210oC for 2-5 min by
Emmel et al, (2003). The best conditions for hemicellulose – recovery was
obtained at 210oC for 2 min, while a lower pretreatment temperature of 200oC
was enough to obtain the highest yield of cellulose conversion (90%) by
enzymatic hydrolysis.

Steam explosion for production of ethanol from several lignocellulosic

materials with Kluyveromyces marxinusn was reported by Ballesteros et al.,
(2004). The popular and eucalyptus chips were treated at 210oC for 4 min, wheat
straw at 190oC for 8 min. Brassica carinata residue at 210oC for 8 min and sweet
sorghum bagasse at 210oC for 2 min. Laser et al. (2002) compared the
performance of LHW and steam pretreatment for sugarcane bagasse, which was
subsequently used for ethanol production by SSF. They performed the treatment
in a 25 l reactor at 170-230oC for 104 min with 1% to 8% solid concentrations. It
has been found that poplars (Populus sp.) representing an important source of
short rotation energy crops steam-explosion was found to be effective method for
improving enzymatic saccharification of cellulose content of poplar wood
(Excoffer et al., 1991).

2.9 PRODUCTION AND OPTIMIZATION OF CELLULASE UNDER

SUBMERGED FERMENTATION

Submerged fermentation system can be defined as the cultivation of

microorganisms in a liquid medium containing soluble carbon source and
nutrients, maintained or not under agitation. Several characters make attractive
system for the microorganism cultivation and production of biological products,
which include:

30
• The mixture of the medium is easy and allows uniform conditions for the
microorganism growth
• Modification of the cultivation conditions like pH, dissolved oxygen,
temperature, agitation and nutrient concentration are easy and fast
• The temperature control is favoured by the high specific heat and thermal
conductivity
• Efficient technologies have already been developed with high automation
grade, diversity and availability of equipments

Countless microorganism strains have been used in the fermentation

processes by submerged cultivation, including bacteria, yeast, fungi and algae.
Fermentation media used in these systems may be synthetically formulated or
hydrolysis of lignocelluloses. Sugars released from cellulose and hemicelluloses
structure can be microbially converted into various products of industrial interest
by SmF system including organic acids, ethanol, glycerol, food additives, butanol
etc ( Mussatto and Teixeira, 2010).

The production of extracellular amylase by Bacillus sp. was optimized in

a submerged fermentation by Vidyalakshmi et al. (2009). The production of the
enzyme was maximum at 10 h after inoculation. The pH of the medium and
incubation temperature was optimized. The maximum productions of enzyme
were obtained at 35°C and pH 7.

Rhizopus oryzae PR7 MTCC 9642, a producer of endoglucanse was found

to produce extra cellular exoglucanase or avicelase when grown on avicel or
micro-crystalline cellulose, when grown in media supplemented with various
cellulosic wastes, of which dried flower showed the best result followed by the
sweet lime peel at optimum pH 8.0 and 5.0 respectively at 37°C. Peptone was
found to be the best nitrogen source for exoglucanase production whereas
amongst ions Mn2+ and Fe2+ could bring a 1.23 fold increase in enzyme
production in sweet lime peel supplemented culture. Under optimized condition,
highest exoglucanse production was achieved at 96 hours of growth. The enzyme
showed optimum activity at pH 5.0 and 40°C and stability at pH range of 5-9 and

31
about 90% activity was retained even after an exposure of 10 min at 80° C
(Mukherjee et al., 2011).

A locally isolated fungus genus, Fusarium verticillioides was reported by

Boonyapranai et al. (2008) as a potential producer of naphthoquinone pigment.
Five variable growth media (type of complex media, carbon source, nitrogen
source, mineral salt and initial pH of the medium) were systematically
manipulated in shake flask culture to improve the yield of total naphthoquinone.
The naphthoquinone production was supported by addition of peptone and yeast
extract but malt extract exhibited the opposite result, the mineral salt of K+, Na+,
Mn2+, Cu2+ and Zn2+, less than 50 mg/l, were necessary for the efficient
napthoquinone production by F. verticillioides. In addition, efficiency of pigment
production at various values of initial medium pH showed that the biosynthesis of
napthoquinone was regarded by initial pH and changing of medium pH. Taken
together, the results indicated that the napthoquinone pigment production for this
particular strain could be maximized by using a basal growth medium consisting
of 20% (w/v) of boiled white potatoes; 20 g/l glucose; 2.5 g/l yeast extract,
supplemented with 5 mg/l of KH2PO4 and adjust initial medium pH at 8.0. The
pigment production under the optimum condition was 13.61 mg/g-cell when
cultured at 30°C with agitation rate at 200 rpm for 7 days.

Shabeb et al. (2010) tested different factors for cellulase productivity by

Bacillus subtilis KO strain using Carboxymethyl cellulose clear zone (CMCZ) and
Dinitrosalicylic acid (DNSA) techniques. Cellulase enzyme revealed its best
production 32 IU/ml by CMCZ techniques and 228 µg/ml broth medium by
DNSA technique as well as 525 µg/ml broth medium of protein content after 24
hr. incubation period. At 45°C, cellulase productivity was 36 IU/ml by CMCZ and
344 µg/ml broth medium by DNSA with 301 µg/ml broth medium of protein
content. Moreover, maximum cellulase productivity was 35 IU by CMCZ and 420
µg/ml broth medium by DNSA with 601µg/ml broth medium protein content
when molasses broth medium was supplemented with cellulose and trypton.

32
 Jaradat et al. (2008) determined the influence of growth conditions and
medium composition on the cellulase enzyme production by Streptomyces sp.
Production of cellulase enzyme by a Streptomyces strain (J2) was detected on
cellulose agar (CA) medium after 4 days of incubation at 28°C that exhibited a
clear zone of 22 mm around the colony. Cellulase production was assayed by
measuring the amount of glucose liberated in µmol/ml/min by using the
dinitrosalicylic acid assay method. The highest crude enzyme activity (432 U/l)
was observed after 3 days of incubation at pH 7 and 60°C in a medium that was
supplemented with 0.5% glucose, 0.2% starch, and 0.2% NH4Cl. However,
enzyme production and activity were strongly decreased at 45°C and acidic pH.
Enzyme production and activity were also inhibited when Streptomyces strain (J2)
was grown in CMC broth supplemented with arabinose and yeast extract as a sole
carbon and nitrogen source, respectively.

Methane producing thermophilic Methanosarcina strain was isolated from

a digester fed with water hyacinths and inoculated with ground termites from the
congo. Optimal growth temperature was 55°C and pH between 6.5 and 7.0,
bacterium will grow on acetate, methanol and methylamines in absence of growth
factors but could not use H2-CO2 or formate, yeast extract and vitamin will
stimulate the growth (Ollivier et al., 1984)

An extremely thermostable cellulase form the thermophilic Eubacterium

Rhodothermus marinus able to produce an extracellular endo-β-1-4-glucanasee
isolate from the submarine hot springs was active on carboxymethylcellulose but
not on avicel, sigmacell 10 cellulose, granular cellulose, p-nitrophenyl-β-D-
cellobioside or 4-methyllumbelliferyl-β-d-cellobioside, were reported by
Hreggvidsson et al., 1996) hydrolysis of carboxymethyl cellulose were glucose,
cellobiose and cellobiotriose as well as mixture of larger polysaccharides,
monomeric molecular weight was about 49,000 and pH optimum was at 7.0, the
enzyme is most thermostable endocellulose reported, retaining 50% of its activity
after 3.8 h at 100°C and 16 h at 90°C.

Al-Tai et al. (1989) reported the cellulase producing actinomycetes had

been isolated from the Iraqi soil Streptomyces sp. strain AT7 releases

33
carboxymethylcelllulose (Cx) and avicelase (C1) enzyme during growth in liquid
medium, maximum activity is found to be 6.0 and 13.6 (measured in mg/ml/hr at
36°C for 2 and 9 days of incubation period Cx retained its activity at the end of
incubation period of 18 days while C1 has lost almost 50% of its activity,
maximum yield of the cell growth obtained at 28°C, 36°C and 42°C were
0.23,0.22 and 0.25 µg/ml.

Rastogi et al. (2009) studied the different cultivated thermophilic and

mesophilic cellulose degrading bacterial diversity a weathered soil like sample
collected from the deep subsurface of the homestake gold mine in lead, south
Dakota USA. Rrs(16S rRNA) gene sequencing revealed from enrichment culture
growing in presence of microcrystalline cellulose as a sole source of carbon, all
phytotypes retrieved from the enrichment culture were belongs to Firmicutes sp.
cellulose degrading mesophilic and thermophilic pure culture will belongs to the
genera Brevibacillus, Paenibacillus, Bacillus and Geobacillus , the mesophillic
isolate has the optimal pH and temperature for carboxymethylcellulase (CMCase)
were 5.5 and 55°C, while for the thermophilic isolates (DUSELR7) they were 5.0
and 75°C, thermophilic isolate DUSELG7 retained 26% CMCase activity at
60°C up to a period of 30 h.

Cellulose and hemicelluloses degrading thermophilic anaerobes were

enumerated in biomat samples of various temperature from two hot springs in the
Hveragerbi area of Iceland, one had a pH 7 and other near the pH 9. Bacteria in
the sample are enumerated by most probable number technique (37-90°C) and
two pH 7 and 9, no growth observed at 80°C in the neutral spring or at 37°C in
the alkaline spring, there are more number of xylanase degrading microorganism
than the cellulose utilizing organisms , both type of organisms are present in the
alkaline spring at 800C and in neutral springs at 37°C, no culture will grew from
the most probable number tubes inoculated with the Hveragerbi samples and
incubated at 90°C or with media at pH 9.0, however xylan degrading culture at
70°C were enriched at pH 9 with samples from some other Icelandic hot spring
(Indra et al., 1993).

34
 Tong et al. (1982) reported the most active cellulase producer of several
thermophilic fungi tested was Thermoascus aurantiacus, optimum growth
temperature for T. Auranticus in liquid medium was 45°C and maximum
cellulase production from the filter paper occurred at 40°C, optimum pH for the
β-glucosidase and carboxymethylcellulase was 70°C, for the filter paper
degrading activity 65°C, maximum activity was found at the pH 5.0 for the filter
paper degrading enzyme and β-glucosidase and pH 4.3 for
carboxymethylcellulase activity.

2.10 PRODUCTION AND OPTIMIZATION OF XYLANASE UNDER

SUBMERGED FERMENTATION

A xylanase producing thermophilic strain Bacillus subtilis was isolated

from the hot spring by Sa-Pereira et al. (2002) optimal xylanase production of
about 12 U/ml achieved at pH 6 and 50°C within 18 h fermentation. At 50°C
xylanase production obtained after 11 h in shake flasks 96,000 U/l/h and in
reactor 104.000 U/l/h, increased temperature to 55°C a high productivity was
obtained in the batch reactor 45,000 U/l/h compared to flask fermentation 12,000
U/l/h , maximum xylanase activity was obtained at 60°C. In phosphate buffer at
pH 6.0 and remain fully stable at 60°Cfor 3 h, with increase in the temperature
activity will be reduced at 90°C, 20% relative activity remains after 14 min,
Protein disulfide reducing agent such as DTT enhanced xylanolytic activity about
2.5 fold, when xylan is used as a substrate the xylanase production will be
decreased instead with tetrahexose as carbon source, xylanase production in
maintained constant for at least 80 h fermentation.

A thermostable xylanase producing thermophilic strain Bacillus circulans

AB 16 in basal medium containing rice straw has been isolated from the garbage
dump. The highest xylanase activity obtained in liquid medium containing
tryptone was 55 IU/ml, Xyl A had a optimum pH of 6 and temperature optima of
75-80°C yield mainly xylobiose with lower amount xylan-oligosaccharide and
xylose while Xyl B also had a pH 6 with temperature optima of 65-70°C, yielded
mainly higher oligosaccharides with lesser amount of xylobiose and negligible
amount of xylose, both (Xyl A and Xyl B) will retained had a optima at pH 6,

35
but retained its activity even at the pH 8, and 90% of inhibition in activity by 1
mM Hg (Dhillon et al., 2000).

Basar et al. (2010) reported the enhancement of thermophilic xylanase

production by recombinant Escherichia coli DH5α was investigated initiated
using shake flask cultures. Effect of the xylanase fermentation on the E. Coli was
investigated in 2 l of stirred tank bioreactor, using the mineral medium gave the
highest growth and xylanase production the optimal glucose and ammonium
sulphate for xylanase production was obtained at 10g/l ad 2g/l, growth of the
xylanase enzyme was inhibited in oxygen limited fermentation, when the
dissolved oxygen tension level was controlled at 20% saturation, xylanase
production was enhance at dot level controlled at 20% saturation, substantially
high xylanase production (1784.57 U/ml) was obtained in fermentation using the
optimal medium, composition and DOT level.

Aspergillus niger morphology and phytase production were investigated

by Papagianni et al. (2001) in submerged and solid-state fermentations, phytase
production increased with increasing shaker speeds from 150 to 300 rpm,
although specific growth rates and phytase production rates were higher at 150
rpm for 72 h from inoculation. Fungal morphology was greatly influenced by
agitation with the morphological forms of small pellets and entangled mycelia
predominating at 150 rpm, while the free filamentous form was obtained at 300
rpm. Upon inoculation of SSF, increased productivities were obtained from
inoculums grown at 150 rpm. A shift towards the filamentous growth form was
observed when guar gum was added to the liquid media, which increased the
viscosity from 2000 cp (medium without gum addition) to 52000 cp (addition of
1 g/L gum). At both shaker speeds, with the effect being more pronounced at 300
rpm, phytase production increased with gum addition. Specific growth rates for
the first 72 h, were higher at 150 rpm, irrespective of gum concentration, while
solid-state fermentations inoculated with these cultures led to higher amounts of
phytase compared to those obtained from 300 rpm.

Samples were collected from the Bulgarian hot springs, two alkali tolerant
thermophilic bacterial strains were isolated by continuous cultivation, similar in

36
their respect of their temperature and pH optimum (70-75°C and pH 6.5-7.0) as
well as their thermostability, xylanolytic activity was resistant to pH 5.5-8.0
(strain SP) and pH 5.5-8.0 (strain BC) and are thermostable at 70°C for 30 min
(Dimitrov et al., 1997).

An extracellular xylanase producing thermophilic Bacillus strain TAR-1

had been isolated from the soil by Nakamura et al. (1995) enzyme was active
over the range of pH 5.0 to 10.0 at 50°C, optimum temperature for activity were
75°C at pH 7.0 and 70°C at pH 9.0, xylanase was stable up to 65°C at pH 9.0 for
30 min in the presence of xylan, the enzyme was purified by the ammonium
sulphate fractionation and anion exchange chromatography.

Two highly thermophilic xylanolytic bacteria related to non xylanolytic T.

thermophilus strain were isolated from the hotest zones of compost piles the
growth rate of X6 on xylan was 0.404 h at 75°C, maximal growth temperature
was 81°C. Xylanase activity was cell bound , was solubilised into the medium by
sonication , optimal pH and temperature is about 100°C and pH 6, xylanoltyic T.
thermophilus strains could contribute to the degradation of hemicelluloses during
the thermogenic phase of industrial compositing (Touzel et al., 2000).

Hot spring in the west of Turkey were investigated for the presence of
thermophilic microorganism by Canakci et al. (2007) based on the rrs (16S
rRNA) gene sequencing analysis isolates will belong to the genus Geobacillus
and grow optimally at 60°C on nutrient agar. It was found that 7 of the strains
have both xylanase and arabinofuranosidase activities, 4 of them has xylanase
activity and remaining has no activity, based on the recN sequencing analysis
isolates 11, I3 and 12 are strains of Geobacillus stearothermophiles and 14.3
was a strain of G. thermodenitrificansisolates 9.1, IT 4.1 and 4.5 are uncertain
and require further analysis, isolates 9.1, 7.1 and 3.3 have highest temperature
optima (80°C) and 7.2, 9.1, AO4, 9.2 and AO17 have the highest pH optima (pH
8) of xylanase, isolates 7.2 AO4, AC 15 and 12 have optimum
0
arabinofuranosidase activities at 75 C and AC15 has the lowest pH of 5.5.

37
 Anuradha et al. (2007) reported three bacterial strains isolated from the
sugarcane field StA. StB and StC were identified as Bacilllus sp. Based on the
morphological, biochemical and physiological characters, isolated on oat spelt
xylan agar medium. Optimal pH and temperature were 9.0 and 55°C for strain
StA and 9.0 and 50°C for strain StB and 8.0 and 55°C for strain Stc, the enzymes
were stable between 30 and 50°C and xylanase activity remained 2 h for the
enzyme for the three strains, enzyme activity was stimulated by isopropanol,
these properties qualify the enzymes to be novel and exhibits favourable potential
for application to bleaching in the paper and pulp industry.

Thermophilic Enterobacter sp. MTCC was isolated from a sediment

sample collected from the Mandovi Estuary, West coast of India, producing
xylanase isolated by ammonium sulphate (80%) fractionation, optimal pH for the
enzyme activity was 9 and at room temperature shows 100% stability for 3 h at
pH 7, 8 and 9, optimal temperature for the enzyme activity was 100°C at pH 9, at
80°C and pH 9, 90% the enzyme activity was retained after 40 min, at 70 and
60°C the enzyme retained 64% and 85% of its activity after 18 h, at 50°C and pH
9 the enzyme remain stable for many days, for xylan when reaction carried out at
100°C and pH 9, the enzyme gave a Km value of 3.3 mg/ml and Vmax value of
5000 µml/min/mg, in the presence of metal ions like Co, Zn, Fe, Cu, Mg, Ca
activity of enzyme increased , growth is decreased when the strong inhibitors are
present Hg and EDTA (Khandeparkar and Bhosle, 2006).

Bai et al. (2010) reported a highly pH adorable and stable xylanase

(XynA4) thermoacidophilic Alicyclobacillus sp. A4 strain was isolated from the
hot spring in Yannun Province, China exhibiting maximum activity at 55°C and
pH 7.0 and broad pH adaptability (>40% activity at pH 3.8-9.4) and stability
>80% activity after the incubation at pH 2.6-12.0 for 1 h at 37°C was highly
thermostable (retaining 90%) activity after incubation at 60°C for 1 h at pH 7.0,
gene Xyn A4 that encode the xylanase was encode and expressed in E.coli , it
will encodes for a 338-residues polypeptides , with molecular weight of 42.5
kDa and these properties make them industrially important enzymes.

38
 Twenty strains of thermophilic fungus were isolated from the soil,
screened for their xylanolytic and cellulolytic enzyme in medium containing
birchwood xylan as substrates. The selected thermophilic fungus, identified as a
strain of Thermomyces lanuginosus grew well on potato dextrose agar and
Mandels and Sternberg medium in which cellulose was supplemented with the
xylan, optimum temperature and pH growth at 55°C and pH 6.0. Sugar and xylan
based substrates as carbon source, xylan and corn cob induced simultaneous
production of high levels of xylanase and pectinase maximum enzyme activity of
5,846 and 840 IU/ml were obtained in liquid Mandel’s and Reese medium with
3% corn cob at pH 6 after 7 days incubation at 50°C and T 150 rpm (Mendoza et
al., 2006).

2.11 PRODUCTION AND OPTIMIZATION OF CELLULASE UNDER

SOLID STATE FERMENTATION

Substantial cost reduction may be possible by exploring ways of cellulose

conversion using microorganisms that produce cellulolytic/ xylanolytic enzymes.
Solid state fermentation is an attractive process to produce cellulase and xylanase
economically due to its lower capital investment and lower operating expresses
(Xen and Cen, 1999). To reduce the cost of enzyme, selection of a cheap and
easily available substrate appears to be essential (Beg et al., 2000 & Senthikumar
et al., 2005). The lignocellulosic biomass especially agricultural wastes are
known to be excellent carbon source for microbial enzyme production (Yang et
al., 2006). It is an attractive method for fungal enzymes production because it
stimulates the natural growth of microorganisms on a moist insoluble substrate in
the absence or near absence of free liquid (Pandey, 2002). This cultivation
technique is acquiring a special relevancy in the field of biotechnology processes,
as an alternative to the traditional submerged fermentation, because has lower
energy requirement, produces less wastewater, gives high product concentrations,
avoids the foaming and has lower risks of contamination (Gessesse and Mamo,
1999 & Suryanarayan, 2003).

Trichoderma harazium , cellulose degrading filamentous fungi, having

tremendous ability to biomass degradation (Ahmed et al., 2009) the partial

39
purification of three cellulose enzymes exoglucanase (EXG), endoglucanase (EG)
and β-glucosidase (BGL), the optimal pH, temperature and incubation time of
cellulase production is found to be 120 h at 25°C, pH 5.5.

The pretreated corn cobs samples were tested for hydrolytic enzyme
production from Aspergillus niger-IR01. In 250 ml conical flask 25 ml of Vogel’s
media with 2% pretreated substrates were used for enzyme production with 2%
inoculum size for 96 h of fermentation period. Maximum CMCase activity (40.1
±1.3 IU/ml) was achieved at 2.5% NaOH treated corn cob for 30 min of
autoclaving time. Highest xylanase and FPase activities of 140.3 ± 1.36 IU/ml
and 10.1 ± 0.54 IU/ml was noted with 0.5% NaOH (for 15 min of steaming time)
and 15min steam hydrolyzed corn cobs (Irfan et al., 2010).

Raza et al. (2011) reprted the production of β-glucosidase for selection of

co-culture of Aspergillus sp., and optimization of cultural conditions for the
biosynthesis of β-glucosidase by solid state fermentation. For this purpose, mono
and co-cultures of Aspergillus niger, A. awamori and Aspergillus oryzae were
tested and co-culture of Aspergillus niger and Aspergillus oryzae gave
comparatively better production of β-glucosidase. The enzyme production was
optimal when agricultural by product wheat bran (10 g) was used as solid
substrate for fungal growth. Among 10 different diluents tested, M-2 containing
(g/l, w/v) KH2PO4 2.0, (NH4)2SO4 1.4, urea 0.3, MgSO4.7H2O 0.3, ZnSO4.7H2O
0.0014, FeSO4.7H2O 0.005, MnSO4 0.0016, CoCl2 0.002, CaCl2 0.002,
polypeptone 1.0 and Tween-80 2.0 ml gave relatively higher enzyme production.
The maximum production of β-glucosidase (2975±5.3 U/g/min) was obtained
after optimization of cultural conditions such as incubation period 72 h, initial pH
5.5, substrate to diluents ratio (1:1) and inoculum size (2 ml of spore suspension
i.e., 3:1 ratio of Aspergillus niger and Aspergillus oryzae).

Gram positive rod shaped and endospore forming

alkalothermoanaerobacterium strain Tepidimicrobium xylanilyticum BT14,
having the ability to bind to avicel, xylan and corn hull, pH and temperature
optima for the growth were 9.0 and 60°C, when grown on the corn hull under the
anaerobic condition, a cellulolytic and xylanolytic enzyme is produced, optimum

40
condition for cellulase and xylanase activities were pH 8.0 and 9.0 at 60°C,
having ability to bind to avicel and xylan, the analysis of native-PAGE and native
zymograms indicated the cellulose binding protein showing both cellulase and
xylanase activities, cohesive like amino acids indicates that the protein shared a
direct relationship with the cellulosome of Clostridium thermocellum the crude
enzyme showed effective degradation of biomass, when grown on corn hull at pH
9.0 and 60°C under anaerobic condition, the strain BT14 produced ethanol and
acetate as the main fermentation products (Phitsuwan et al., 2010).

Production of substantial extra cellular cellulase on cellulosic substrates

(wheat straw, wheat bran and corn straw), 2% (w/v) wheat straw, wheat bran and
corn straw were used as the carbon source and urea are used as a nitrogen source,
experiment is conducted in following condition pH 5.0, stirring speed 300 rpm,
temperature 45°C and aeration 50%, sample were withdrawn after every 12 h.
Endo-β-1-4- glucanase activity on wheat straw, wheat bran and corn straw were
25.1, 1.62 and 5.10 U/ml and extra cellular protein contents were 1.48, 1.17 and
2.88 mg/ml, specific activity after the ammonium sulphate precipitation and fast
protein liquid chromatography was 2.1 and 3.3 U/mg of protein. Optimum
temperature of the CMCase were 60°C and from pH 6.0-7.5 (Raza and Shafiq-
Ur-Rehman. 2009).

The ability of Aspergillus terreus for the production of cellulolytic

enzymes and reduction of lignocellulose contents of rice straw in solid state
fermentation was reported by Jahromi et al. (2011). 8 days fermentation was
appropriate, with enzymes activities as follows: FPase = 410.76 U/gdm, CMCase
= 351.96 U/gdm β-glucosidase = 16.37 U/gdm, xylanase = 6166.01 U/gdm and
amyloglucosidase = 425.04 U/gdm (with maximum 993.71 U/gdm on day 6). In
addition, the solid state fermentation significantly (P<0.01) reduced the
concentrations of NDF, ADF, cellulose and hemicellulose in the rice straw by
19.96, 13.8, 16.32 and 32.87%, respectively. The high degradation of the
hemicellulose was reflected by the high activity of xylanase enzyme, which
hydrolyses xylan in hemicellulose to xylose. Higher reducing sugar and microbial
cell mass productions were also obtained after 8 days fermentation. Present data

41
showed that A. terreus was capable of producing high quantity of cellulolytic
enzymes for the reduction of lignocellulose contents of biomass in a shorter
incubation time when compared with the previously reported for biological
treatment of agricultural by-products using white rot fungi.

Odeniyi et al. (2009) tested Bacillus coagulans strain isolated from palm
fruit husk for abilities to hydrolyse plant structural polysaccharides through the
depolymerising activities of carboxymethylcellulase and polygalacturonase. The
two enzymes were produced using solid substrate fermentation. Both had a
working pH range of 4-9 with an optimum pH of 6.0 for the
carboxymethylcellulase and 7.0 for the polygalacturonase. The respective
enzymes remained active when allowed to stand at 27°C for 1 h over a wide pH
and temperature range maintaining maximum activity at an optimum temperature
of between 50°C and 60°C respectively. The carboxymethylcellulase still retained
full activity after being allowed to stand at 60°C for 10 min while the
polygalacturonase retained full activity at 80°C for 5 min and had 50% activity at
70°C at 30 min. All enzymatic activities were fully inhibited by Mercury ions at
1.0 mM concentration.

2.12 PRODUCTION AND OPTIMIZATION OF XYLANASE UNDER

SOLID STATE FERMENTATION

Suyanto et al. (2003), new thermophilic fungus, Chaetomium sp. nov MS-
017, exhibiting rapid growth on POMF was isolated from rotted wood. POMF is
a fibrous, natural hard material discarded in enormous amounts from palm oil
mills in tropical plantation. MS-017 produced 855 g of decomposed product from
the 1,000 g of intact POMF in 12 days under optimized solid state condition,
indicating the higher degradation rate of the POMF by the MS-017.TE
decomposition rate of POMF was 23% (w/w) and the cell yield calculated from
consumed POMF was as high as 36% (w/w).

Paecilomyces thermophila J18 produces an extracellular xylanase free

cellulose on the lignocellulosic material under the solid state fermentation (SSF),
strain grew well at 50°C, production of xylanase will be enhanced on wheat straw
by optimization of the particle size of the wheat straw, nitrogen source, initial

42
moisture level, growth temperature and initial pH of the culture medium (Yang
et al., 2006), xylanase exhibited remarkable stability and retained more than 50%
of its original activity at 70°C for 4 days at 70°C , pH 7.0-8.0, under the
optimized condition yield as high as 18580 U/g.

A highly potential and industrially important xylanase degrading fungal

strain has been isolated from the pitchavaram mangroves by Muthezhilan et al.
(2007) in secondary screening based on the diameter of the clear zone formation
in the oat spelts xylan agar plates, Penicillium oxalicum was selected and
optimized from xylanase production in solid state fermentation using cheap
sources like wheat bran, rice bran, sesame oil cake and wood husk. Maximum
xylanase activity is found in the wheat bran, optimum pH and temperature for the
xylanase activity were 8 and 45°C at 3% salt concentration, in purification of the
enzyme 80% ammonium sulphate saturation is found to give the maximum
xylanase activity.

Fusarium verticillioides, was evaluated as a potential producer of

naphthoquinone pigment. Five variable growth media (type of complex media,
carbon source, nitrogen source, mineral salt and initial pH of the medium) were
systematically manipulated in shake flask culture to improve the yield of total
naphthoquinone. The naphthoquinone production was supported by addition of
peptone and yeast extract but malt extract exhibited the opposite result.
Substantially, the mineral salt of K+, Na+, Mn2+, Cu2+ and Zn2+, less than 50 mg/l,
were necessary for the efficient naphthoquinone production by F. verticillioides.
In addition, efficiency of pigment production at various values of initial medium
pH showed that the biosynthesis of naphthoquinone was regarded by initial pH
and changing of medium pH. Taken together, the results indicated that the
naphthoquinone pigment production for this particular strain could be maximized
by using a basal growth medium consisting of 20% (w/v) of boiled white
potatoes; 20 g/l glucose; 2.5 g/l yeast extract, supplemented with 5 mg/l of
KH2PO4 and adjust initial medium pH at 8.0. The pigment production under the
optimum condition was 13.61 mg/g-cell when cultured at 30°C with agitation rate
at 200 rpm for 7 days (Boonyapranai et al., 2008).

43
 The ability to produce xylanolytic enzyme in submerged (SmF)
fermentation by Pleurotus eryngii and Flamulina velutipes was checked first time
by Simair et al. (2010). The cultivation in identical culture conditions revealed
wide differences among xylanase production by both species. Among the carbon
sources used, the maximum xylanase was produced by Flamulina velutipes 5.3
IU/ml (xylose) and Pleurotus eryngii 6.83 IU/ml (starch) in comparison to other
carbon sources. This study pointed out that the both organisms are capable to
produce sufficient amount of xylanolytic enzymes.

Seventy fungal strains were isolated from soils collected from different
parts of southern Kerala, India. The strains were screened for xylanase production
using Czapek’s agar medium. On the basis of clearing zones formed, 34 fungal
strains were selected and identified. Solid state and submerged fermentation were
done to identify strains that could produce maximum amount of xylanase, as well
as to identify those strains that could produce cellulase-free xylanase under these
conditions. All strains produced cellulase along with xylanase in solid state
fermentation, while 70% of the strains produced cellulase-free xylanase during
submerged fermentation (Nair et al., 2008).

44
 Chapter-3

MATERIALS AND METHODS

The methodology followed to achieve the objectives of study was divided
into following main sections:

3.1 Isolation and screening of cellulolytic and xylanolytic

microorganisms from hot springs
3.2 Identification of thermostable cellulolytic and xylanolytic enzyme
producing isolates
3.3 Production and optimization of cellulase and xylanase under
submerged fermentation
3.4 Production and optimization of cellulase and xylanase under solid
state fermentation

3.1 ISOLATION AND SCREENING OF CELLULOLYTIC AND

XYLANOLYTIC MICROORGANISMS FROM HOT SPRINGS OF
HIMACHAL PRADESH

3.1.1 Collection of hot water samples from hot springs:

Water sample were collected in the sterile container from the hot springs of
Himachal Pradesh, located in Distt. Mandi (Tattapani) and Distt. Kullu
(Manikaran and Vashist) and were kept refrigerated until processed further, the
temperature and pH of the sites vary depend upon the sites of collection of water
samples (Fig 1).

3.1.2 Physiochemical analysis of water:

Following parameters were evaluated for various physio-chemical

analysis of the hot water sample from the hot springs

3.1.2a. Temperature

The temperature of the hot springs were recorded at the site of collection
3.1.2.b pH

pH of the water sample was recorded using pH meter in the laboratory.

3.1.2 c Chloride (APHA, 1976)

Reagents:

• Silver nitrate (0.141 N)

• Potassium chromate indicator (100.0 gm/l)

Procedure

Water sample were assayed titrametrically.10ml of sample was taken in

the flask and 5ml of chromate reagents was added to it. The yellow coloured
solution was then titrated against solution of silver nitrate (0.141N) till the
appearance of brick red color. A blank titration was also done.

Calculation

ml of titrant x N x 35.46 x 103

Chloride (mg/l) =
ml of sample

3.1.2 d Sulphate (APHA, 1976)

Reagents
• Barium chloride (20-30 crystal)
• NaCl-HCl (100.0 gm NaCl added to 800 ml of distilled water to which
200 ml HCl was added and then the volume was made up to 1 litre)
• Conditioning reagent (glycerol-ethanol solution; mixed 50 ml glycerol in
100 ml ethanol)
• 0.147 gm anhydrous Na2SO4 in one litre of distilled water: 1ml of solution
contains 100.0 µg SO4

Procedure

Diluted 10ml of sample to 100 ml. 5ml of the conditioning reagents was
added to the flask with the constant stirring using a magnetic stirrer. A few

46
Fig. 1. Hot springs of district Mandi and Kullu
crystal of BaCl2 was added and mixed for 1 minute. Immediately absorbance was
read at 420 nm against a blank. Estimated the sulphate concentration of the
sample by comparing the sulphate standard through entire procedure. Sulphate
was expressed then in mg/ml.

Calculation
MgSO4 x 1000
mg/1 SO4 =
ml of sample

3.1.2 e Total hardness (APHA, 1976)

Reagents

• Standard EDTA titrate (0.01 M)

• Ammonium buffer solution, 114 ml concentrated NH4OH was added 13.5
gm of NH4Cl and made the final volume to 200 ml
• Erichrome Black T indicator dipped 5.0 gm in 100 ml of 80% ethyl
alcohol.

Procedure

To 50 ml of water sample, 1 ml of ammonium buffer and 5 drops of

indicator was added. The wine red colored solution was then titrated against the
standard EDTA solution (0.01 M) till the blue color appeared.

Calculation

A x 1.05 x 1000
Total hardness as mg/l as CaCO3 =
ml of sample

Where;

A = ml of titrant used

3.1.2 f Calcium hardness (APHA, 1976)

Reagents

• Standard EDTA (0.01M)

• Sodium hydroxide solution (8.0%)
• Murexide indicator mixture

47
Procedure

To 50 ml of the sample, added 1 ml NaOH solution and pinch of

murexide indicator. Added EDTA titrant slowly with continuous stirring till the
pink coloured solution changed to purple. Checked to the end point by adding 1
to 2 drops of titrant in excess till no further change in colour occurred.

Calculation

A x B x 1000
Calcium hardness as mg/l CaCO3 =
ml of sample

A x B x 400.8
Calcium hardness as mg/ l Ca 2+ =
ml of sample

Where;

A = ml of titrant
B = mg CaCO3 equivalent to 1.00 ml EDTA titrant at calcium
indicator end point B= 1.05

3.1.2 g Magnesium Content (APHA, 1976)

Procedure

If the calcium hardness as calcium carbonate is known, the magnesium

contents can be calculated as follows:

Magnesium in mg/ l = (Total hardness- Calcium Hardness) × 0.243

3.1.2 h Total Phosphorus (APHA, 1976)

Reagents

• Perchloric acid (HClO4.2H2O):70-72%

• 1N Sodium hydroxide
• Ammonium molybdate reagents: 25.0 gm of ammonium molybdate
reagent was added to 175 ml distilled water. Continuously added 280 ml
concentrated H2SO4 to 400 ml of distilled water and cooled it. Then

48
 ammonium molybdate solution was added to it and the mixture was
diluted to 1litre
• Phenolphthalein indicator solution
• Stannous chloride reagents: dissolved 2.5 gm of fresh SnCl2 in 100 ml,
heated water bath and continuously stirred with glass rod
• Standard phosphate solution: dissolved 219.5 mg KH2PO4 in distilled
water and diluted it to 100 ml; 1 ml; 50.0 µg PO4

Procedure

Evaporated 20 ml of sample in a flask and the residue was dissolved in

1ml perchloric acid. It was heated till the residue becomes colorless. Fumed off
the perchloric acid by heating and then cooled it to the room temperature. A drop
of aqueous phenolphthalein solution was added to it. Neutralized this solution
titrametrically with 1 N NaOH till slight pink colour was observed as end point.
The final volume of this solution was made to 50 ml. To 25 ml of this 1 ml of
ammonium molybdate solution and 0.12 ml stannous chloride were added and
blue colour appeared gradually. Absorbance was recorded at 690 nm after 10 min
against a blank.

The phosphate concentration was estimated by plotting against the

standard curve. Prepared standard curve by using KH2PO4 as standard and
adding 1 ml of ammonium molybdate and 0.12 ml stannous chloride in 25 ml
KH2PO4 solution. Absorbance was noted at 690 nm and amount of total
phosphorous was then expressed in mg/ml.

Calculation

mg P x 1000
mg/l Phosphorus =
ml of sample

3.1.3 Enrichment of water sample from hot springs

Two protocol were used for the isolation of the thermophilic cellulolytic
and hemicellulolytic microorganisms

49
• Direct isolation: the collected hot water sample were suspended and
serially diluted in the sterile distilled water upto 10-8 , 100 µL of each
diluted sample were spread over the agar plate and incubated at 50oC for
1-2 days ,
• Enrichment technique: enrichment of the hot water sample collected
from thermal springs of Himachal Pradesh, hot water sample (10%) was
inoculated in the 45 ml of modified thermus broth and then kept for the
incubation at 500C for 3 or 5 days.

3.1.4 Isolation of cellulolytic and xylanolytic thermophilic microorganisms

One ml of enriched compost sample was serially diluted from 10-1 to 10-8
times using sterilized 9 ml diluted blanks. The diluents (0.1 ml) were placed on
the surface of GYM (pH 7.2) in a petriplate and evenly spreader with the help of
spreader and the plates were incubated at 50°C for 2-3 days. The pure cultures are
obtained by streaking method for bacteria and bit method for fungus. These pure
line cultures of all isolates were maintained on nutrient agar slants (pH-6.8) and
preserved in refrigerator at 40C for further use. The periodic subculturing was
done once in month.

Chemical composition of GYM medium (Hosoya et al.,1998)

Glucose - 4g; Yeast extract - 4g; Malt extract - 10.0g; CaCO3 - 2.0 g;
Cellulose powder - 1% and Agar - 12.0g; Distilled water - 1000 ml and pH -7.2

3.1.5 Morphological and physiological characteristics of cellulase and

xylanase producing isolates

The cellulolytic and xylanolytic microorganisms isolated from different

water samples were characterized on the basis of morphological, cultural and
physiological characteristics. Isolates were classified into bacteria and fungi
accordingly.

3.1.5.1 Phenotypic characterization

The conventional morphological and biochemical tests as described by

Aneja (2003) was performed which includes:

50
3.1.5.1a Morphological test:

Main morphological characterstics: cell morphology, gram reaction, shape

and culture conditions were noted down.

3.1.5.1b Physiochemical and Biochemical test:

Main morphological and biochemical tests performed with each of the

isolates were catalase, urease, H2S production, MR VP test, gelatine hydrolysis,
amylase producing test and fermentation of carbohydrate.

3.1.6 Screening of cellulolytic and xylanolytic microorganisms:

The microbial isolates of section 3.1.4 were screened for the production of
extracellular enzymes i.e. CMCase, FPase, β-glucosidase and xylanase.

3.1.6.1 Production of extracellular enzymes- cellulase and xylanase by

microbial isolates

Each bacterial culture isolate was grown in 50ml of nutrient broth at

35±20C from 24 h. As soon as the substaintial growth was observed in the broth.
The optimcal density was set to 1.0 using autoclaved distilled water. Whereas for
fungus, different fungal culture were grown in malt extract media petriplates and
were kept for 5-7 days in incubator for full plate growth. As soon as full plate of
fungus was observed, 10 ml of autoclaved distilled water was poured and
scratched. The spore suspension thus formed contained 1×107 spores/ml.

5ml of inoculum was added to each 45 ml of Reese medium’s broth

containing 1% cellulose for cellulase and 1% xylose for xylanase in 250ml of
Erlenmeyer flasks and the flasks were incubated for 5 days at 50±20C for bacteria
and at 50±20C for fungus at 120rpm.

After the incubation at 50°C for 5 days the culture contents were
centrifuged at 10,000 rpm for 15 min (40C), supernatants were collected. The
following quantitative tests were performed with the supernatants to screen out
the hypercellulolytic and hyperxylanolytic producers among different isolates.

51
3.1.6.1.1 Cellulase Assays

3.1.6.1.1.1 CMCase assay (Grajek, 1987)

Reagents:

• 1 % of CMCase in acetate buffer (0.055M , pH 5.0)

• Dinitrosalicylic acid (DNSA) reagents:
• NaOH: 1.0 g, phenol:0.1g, sodium potassium tartarate: 20.0 mg, sodium
metasulphate:0.05 g, DNSA reagents: 1.0 g and distilled water:100ml
• Standard solution of glucose (0.4 mg/ml)

Procedure

The reaction mixture containing 0.5 ml of 1% CMC in acetate buffer

(0.05M, pH 5.5) and 0.5 ml of diluted enzyme (supernatant). Now the reaction
mixture was incubated at 50°C for 30 min. After the incubation, added 3ml of
DNSA, then tubes were immersed in the boiling water for 15 min and removed
when the colour development was completed. Control was run with all
components except the enzyme was not added. Tubes were cooled at room
temperature and O.D was read at 540 nm in spectrophotometer against the blank
(1ml distilled water and 3 ml of DNSA). The standard curve was prepared from
the stock solution of glucose (0.4 mg/ml), enzyme activity was expressed in
terms of International Unit (IU) and Specific activity (SA).

• One International Unit (IU) of enzyme activity represents µmoles of

glucose released/min/ml of enzyme.
• Specific Activity (SA) represents µmoles of glucose released/min/mg of
protein.

3.1.6.1.1.2 Filter Paperase (FPase) assay (Grajek, 1987)

Reagents

• Strips of filter paper (Whattman filter paper no.1)

• 0.055 M actetate buffer (pH 5)
• Dinitrosalicylic acid (DNSA) reagent
• Standard solution of glucose (0.4 mg/ml)

52
Procedure

To 50 mg of filter paper strips (Whattman no.1), 0.5 ml of acetate buffer

(0.05M, pH 5) and 0.5ml of culture supernatant were added. Reaction mixture
was then kept for the incubation at 50°C for 30 min. After the incubation 3ml of
DNSA reagents was added. Tubes were then immersed in the boiling water bath
and removed after 15 min when colour developed was complete. Control was run
with all components except the enzyme. Tubes were cooled at room temperature
and O.D. was read at 540 nm in spectrophotometer against a reagent blank i.e. 1
ml distilled water and 3ml of DNSA reagent. The standard curve was prepared
from the stock solution of glucose (0.4 mg/ml), The enzyme activity was
expressed in terms of International unit (IU) and Specific activity (SA).

• One International Unit (IU) of enzyme activity represents µmoles of

glucose released/min/ml of enzyme.
• Specific Activity (SA) represents µmoles of glucose released/min/mg of
protein.

3.1.6.1.1.3 β-Glucosidase assay (Grajek, 1987)

Reagents

• 1mM p-nitrophenyl β- D- glucopyranoside in 0.055 M acetate buffer

(pH 0.5)
• 2 M sodium carbonate solution
• Standard solution of p-nitrophenol (80 µg/ml)

Procedure

Reaction mixture contained 0.9 ml of p-nitrophenol β-D-glucoyranoside

in 0.05M of acetate buffer and 0.1ml of supernatant. After incubation at 50° C for
10 min, 2 ml of 2 M of sodium carbonate was added into the reaction mixture to
stop the reaction and heated in the boiling water bath for 15 min and after heating
it was diluted with 10ml of distilled water. p-nitrophenol liberated was
determined from the absorbance at 420 nm against the blank, 1 ml of distilled
water and 2 ml of 2 M of sodium carbonate. The standard curve was prepared

53
from the stock solution of p-nitrophenol β-D-glucopyranoside (80µg/ml). the
enzyme activity was expressed in terms of International Units(IU) and Specific
activity (SA).

• One International Unit (IU) of enzyme activity represents µmoles of p-

nitrophenyl β-D- glucopyranoside released/min/ml of enzyme.
• Specific Activity (SA) represents µmoles of p-nitrophenyl β-D-
glucopyranoside released/min/mg of protein.

3.1.6.1.2 Xylanase assay (Miller, 1959)

Reagents

• 1.0 g Oat spelt xylan in 100 ml of 0.05 M citrate buffer at pH 4.0

• DNSA reagent
• Standard solution of xylose (0.4 mg/ml)

Procedure

To 0.5 ml of xylan solution (which is incubated overnight at 37°C),

centrifuged and clear supernatant was used, 0.3 ml of citrate buffer (pH 5) was
added and 0.2 ml of enzyme. The control was run with all components except the
enzyme. The reaction mixture was incubated at 45°C for 10 min and then 3 ml of
DNSA reagent was added and the mixture was then heated on boiling water bath
for 15 min, after cooling down at room temperature, absorbance of reaction
mixture was read at 540 nm.

• One International Unit of enzyme activity represents µmoles of xylose

released /min/ml of enzyme.
• Specific activity (SA) represents µmoles of xylose released/min/mg of
protein.

3.1.6.1.3 Protein Assay (Lowery et al., 1951)

• 1N Folin ciocalteau’s phenol reagent

• 2% Sodium carbonate in 0.1 M sodium hydroxide solution

54
• 1% Sodium potassium tartarate solution
• 1% Copper sulphate solution
• Lowry’s alkaline reagent was prepared by mixing 98 ml of (2) with 1ml
of (3) and 1ml of (4), it was made fresh at the time of use only
• Bovine serum albumin (BSA) as standard (10-100 µg/ml)

Procedure
To 0.1 ml of culture supernatant, 2.5 ml of Lowry’s alkaline reagent was
added, mixed and allowed to stand for 10 min. Diluted (1N) Folin ciocalteau’s
reagent (0.25 ml) was added. The contents were shaken quickly and allowed to
stand for 30 min for maximum color development, Absorbance of reaction
mixture was read at 670 nm against a reagent blank. The contents of protein in
culture were estimated from standard curve which was prepared by using Bovine
serum Albumin (BSA) in concentration of 10-100 µg/ml.

3.2 IDENTIFICATION OF HYPERCELLULOLYTIC AND

HYPERXYLANOLYTIC ENZYME PRODUCING ISOLATES
Among fungi F1 showing highest cellulolytic and xylanolytic activity
were selected further for production and qualitative estimation of enzyme. This
was identified as Thermomyces sp. depending upon their morphological and
physiological characteristics.

. Among the bacteria N1 was found to be the highest producer of xylanase.

This was tentatively identified as Bacillus sp. on the basis of their morphological,
physiological and biochemical characteristics. Their molecular characterization
using rrs (16S rRNA) was performed for confined identification.

3.2.1 Molecular characterization using rrs (16S rRNA) PCR technique

(www.banglore.com.GenNeiTM)
Isolate N1 was further identified at genomic level using rrs (16S rRNA)
technique as given below.

3.2.1.1 Isolation of genomic DNA (Genei DNA Isolation Kit)

The pure culture of bacterial strain N1 was inoculated in 10 ml of nutrient

broth and grown at 35±2ºC for 18 hrs. Isolation of total genomic DNA from the
culture was carried out by the following procedure:

55
 1ml of culture was centrifuged at 10,000 rpm for 10 min. Supernatant was
discarded. Bacterial pellet was resuspended in 100 µl of bacterial lysis buffer.
Incubated at 37ºC for 30 min. 180 µl of lysis buffer I and 20 µl of Proteinase K
was added and mixed thoroughly by vortexing. Incubated at 55ºC for 3 h.
Vortexed the sample at intervals for better lysis. The sample was spun at 10,000
rpm in a micro-centrifuge for 5 min and the supernatant was decanted carefully to
a fresh vial. 200 µl of lysis buffer II was added to the supernatant, mixed
thoroughly by vortexing and incubated at 70ºC for 20 min. 4µl of RNAase A
(100 mg/ml) was added, mixed by vortexing and incubated at room temperature
for 5 min. The sample was spun at 10,000 rpm in a micro-centrifuge for 5 min
and the supernatant was decanted carefully to a fresh vial. 200 µl of distilled
ethanol was added to the supernatant and was mixed thoroughly by vortexing.
Spin column was kept in a 2 ml collection tube and the sample ethanol mixture
was added to the column. Centrifuged at 10,000 rpm for 1 min. Collection tube
was discarded with flow through. The spin column was kept in a fresh 2 ml
collection tube and 500 µl of wash buffer I was added. Spun at 10,000 rpm for 1
min. Collection tube was discarded with washed sample. The spin column was
kept in a fresh 2 ml collection tube and 500 µl of wash buffer II was added.
Centrifuged at 10,000 rpm for 5 min. Washed fraction was discarded and the
collection tube was retained for further steps. The empty column was spun at
10,000 rpm for 2 min to ensure the removal of wash buffer. The spin column was
placed in a 1.5 ml tube and 200 µl of prewarmed elution buffer was added. It was
further incubated at room temperature for 5 min and centrifuged for 2 min to
elute the DNA.

3.2.1.2 PCR amplification of rrs (16S rRNA) region

PCR amplification was done to confirm the identity of the bacterial strain,
the small subunit rrs (16S rRNA) genes were amplified from the genomic DNA
with 16SF (5’AGAGTTTGATCCTGGTCAG3’) and 16SR
(5’TACCTTGTTACGACTT3’) primers to get an amplicon size of 1500 bp.
Amplification was carried out in 20µl reaction volume consisting of 10 X buffer,
2.0 µl; 2mM dNTPs, 2.0 µl; 3U/µl Taq DNA polymerase, 0.2 µl; 100ng/µl of

56
each primer, 1µl; template DNA, 1µl and sterilized distilled water 12.8 µl in a
ASTEC thermalcycler using the PCR conditions 95ºC for 2 min (denaturation),
58ºC for 1 min (annealing) and 52ºC for 3 min (extension). The total number of
cycles were 35 with the final extension at 72ºC for 10 min. The amplified product
(20 µl) was size separated on 1.0% agarose gel prepared in 1% TAE buffer
containing 0.5µg/ml ethidium bromide and photographed with the gel
documentation system (alpha Imager 2200). A 100 bp DNA ladder (Genei) was
used as molecular weight size markers.

3.2.1.3 Purification of the PCR product

The PCR product (1500 bp) was purified from contaminating products by
electroelution of the gel slice containing the excised, desired fragment with
Qiaquick gel extraction kit (Qiagen, USR). The elution was carried out in 30 µl
of nuclease free water.

3.2.1.4 Nucleotide sequencing

The PCR amplicon obtained by amplifying PCR product was diluted in

Tris buffer (10 mM, pH 8.5). Dilution used was 1:1000 in order to obtain the
DNA concentration required for sequencing (30 mg/ µl), the sequencing required
8µl DNA. The primer used in sequencing reaction was 16SF
(5’AGAGTTTGATCCTGGTCAG3’) at a concentration of 3 µM. Sequencing
was then performed using an automated sequencer (ABI PRISM 310, Applied
Biosystems, USA).

3.2.1.5 BLASTN Analysis

Translated nucleotide sequence was then analyzed for similarities by

BLASTN tool (www.ncbi.nlm.nih.gov:80/BLAST)

After molecular characterization on the basis of rrs (16S rRNA) PCR

technique N1 bacterial isolates was identified as Paenibacillus sp. N1.

57
3.3 PRODUCTION AND OPTIMIZATION OF XYLANASE FROM
HYPERXYLANOLYTIC BACTERIAL STRAIN Paenibacillus sp.
N1 UNDER SUBMERGED FERMENTATION

Various fermentation variables viz. medium, incubation time, pH,

temperature, inoculum size, amino acid, carbon, nitrogen source and additives
were studied to monitor their effect on xylanase production.

3.3.1 Effect of different media

3.3.1.1 Composition of five different media:

3.3.1.1.1 TGY-Medium (Garg et al., 2009)

Tryptone - 0.5%, K2HPO4 - 0.1%, Glucose - 0.1%, Yeast Extract-0.5%,

pH 7.1.

3.3.1.1.2 Basal Salt Medium (Dhillon and Khanna, 2000)

(NH4)2SO4 - 6.0g, KH2PO4 - 3.0g, NaCl - 0.5g, NH4Cl - 1.0 g, 1M MgSO4

- 0.2 ml, 1M CaCl2 - 0.1 ml, Yeast Extract - 0.5%./1000 ml, pH- 7.1.

3.3.1.1.3 Reese Medium (Singh et al., 2010)

NH4SO4 - 0.14%, KH2PO4 - 0.2%, MgSO4 - 0.03%, CaCl2 - 0.03%, Fe2SO4

- 0.0005%, Peptone - 0.1%, Urea - 0.03, ZnCl2 - 0.00017, CoCl2 - 0.0002, pH 9.

3.3.1.1.4 Emerson Medium (Garg et al., 2009)

Yeast Extract - 0.55%, K2HPO4 - 0.1%, MgSO4.7H2O - 0.02%, Peptone -

0.5%, pH 7.1.

3.3.1.1.5 Nakamura Medium (Nakamura et al., 1995)

Peptone - 0.5%, Yeast Extract - 0.5%, K2HPO4 - 0.1%, MgSO4 - 0.02%,

Oat spelts - 0.5%, pH 6.

3.3.1.1.6 Xylan Medium (Cacais et al., 2001)

Peptone-1%, Yeast extract-0.5%, xylan-0.5%, NaCl-0.3%, KH2PO4-0.1%,

MgSO4.7H2O-0.02%, Na2CO3-1%, pH 7.5.

58
3.3.1.2 Inoculum preparation

Each bacterial isolate was grown in 100 ml of nutrient broth and was
incubated at 35± 20C for 24 h, as soon as the substantial growth was observed in
the broth, as the optical density was set at 1.0 using sterilized distilled water.

3.3.1.3 Production of extracellular xylanase

5ml of 1.0 O.D. inoculum was added to each 45ml of each above
mentioned different media in 250ml of Erlenmeyer flasks and then flasks were
incubated at 35±2 °C under shaking condition for 5 days.

After incubation the culture contents were centrifuged at 10,000 rpm for
15 min (4°C). The supernatant was collected. The following quantitative test was
performed to find out the best media for xylanase production.

3.3.1.4 Enzyme assay

3.3.1.4.1 Xylanase activity

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.3.1.5 Protein Estimation

The protein content was estimated in the culture supernatant according to

Lowry’s Method (1951) as given in 3.1.6.1.3.

3.3.1.6 Statistical analysis

The data collected for different parameters were subjected to Completely

Randomized Design (Gomez and Gomez, 1976). The statistical analysis based in
mean value of replications used for each treatment was made using analysis of
variance (ANOVA) for Completely Randomized Design.

Basal medium showing the maximum xylanase production for

Paenibacillus sp. N1 strain was selected for the next experiment.

59
3.3.2 Effect of incubation time

Optimization of the incubation time for xylanase production from

Paenibacillus sp. N1 was done by varying the incubation days viz 1, 2, 3, 4, 5, 6
days. Rest of the conditions were same as in section 3.3.1.1.2.

3.3.2.1 Inoculum preparation

Same as described in section 3.3.1.2.

3.3.2.2 Production of extracellular xylanase

Same as described in section 3.3.1.3.

3.3.2.3 Enzyme assay

3.3.2.3.1 Xylanase assay

Activity of culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.3.2.4 Protein estimation

The protein content was determined in the culture supernatants according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.1.2.5 Statistical method

Same as in section 3.3.1.6.

Xylanase production from Paenibacillus sp. N1 was maximum on the 3rd

day and was selected for the next experiment

3.3.3 Effect of pH
Xylanase production from Paenibacillus sp. N1 was determined by
changing the initial pH of the medium from 5.0, 5.5......................10 using
optimized incubation time from previous experiment and rest of the conditions
were same as in section 3.3.1.1.2.

3.3.3.1 Inoculum preparation

Same as described in section 3.3.1.2.

60
3.3.3.2 Production of extracellular xylanase

Same as described in section 3.3.1.3.

3.3.3.3 Enzyme assay

3.3.3.3.1 Production of xylanase

Same as described in section 3.1.6.1.2.

3.3.3.4 Protein estimation

The protein content was estimated in the culture supernatant according to

Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.3.5 Statistical method

Same as in section 3.3.1.6.

Xylanase production shown highest at pH 9 was used further.

3.3.4 Effect of Temperature

The optimum temperature for the xylanase production from Paenibacillus

sp. N1 was determined by incubating the culture at different temperature ranges
i.e. 30, 35, 40, 45, 50, 55°C using optimized pH from the previous experiment
and rest of the conditions were same as in section 3.3.1.1.2.

3.3.4.1 Inoculum preparation

Same as described in section 3.3.1.2.

3.3.4.2 Production of extracellular xylanase

Same as described in section 3.3.1.3.

3.3.4.3 Enzyme assay

3.3.4.3.1 Xylanase assay

Activity of culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

61
3.3.4.4 Protein estimation

The protein content was determined in the culture supernatant according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.4.5 Statistical method

Same as in section 3.3.1.6.

Maximum xylanase production from Paenibacillus sp. N1 was observed

at temperature 50°C and based on this it has been carried forward for another
parameter.
3.3.5 Effect of the inoculum size

Inoculum size was optimized for maximum xylanase production from

Paenibacillus sp. N1 strain by varying the sizes viz. 2.5%, 5%, 7.5%, 10%, 12.5%
and 15% using optimized conditions from the previous experiments.

3.3.5.1 Inoculum preparation

Same as described in section 3.3.1.2.

3.3.5.2 Production of extracellular xylanase

Same as described in section 3.3.1.3.

3.3.5.3 Enzyme assay

3.3.5.3.1 Xylanase assay

Activity of culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.3.5.4 Protein estimation

The protein content was determined in the culture supernatant according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.5.5 Statistical method

Same as in section 3.3.1.6.

62
 Inoculum size of 12.5% exhibiting maximum xylanase production was
chosen further.

3.3.6 Effect of different amino acids

Xylanase production from Paenibacillus sp. N1 was optimized by adding

different amino acids like phenylalanine, tryptophan, aspargine, glutamic acid,
cystiene and aspartic acid (0.02%) utilizing optimized parameters from the
previous experiments.

3.3.6.1 Inoculum preparation

Same as described in section 3.3.1.2.

3.3.6.2 Production of extracellular xylanase

Same as described in section 3.3.1.3.

3.3.6.3 Enzyme assay

3.3.6.3.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.3.6.4 Protein estimation

The protein content was determined in the culture supernatant according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.6.5 Statistical method

Same as in section 3.3.1.6.

Xylanase production from Paenibacillus sp. N1 was observed to be

highest when tryptophan was added into the medium and thus was finalised for
the next experiments.

3.3.7 Effect of different carbon sources

Effect of different carbon sources on xylanase production were optimized

by adding different carbon sources at a concentration of 1% viz. sucrose, maltose,

63
dextrose, lactose, fructose, arabinose, ribose and xylose and keeping rest of the
conditions optimized in the previous experiments.

3.3.7.1 Inoculum preparation

Same as described in section 3.3.1.2.

3.3.7.2 Production of extracellular xylanase

Same as described in section 3.3.1.3.

3.3.7.3 Enzyme assay

3.3.7.3.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.3.7.4 Protein estimation

The protein content was determined in the culture supernatant according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.7.5 Statistical method

Same as in section 3.3.1.6.

Highest xylanase production from Paenibacillus sp. N1 was observed

with xylose and thus used as a carbon source into the medium for next process
factor.

3.3.8 Effect of different nitrogen sources

Xylanase production from Paenibacillus sp. N1 was determined by adding

different nitrogen source viz. yeast extract, peptone, beef extract, casein,
tryptone, NH4NO3, NH4NO2, (NH4)H2PO4, (NH4)HPO4 etc into the medium at a
concentration of 0.5% using optimized conditions from the previous experiment.

3.3.8.1 Inoculum preparation

Same as described in section 3.3.1.2.

64
3.3.8.2 Production of xylanase

Same as described in section 3.3.1.3.

3.3.8.3 Enzyme assay

3.3.8.3.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.3.1.8.4 Protein estimation

The protein content was determined in the culture supernatant according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.8.5 Statistical method

Same as in section 3.3.1.6.

(NH4)2HPO4 was reported the best nitrogen source used further.

3.3.9 Effect of different additives

To find out the optimum condition for xylanase production different

additives like PEG 2000, Tween 20, Tween 80 and SDS were added into the
medium at concentration of 10 µg/ml.

3.3.9.1 Inoculum preparation

Same as described in section 3.3.1.2.

3.3.9.2 Production of xylanase

Same as described in section 3.3.1.3.

3.3.9.3 Enzyme assay

3.3.9.3.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

65
3.3.9.4 Protein estimation

The protein content was determined in the culture supernatant according

to Lowry’s Method (1951) as given in section 3.1.6.1.3.

3.3.9.5 Statistical method

Same as in section 3.3.1.6.

3.3.10 Percent (%) increase in enzyme activity after optimization of

different parameters
Final enzyme activity – initial enzyme activity
% increase in enzyme activity = x 100
Initial enzyme activity

3.4 PRODUCTION AND OPTIMIZATION OF CELLULASE AND

XYLANASE FROM HYPERCELLULOYTIC AND
XYLANOLYTIC FUNGUS Thermomyces sp. F1 UNDER SOLID
STATE FERMENTATION

3.4.1 Selection of lignocellulosic biomass for cellulase and xylanase

production

Rice straw (Oryza sativa) is being an agricultural waste that has been
generated in the bulk can be used as the agricultural residue for the production of
cellulase and xylanase enzyme. Rice straw was collected and chipping of biomass
was done to get the small pieces. The chipped pieces were dried in oven at 50°C.
After drying the chips of each were ground to fine particles of 2 mm mesh size.
The completely dried biomass was stored in the air tight containers for further
use.

3.4.2 Pretreatment of the lignocellulosic biomass

3.4.2.1 Microwave irradiation

100 g of selected biomass (Oryza sativa) was taken in a beaker and was
microwave (Godrej make) irradiated at 250 V and 50 Hrtz for 2 min (biomass
were mixed properly after interval of 1 min) and kept in air tight container for
further use.

66
3.4.3 Production of cellulase by utilizing lignocellulosic biomass i.e.
rice straw (Oryza sativa) as a substrate
Production of cellulase under solid state fermentation was carried out in
Vogel’s medium at pH 5.5, moisture level of 1:4 and temperature 500C.
3.4.3.1 Inoculum preparation

Added 10% of seven days old fungal culture scratched with 10ml of
autoclaved distilled water and fixing their inoculum size 1×107 spores/ml.

3.4.3.2 Production of enzyme

To each 5 g of untreated and each pretreated biomass, 20 ml of moistening

agent (Vogel’s medium for Thermomyces sp. F1) i.e. in ratio of 1:4 (substrate :
moistening agent) was added in 250 ml Erlenmeyer flask. It was autoclaved.
After autoclaving, 2ml of fungal inoculum size 1×107 spores/ml was added and
incubated at 50±2°C for 8 days in static phase.

3.4.3.3 Extraction of cellulase by repeated extraction method (Bollag &

Edelstein, 1991)

To 5 g of each untreated and pretreated biomass, 50 ml of phosphate

buffer (0.1M, pH 6.9) with 0.1% Tween-80 was added in 250 ml Erlenmeyer
flask. The contents were kept in the shaker for 1 h at 120 rpm and then filtered
through muslin cloth. The process was repeated twice with 25 ml of phosphate
buffer. After filtration, contents were centrifuged at 5,400 rpm for 10 min at 4°C.
The supernatant was collected for further studies.

3.4.3.4 Enzyme assays

3.4.3.4.1 Cellulase activity

Activity of the culture supernatant was determined by the same method as

described in section

CMCase assay as given in section 3.1.6.1.1.1.

FPase activity mentioned in section 3.1.6.1.1.2.
ß-glucosidase activity as given in section 3.1.6.1.1.3.

67
3.4.3.5 Protein estimation

The protein content was estimated in the culture supernatant according to

Lowry’s Method as given in section 3.1.6.1.3.

3.4.3.6 Statistical analysis

Same as described in section 3.3.1.6.

3.4.4 Optimization of moisture level for cellulase production
Cellulase production from Thermomyces sp. F1 was determined by
changing the moisture level of the substrate from 1:2, 1:4, 1:6 and 1:8. Using
Vogel’s medium as a moistening agent, at pH 5.5.

3.4.4.1 Inoculum preparation

Same as described in section 3.4.3.1.

3.4.4.2 Production of enzyme

Same as described in section 3.4.3.2.

3.4.4.3 Extraction of cellulase by repeated extraction method (Bollag &

Edelstein, 1991)
Same as described in section 3.4.3.3.

3.4.4.4 Enzyme assays

3.4.4.4.1 Cellulase assay

Activity of the culture supernatant was determined by the same method as

described in section:

CMCase activity as given in section 3.1.6.1.1.1.

FPase activity as given in section 3.1.6.1.1.2.
β-glucosidase activity as given in section 3.1.6.1.1.3.

3.4.4.5 Protein estimation

As mentioned in section 3.1.6.1.3.

68
3.4.4.6 Statistical analysis

Same as described in section 3.3.1.6.

Moisture level of 1:6 revealing highest cellulase was chosen for another
experiment.

3.4.5 Optimization of temperature for cellulase production

Optimum temperature for cellulase production from Thermomyces sp.F1

was determined by incubating the cultures at different temperature ranges from
viz 30, 35, 40, 45, 500C using optimized moisture from the previous experiments
and rest of the conditions were same.

3.4.5.1 Inoculum preparation

Same as in section 3.4.3.1.

3.4.5.2 Production of enzyme

Same as in section 3.4.3.2.

3.4.5.3 Extraction of cellulase by repeated extraction method (Bollag &

Edelstein, 1991)

Same as in section 3.4.3.3.

3.4.5.4 Enzyme assays

3.4.5.4.1 Cellulase assay

Activity of the culture supernatant was determined by the same method as

described in section:

CMCase activity as given in section 3.1.6.1.1.1.

FPase activity as given in section 3.1.6.1.1.2.
β-glucosidase activity as given in section 3.1.6.1.1.3.

3.4.5.5 Protein estimation

As mentioned in section 3.1.6.1.3.

69
3.4.5.6 Statistical analysis

Same as in section 3.3.1.6.

Maximum cellulase production from Thermomyces sp. F1 was observed at

temperature 500C and based on this it has been selelected for next parameter.

3.4.6 Optimization of incubation time on cellulase production

Incubation time was optimized for maximum cellulase production from

Thermomyces sp. F1 at different period of time i.e. 4, 5, 6, 7, 8, 9, 10 day using
optimized conditions of the previous experiments.

3.4.6.1 Inoculum preparation

Same as in section 3.4.3.1.

3.4.6.2 Production of enzyme

Same as in section 3.4.3.2.

3.4.6.3 Extraction of cellulase by repeated extraction method (Bollag &

Edelstein, 1991)

Same as in section 3.4.3.3.

3.4.6.4 Enzyme assays

3.4.6.4.1 Cellulase assay

Activity of the culture supernatant was determined by the same method as

described in section:

CMCase activity as given in section 3.1.6.1.1.1.

FPase activity as given in section 3.1.6.1.1.2.
β-glucosidase activity as given in section 3.1.6.1.1.3.

3.4.6.5 Protein estimation

As mentioned in section 3.1.6.1.3

70
3.4.6.6 Statistical analysis

Same as in section 3.3.1.6.

3.4.7 Production of xylanase by utilizing lignocellulosic biomass i.e.

rice straw (Oryza sativa)

The production of xylanase under solid state fermentation was carried out
at temperature of 500C, in Vogel’s medium at pH 5.5.

3.4.7.1 Inoculum preparation

Added 10% of seven days old fungal culture scratched with 10ml of
autoclaved distilled water and fixing their inoculum size 1×107 spores/ml.

3.4.7.2 Production of enzyme

To 5 g of each untreated and each pretreated biomass, 20 ml of moistening

agent i.e. in ratio of 1:4 (substrate: moistening agent) was added in 250 ml
Erlenmeyer flask. After autoclaving, the flasks were inoculated with 1×107
spores/ml was added and incubated at 50±2°C for 5 days in static phase.

3.4.7.3 Extraction of xylanase by repeated extraction method (Bollag &

Edelstein, 1991)

To 5 g of each untreated and pretreated biomass, 50 ml of phosphate

buffer (0.1M, pH 6.9) with 0.1% Tween-80 was added in 250 ml Erlenmeyer
flask. The contents were kept in the shaker for 1 h at 120 rpm and then filtered
through muslin cloth. The process was repeated twice with 25 ml of phosphate
buffer. After filtration, contents were centrifuged at 5,400 rpm for 10 min at 4°C.
The supernatant was collected for further studies.

3.4.7.4 Enzyme assay

3.4.7.4.1 Xylanase activity

Same as described in section 3.1.6.1.2

3.4.7.5 Protein estimation

As mentioned in section 3.1.6.1.3.

71
3.4.7.6 Statistical analysis

Same as described in section 3.3.1.6.

3.4.8 Optimization of moisture level for xylanase production

Xylanase production from Thermomyces sp. F1were determined by

varying the moisture level viz 1:2, 1:4, 1:6 and 1:8 using optimized conditions
from the previous experiment 3.4.3.

3.4.8.1 Inoculum preparation

Same as in section 3.4.7.1

3.4.8.2 Production of enzyme

Same as in section 3.4.7.2

3.4.8.3 Extraction of xylanase by repeated extraction method (Bollag &

Edelstein, 1991)

Same as in section 3.4.7.3.

3.4.8.4 Enzyme assay

3.4.8.4.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.4.8.5 Protein estimation

As mentioned in section 3.1.6.1.3.

3.4.8.6 Statistical analysis

Same as in section 3.3.1.6.

Maximum xylanase production observed at moisture level of 1:6 was

selected further.

3.4.9 Optimization of the temperature for the xylanase production

Optimum temperature for xylanase production from Thermomyces sp.F1
was determined by incubating the cultures at different temperature

72
ranges viz. 30, 35, 40, 45, 500C using optimized moisture level from the previous
experiment and rest of the conditions were same as in previous experiment.

3.4.9.1 Inoculum preparation

Same as in section 3.4.7.1.

3.4.9.2 Production of enzyme

Same as in section 3.4.7.2.

3.4.9.3 Extraction of xylanase by repeated extraction method (Bollag &

Edelstein, 1991)

Same as in section 3.4.7.3.

3.4.9.4 Enzyme assay

3.4.9.4.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.4.9.5 Protein estimation

As mentioned in section 3.1.6.1.3.

3.4.9.6 Statistical analysis

Same as in section 3.3.1.6.

Xylanase production highest at temperature 500C was used further.

3.4.10 Optimization of incubation time for xylanase production

Incubation time was optimized for the maximum xylanase production by

varying the incubation days i.e. 2, 3 ,4 ,5 ,6 ,7 and rest of the condition were
same as optimized in the previous experiment.

3.4.10.1 Inoculum preparation

Same as in section 3.4.7.1.

73
3.4.10.2 Production of enzyme

Same as in section 3.4.7.2.

3.4.10.3 Extraction of xylanase by repeated extraction method (Bollag &

Edelstein, 1991)

Same as in section 3.4.7.3.

3.4.10.4 Enzyme assay

3.4.10.4.1 Xylanase assay

Activity of the culture supernatant was determined by the same method as

described in section 3.1.6.1.2.

3.4.10.5 Protein estimation

As mentioned in section 3.1.6.1.3.

3.4.10.6 Statistical analysis

Same as in section 3.3.1.6.

3.5 PERCENT (%) INCREASE IN ENZYME ACTIVITY AFTER

OPTIMIZATION OF DIFFERENT PARAMETERS

Final enzyme activity – initial enzyme activity

% increase in enzyme activity = x 100
Initial enzyme activity

74
 Chapter-4

RESULTS AND DISCUSSION

Globally distributed hot springs have concentrated on cultivating and
isolating extremophiles viz. thermophilic, alkalophilic and acidophilic strains
(Belkova et al., 2007). In the present study thermophilic microorganisms were
isolated from thermal hot water springs of Himachal Pradesh and studied for their
ability to produce cellulase and xylanase enzymes. In Himachal Pradesh-
Tattapani (Distt. Mandi), Manikaran and Vashist (Distt. Kullu) are the popular hot
water spring pockets which are scarcely being explored for their microbial
diversity.

4.1 PHYSIOCHEMICAL ANALYSIS OF WATER FROM HOT

SPRINGS

Physiochemical characteristics of Tattapani, Manikaran and Vashist

thermal springs include the study of temperature, pH, conductivity, chloride,
sulphate, calcium, magnesium and total hardness. Most of the physiochemical
characterization of the water samples from these hot pockets has been enlisted in
Table 1.

Tattapani hot spring located near Sunni in Distt. Mandi on the right bank
of Satluj river at an elevation of 625 m above sea level showed temperature in the
range of 530C to 600C. All the four sites where the water samples were collected
showed the pH in the neutral range i.e. 6.96.

Beas and Parbati valley accounts for 15 of the 30 thermal springs in

Himachal Pradesh. In the Parbati valley, Manikaran and in Beas valley, Vashist
are the most significant amongst all. The temperature of hot springs varied from
990C at Manikaran and 450C in Vashist. Maximum temperature (990C) was
reported among these three hot springs. pH of the hot springs both in Manikaran
and Vashist fell into neutral/near neutral range i.e. 7.67 and 6.59 respectively.
 Physiochemical analysis of the water from Manikaran and Vashist had
total hardness i.e. 164 mg/ml and 60 mg/l are considered as soft and moderant
water when compared with tap water having total hardness of 0-100 mg/l as soft
water and 100-200 mg/l as moderate water (www.idph state.il us/envhealth/
pdf/drinkingwater.pdf). Whereas water from Tattapani contained total hardness of
530.000 mg/l and thus considered as extremely hard water containing
bicarbonates, sulphates, chloride, and nitrates of Ca and Mg. Maximum
permissible limit for total hardness is 600 mg/l as per Indian standards (Kumar et
al., 2010).

Table 1. Physiochemical analysis of water sample from hot springs of

Himachal Pradesh

Parameter Vashisht Manikaran Tattapani Mean S.E. (Mean)

pH 6.590 7.671 6.960 7.071 0.321
Conductivity 782.000 655.000 975.001 804.00 93.030
Total Hardness (mg/l) 60.000 164.000 530.000 399.671 289.231
2+
Ca (mg/l) 8.011 40.031 148.120 65.390 42.391
-
Cl (mg/l) 122.200 123.260 385.761 210.41 87.680
2+
Mg (mg/l) 9.721 15.550 38.870 21.380 8.911
2-
SO4 (mg/l) 57.500 31.681 90.800 59.990 17.110

2-
PO4 0.280 0 0 0.091 0

Temperature 45.000 99.000 52 .000 65.330 16.950

Turbidity 0.021 0.032 0.033 0.033 0.033
Colour Transparent Transparent Transparent - -

Chloride contents were very high in water of Tattapani (385.761 mg/l) as

compared to chloride content in Manikaran (123.260 mg/l) and Vashist (122.200
mg/l). Chloride concentration above 250 mg/ml can produce a distinct taste in
drinking water (www.idph state.il us/evehealth/pdf/drinking water/pdf).

The chemical characterization of the water sample collected from the hot
springs showed that there was a higher concentration of chemicals in Tattapani as
compared to Manikaran and Vashist. Sulphur is a mineral naturally occurring in
hot springs and volcanic craters. The "rotten egg" smell of sulphur mineral baths

76
is caused by sulfur dioxide gas escaping into the air. Sulphur content were more in
water of Tattapani (90.800 mg/l) as compared to water sample of Vashist (57.500
mg/l) and Manikaran (31.681 mg/l) while a value more than 500 mg/l causes
abrupt taste and many corrode distribution in network pipes (Kumar et al., 2010).

Hyperthermophilic microflora required the sulphur for their growth. Most

of the sequences of the bacteria and archaea libraries found in the sulfur mat
belong to groups that are known to use reduced or oxidized sulfur compounds for
growth and verifies the chemical composition of the hot spring, i.e., high sulphide
(Skirnisdotti et al., 2000). Several features make these hot water pockets suited
for studies of microbial community ecology: (i) they are accessible; (ii) they have
high biomass, thus enabling sophisticated molecular analysis; (iii) they are
relatively stable systems; (iv) they exhibit well-defined environmental gradients
(e.g., temperature, light) and are geographically separated (Ward and Castenholz,
2000). Microorganisms present in the hot water springs have a range of diversity
varying from neutrophilic (Dimitrov et al., 1997), acidophilic (Tan et al., 2001) to
alkalophilic (Singh et al., 2010). Presence of sulphur and other inorganic material
in the hot water springs have rendered them more suitable for the growth of a
variety of microorganisms.

4.2 ISOLATION OF CELLULOLYTIC AND XYLANOLYTIC

BACTERIA FROM HOT SPRINGS OF HIMACHAL PRADESH

In total, 63 isolates were isolated from the hot springs of Himachal

Pradesh including Tattapani (Distt. Mandi), Manikaran and Vashist (Distt. Kullu)
as enlisted in Table 2a, 2b, 2c and 2d. All the isolates were aerobic in nature
capable of producing cellulase and xylanase and these grew at temperature above
500C. Out of total isolates 27 strains were isolated from the Tattapani and most of
them were rod shaped (74.000%) and gram positive (96.292%). The isolates from
Manikaran and Vashist were mostly gram positive (92.591% and 100.000%)
some were rod shaped (59.250% and 63.633% respectively), and coccobacilli
i.e.7.400% (Tattapani), 25.920% (Manikaran) and 27.270% (Vashist).

77
Table 2a. Isolation of cellulolytic and xylanolytic bacteria from hot springs
of Tattapani and their morphological and cultural characteristics

Sr. Isolate Cell morphology Gram staining

no. no. Colour Form Elevation Margin Gram Shape
(+ve/-ve)
1 T1 White Filamentous Umbonate Undulated + Rod

2 T2 White Rhizoid Umbonate Lobulate + Rod

3 T3 White Irregular Convex Entire + Coccobacillus

4 T4 white Irregular Flat Undulated + Round

5 T5 White Circular Flat Erose + Rod

6 T6 Creamy Circular Undulated Entire + Rod

7 T7 Creamy Punctiform Convex Entire + Rod

8 T8 Creamy Punctiform Flat Entire + Round

9 T9 White Irregular Umbonate Entire + Coccobacillus

10 T10 White Irregular Flat Undulated + Rod

11 T11 White Irregular Umbonate Erose + Rod

12 T12 White Punctiform Flat Entire + Round

13 T13 White Punctiform Umbonate Entire + Rod

14 T14 White Irregular Flat Entire + Rod

15 T15 Dirty White Punctiform Convex Entire + Rod

16 T16 Creamy Punctiform Flat Entire + Rod

17 T17 Creamy Circular Flat Enitre + Rod

18 T18 White Irregular Flat Undulated + Rod

19 T19 White Irregular Flat Undulated + Rod

20 T20 Dirty White Filamentous Umbonate Lobulate + Round

21 T21 Creamy Circular Convex Entire + Rod

22 T22 White Circular Umbonate Entire + Rod

23 T23 White Circular Raised Entire + Rod

24 T24 Dirty Irregular Umbonate Undulated + Rod

25 T25 Creamy Punctiform Convex Entire + Rod

26 T26 Light orange Circular Flat Undulated + Round

27 T27 Creamy Punctiform Flat Entire + Rod

Isolation at 500C

78
Table 2b. Isolation of cellulolytic and xylanolytic bacteria from hot springs
of Manikaran and their morphological and cultural characteristics

Sr. Isolate Cell Morphology Gram Staining

No. no. Colour Form Elevation Margin Gram Shape
+ve/-ve
1 M1 White Circular Flat Erose + Round

2 M2 White Irregular Umbonate Undulated + Rod

3 M3 Creamy Irregular Umbonate Undulated + Rod

4 M4 Creamy Irregular Flat Undulated + Rod

5 M5 White Circular Flat Entire + Rod

6 M6 White Circular Umbonate Erose + Rod

7 M7 White Irregular Umbonate Lobulate + Cocoobacillus

8 M8 Creamy Irregular Umbonate Undulated + Coccobacillus

9 M9 Creamy Irregular Umbonate Erose + Cocobacillus

10 M10 White Irregular Umbonate Undulated - Round

11 M11 White Irregular Raised Entire + Coccobacillus

12 M12 White Circular Umbonate Undulated + Rod

13 M13 White Irregular Flat Erose + Rod

14 M14 Creamy Irregular Flat Undulated + Rod

15 M15 Dirty white Circular Flat Erose + Rod

16 M16 Creamy Irregular Umbonate Lobulate + Rod

17 M17 White Irreglar Flat Erose - Rod

18 M18 White Irregular Umbonate Erose + Rod

19 N1 Creamy Irregular Flat Erose + Cococobacilli

20 N2 white Circular Flat Entire + Coccobacillli

21 N3 White Irregular Umbonate Lobulated + Rod

22 N4 Creamy Circular Flat Entire + Rod

23 N5 Creamy Irregular Flat Entire + Coccobacillii

24 N6 Creamy Circular Raised Erose + Round

25 N7 White Irregular Flat Lobulate + Rod

25 N8 White Irregular Umbonate Erose + Rod

27 N9 Creamy Irregular Flat Lobulate + Rod

Isolation at 50 0C

79
Table 2c. Isolation of cellulolytic and xylanolytic bacteria from hot springs
of Vashisht and their morphological and cultural characteristics

Cell morphology Gram staining

Isolate
Sr. No. Colour Form Elevation Margin Gram Shape
no.
(+ve/ - ve)
1 V1 Creamy Punctiform Convex Entire + Rod
2 V2 White Circular Umbonate Entire + Coccobacilli
3 V3 White Irregular Flat Undulated + Rod
4 V4 Creamy Irregular Flat Erose + Rod
5 V5 Dirty white Irregular Umbonate Undulated + Round
6 V6 Creamy Irregular Flat Undulated + Coccobacilli
7 V7 White Irregular Flat Undulated + Rod
8 V8 White Irregular Flat Lobulated + Rod
9 V9 White Irregular Flat Erose + Rod
10 V10 White Irregular Umbonate Undulated + Coccobacilli
11 V11 White Punctiform Flat Entire + Rod

Isolation at 500C

Table 2d. Percentage analysis of morphological and cultural characteristics

of bacterial isolates from hot springs of Himachal Pradesh

Tattapani Manikaran Vashisht Overall

percentage
Colour White 55.551 55.551 63.631 56.921
Creamy 29.622 40.700 27.271 33.821
Dirty white 11.440 3.700 9.092 7.832
Yellow 3.700 0 0 1.541
Form Punctiform 29.621 0 18.181 15.382
Circular 25.920 29.621 9.092 24.611
Irregular 33.332 70.372 72.720 55.382
Filamentous 7.430 0 0 3.071
Rhizoidal 3.721 0 0 1.540
Elevation Flat 44.411 48.140 63.630 49.211
Raised 7.412 7.400 0 6.150
Convex 18.513 0 9.091 9.220
Umbonate 29.622 44.441 27.270 35.381
Margin Entire 59.251 18.510 27.270 36.922
Erose 7.443 37.033 18.181 21.551
Filamentous 0 0 0 0
Undulated 25.922 25.920 45.450 29.231
Lobulate 7.444 18.510 9.091 12.321
Gram (+/-ve) (+)ve 96.292 92.591 100.000 95.381
(-)ve 3.700 7.400 0 4.611
Shape Rod 74.000 59.250 63.633 66.120
Circular 18.511 14.811 9.091 15.380
coccobacilli 7.400 25.920 27.272 18.460

80
 Table 3a, 3b, 3c represent the biochemical characteristics of 65 bacterial
isolates from the hot springs of Himachal Pradesh. Different biochemical tests viz
cellulase, amylase production, gelatin test, carbohydrate metabolism, fermentation
of carbohydrate were profound with them for the characterization of bacterial
isolate. All the isolates from the different hot springs are capable of cellulase and
amylase production. Out of 65 isolates, 27 isolates showed positive for urease
whereas 12 of the isolates are catalase negative. Depending upon their
morphological and cultural characteristics they were identified as Bacillus spp.
and Coccus spp.

Thermal springs represent extreme niches that have maintained some

degree of pristine quality and their biotechnological potential has remained
unrealized. Several attempts were made for isolation of microfauna from the hot
springs (Reysenbach et al., 2000a & Skimisodottir et al., 2000). Xylan degrading
alkali tolerant thermophiles have been isolated from hot spring in Bulgaria
(Dimitrov et al., 1997) and Portugal (Bataillon et al., 1998). Manikaran thermal
spring located in Himachal Pradesh is famous for its hot water whose temperature
is near boiling point thus it could serve as the good source for isolation of
thermotolerant cellulolytic/xylanolytic bacteria. These springs have already been
reported to be a good source of amylolytic bacteria (Sodhi et al., 2005) but so far
few report was available regarding potential cellulolytic and xylanolytic
microorganisms (Singh et al., 2010).

110 alkalo-tolerant thermophilic bacteria were isolated from 17 samples

(water and sediments) collected from Manikaran. Among them 70 showed the
production of xylanase and were further screened for growth and production of
xylanase at different temperature ranging from 40-750C. Isolates that showed the
maximum production of xylanase at high temperature were selected for
qualitative estimation in modified Reese mineral liquid medium containing wheat
bran and maximum xylanase activity was reported by isolate H-7 followed by H-9
and R-9 and was identified as Panenibacillus ehemensis, Bacillus
cereus/B.thurengenisis and B. subtilis (Singh et al., 2010).

81
Table 3a. Biochemical characteristics of cellulolytic and xylanolytic bacterial isolates from Tattapani

Isolate Cellulase Amylase Casein Carbohydrate Fermentation Citrate Gelatin MR VP Urease Catalase Identification
Test Test Test metabolism of glucose Synthesis Hydrolysis test test
T1 + + - + - - + - - - - Bacillus
T2 + + - + - - + - - - - Bacillus
T3 + + - + - - + + - - - Coccus
T4 + + - + - - + + - - - Coccus
T5 + + + + + + + + - - - Coccus
T6 + + - - + + + + - - - Bacillus.
T7 + + - - + - + + - + - Bacillus
T8 + + - - + - + + - - - Bacillus
T9 + + + - + - + + - - - Bacillus
T10 + + - - + - + + - - - Coccus
T11 + + - - + - + + - - - Coccus
T12 + + - - - + + + - + + Bacillus
T13 + + + + - + + + + - + Bacillus
T14 + + - - - - + + + - + Bacillus
T15 + + + - - - + + + - - Coccus
T16 + + - - + - + + + - - Coccus
T17 + + - + + - - + + + - Bacillus
T18 + + - - + - - + + - - Bacillus
T19 + + + - + - - - - - - Bacillus
T20 + + + - + + - - - - - Coccus
T21 + + + - + + + - - + + Coccus
T22 + + - - + - + - - + - Bacillus
T23 + + - - + - + - - - + Bacilllus
T24 + + + + + - + + - + - Bacilus
T25 + + + + - + + + - + - Coccus
T26 + + + + - + + + - + - Coccus
T27 + + + + - + + + - - - Bacillus
F2 + + - - + + + + - - - Bacillus
Table 3b. Biochemical characteristics of cellulolytic and xylanolytic bacterial isolate from Manikaran
Isolate Cellulase Amylase Casein Carbohydrate Fermentation Citrate Gelatin MR VP Urease Catalase test Identification
Test Test Test metabolism of glucose synthesis hydrolysis test
M1 + + - - + + + - + + - Bacillus
M2 + + - - + + + - + + - Coccus
M3 + + - - + + + - + + - Bacillus
M4 + + - - + + + - + + - Bacillus
M5 + + - - + + + - + + - Bacillus
M6 + + + + + + + + - - - Coccus
M7 + + + + + + + + - - - Coccus
M8 + + + + + + + + - + - Bacillus
M9 + + + + + - + + - + - Bacillus
M10 + + + - + - + + + + + Bacillus
M11 + + + + + - + + + + - Coccus
M12 + + + - - - + + - + - Coccus
M13 + + + - - - + + - - - Bacillus
N1 + + - - + - - + - - - Bacillus
N2 + + - - + + + + - - - Bacillus
N3 + + + - + + + + - - + Coccus
N4 + + + - + + + + - + - Coccus
N5 + + + - + + + + - + - Bacillus
N6 + + + - + + + + - + + Bacillus
N7 + + + - - + - + - + - Bacillus
N8 + + + - - + - + - + - Bacillus
N9 + + + + - + - + + - - Bacillus
M14 + + + + - + - + + - - Bacillus
M15 + + + + - - - + + - + Coccus
M16 + + + + + - + + + - - Coccus
M17 + + + + + - + + + + - Coccus
M18 + + - - + - + + + + - Bacillus
F1 + + + - + - + + + + + Bacillus
Table 3c. Biochemical characteristics of cellulolytic and xylanolytic bacterial isolate from Vashisht
Isolate Cellulase Amylase Casein Carbohydrate Fermentation Citrate Gelatin MR VP Urease Catalase Identification
Test Test Test metabolism of glucose synthesis hydrolysis test test
V1 + + - - + - + - - - - Bacillu

V2 + + - - + - + - - - - Bacilus

V3 + + + - + - + + - - - Bacillus

V4 + + + + + + + + - - + Bacillus

V5 + + + + + + + + - - - Bacillus

V6 + + + + + + - + - - - Coccus

V7 + + + + + - + + + - - Coccus

V8 + + + + + - + + + + + Bacillss

V9 + + + + + + + + + + - Bacillus

V10 + + + + + - - + + - - Bacillus

V11 + + - + + - + - - - - Bacillus
 A xylanolytic thermophilic bacterium (IT-08) was isolated from Gunung
hot spring after two days of enrichment in Modified Thermos Medium (MTM)
supplemented with 0.5% oat spelt xylan, and was active at temperature from 40-
1000C, at pH value between 4.0 and 9.0, while optimum xylanase activity was
obtained at 800C and pH 6.0 ( Tan et al., 2001).

Two alkali tolerant thermophilic bacterial strains were isolated by

continuous cultivation from samples collected near Bulgarian hot springs by
Dimitrov et al. (1997). Both isolates have xylanolytic activity with optimal
temperature i.e. 70-750C and pH 5.5 -8.0. Xylanolytic activity was resistant to pH
5.5-8.0 (for strain SP) and 6.0-7.5 (for strain BC). Similarly Touzel et al. (2000)
isolated an aerobic, thermophilic, xylanolytic spore forming bacterium, XETP
from farm soil situated underneath a manure heap in northern France. It grows at
temperature range of 630C and pH range of 6.5-8.5.

4.3 ISOLATION OF CELLULOLYTIC AND XYLANOLYTIC

THERMOPHILIC FUNGI FROM HOT SPRINGS OF HIMACHAL
PRADESH

Two fungal isolates capable of producing cellulase and xylanase were

isolated from the hot springs of Himachal Pradesh. Their morphological and
cultural characteristics have been shown in Table 2e, Fungus F1 light brown in
color having rough surface and septate hyphae had been isolated from the
Manikaran whereas F2 fungus having septate and white colored hyphae were
isolated from hot springs of Tattapani.

Table 2e. Isolation of cellulolytic and xylanolytic fungi isolated from the hot
springs of Himachal Pradesh and their morphological and
cultural characterization
Fungal Source Mycelium Spore
isolate Colour Texture Identification
F1 Manikaran Short Light Rough Thermomyces
hyphae brown sp.
F2 Tattapani Short White Smooth Determomyces
hyphae sp.
Isolation at 50 0C

85
 The geothermal sites near neutral and alkali thermal springs in Tengchong
Rehai National Park were examined by Pan et al. (2010) through cultivation-
dependent approach to determine the diversity of thermophilic fungi in these
environments. In total, 102 strains were isolated and identified as Rhizomucor
miehei, Chaetomium sp, Talaromyces thermophilus, Talaromyces
byssochlamydoides, Thermoascus aurantiacus, Miehe var. levisporus,
Thermomyces lanuginosus, Scytalidium thermophilum, Malbranchea flava,
Myceliophthora sp., Myceliophthora sp., Myceliophthora sp. and Coprinopsis sp.,
Two species, T. lanuginosus and S. thermophilum were the dominant species
among them, Geothermal soil near Amphitheater Springs in Yellowstone National
Park were characterized by high temperatures (up to 70°C), high heavy metal
content, low pH values (down to pH 2.7), sparse vegetation and limited organic
carbon. Two of these species were thermophilic and six were thermotolerant.

Underground coal mine soil, bird nest materials, vermicompost, cow dung,
poultry litter, decomposting pits are prepared from agrowaste, municipal waste
and zoo dump materials and industrial waste, were used to isolate 46 thermophilic
fungi. Isolates of 446 species belonging to 13 genera on different substrates
collected from different places of Andhra Pradesh, belonged to Humicola
lanuginosus and Aspergillus fumigates are found as a thermo-tolerant (Rajavaram
et al., 2010).

4.4 SCREENING OF HYPERCELLULOLYTIC AND

HYPERXYLANOLYTIC BACTERIA FROM HOT SPRINGS OF
HIMACHAL PRADESH
Production of extracellular enzyme i.e. cellulase and xylanase from
different bacterial isolates are depicted in table 4a, 4b, 4c and fig 1, fig 2 and fig 3
respectively. Among the different isolates, N1 strain isolated from Manikaran
showed the appreciable xylanase production i.e. 24.600 IU/ml maximum followed
by M13 (23.000 IU/ml) and M12 (21.00 IU/ml) and minimum was observed in T11
(2.100 IU/ml). Cellulase production was found minimally low in all the bacterial
isolates. Highest cellulase activity was exhibited by V9 was 0.357 IU/ml (FPase
0.023 IU/ml, CMCase 0.164 IU/ml and β-glucosidase 0.170 IU/ml) while least
cellulase activity was noted in M9 0.022 IU/ml (FPase 0.018 IU/ml, CMCase
0.004IU/ml).

86
Table 4a. Screening of bacterial isolates from hot springs of Tattapani for hypercellulase and hyperxylanase production

Isolates Protein FPase CMCase β -Glucosidase Total Total Protein Xylanase activity
(mg/ml) cellulase specific (mg/ml)
*Enzyme **Specific *Enzyme **Specific *Enzyme **Specific activity *Enzyme **Specific
activity activity activity activity activity activity activity activity
T1 0.492 0.220 0.046 0.044 0.090 0.000 0.000 0.067 0.550 2.090 4.570 1.730
T2 0.456 0.052 0.114 0.014 0.042 0.000 0.000 0.066 0.156 2.360 3.000 0.980
T3 0.886 0.031 0.036 0.152 0.146 0.040 0.079 0.255 0.260 2.140 7.380 3.400
T4 0.139 0.096 0.680 0.082 0.059 0.028 0.180 0.211 1.450 1.430 2.700 1.860
T5 0.881 0.043 0.048 0.019 0.038 0.000 0.000 0.062 0.086 2.290 10.200 4.420
T6 0.862 0.033 0.038 0.001 0.013 0.000 0.000 0.034 0.051 2.360 6.000 2.540
T7 0.453 0.012 0.200 0.078 0.160 0.120 0.320 0.210 0.680 2.430 3.500 1.380
T8 0.132 0.082 0.630 0.038 0.260 0.190 1.300 0.310 2.190 1.730 15.600 1.400
T9 0.566 0.068 0.117 0.048 0.070 0.000 0.000 0.116 0.187 2.340 3.800 1.460
T10 0.785 0.084 0.104 0.020 0.020 0.040 0.054 0.150 0.178 2.590 2.900 0.913
T11 0.519 0.152 0.269 0.034 0.060 0.000 0.000 0.186 0.320 2.590 2.100 8.100
T12 0.298 0.020 0.067 0.044 0.142 0.000 0.000 0.064 0.180 1.740 3.030 5.470
T13 0.836 0.082 0.099 0.044 0.050 0.040 0.047 0.166 0.196 2.180 3.170 1.570
T14 0.220 0.158 0.670 0.076 0.330 0.000 0.000 0.234 1.000 2.940 3.600 3.500
T15 0.760 0.080 0.092 0.034 0.040 0.320 0.430 0.127 0.560 1.980 7.200 3.810
T16 0.361 0.025 0.069 0.044 0.118 0.170 0.470 0.239 0.657 2.150 8.200 3.780
T17 0.259 0.064 0.240 0.034 0.013 0.013 0.146 0.111 0.390 2.430 10.500 4.180
T18 0.419 0.074 0.173 0.022 0.050 0.215 0.550 0.311 0.770 1.340 13.200 13.060
T19 0.340 0.020 0.058 0.038 0.090 0.000 0.000 0.058 0.148 0.850 4.200 4.200
T20 0.019 0.068 3.400 0.049 2.420 0.000 0.000 0.117 5.82 3.760 10.000 2.780
T21 0.988 0.120 0.119 0.014 0.014 0.000 0.000 0.133 0.130 1.320 3.600 2.650
T22 0.488 0.040 0.080 0.036 0.069 0.070 0.143 0.146 0.290 2.030 3.500 1.570
T23 0.369 0.260 0.070 0.026 0.068 0.015 0.038 0.335 0.800 2.150 10.200 4.840
T24 0.512 0.240 0.049 0.032 0.065 0.000 0.000 0.056 0.114 1.530 12.000 7.720
T25 0.432 0.082 0.180 0.074 0.160 0.019 0.036 0.175 0.370 2.430 3.500 1.440
T26 0.560 0.100 0.200 0.074 0.129 0.000 0.000 0.174 0.320 1.050 9.200 9.500
T27 0.406 0.020 0.049 0.018 0.011 0.109 0.340 0.130 0.400 1.980 8.200 4.370
* Enzyme activity (IU): µmoles of reducing sugar released/min/ml of enzyme.
**Specific activity: enzyme activity/mg of protein.
Table 4b. Screening of bacterial isolates from the hot springs of Manikaran for hypercellulase and hyperxylanase production
Isolates Protein FPase CMCase β-Glucosidase Total Total Protein Xylanase activity
(mg/ml) *Enzyme **Specific Enzyme Specific Enzyme Specific cellulase specific (mg/ml) Enzyme Specific
activity activity activity activity activity activity activity activity activity
M1 0.430 0.034 0.079 0.012 0.027 0.098 0.227 0.144 0.333 1.300 17.200 13.200
M2 0.216 0.042 0.053 0.016 0.020 1.580 0.200 0.216 0.273 0.730 3.500 4.700
M3 0.092 0.034 0.098 0.026 0.029 0.032 0.043 0.092 0.170 1.200 9.200 7.600
M4 0.550 0.104 0.189 0.016 0.021 0.011 0.021 0.131 0.229 1.140 13.200 9.000
M5 0.305 0.090 0.068 0.059 0.042 0.156 0.123 0.305 0.233 0.930 8.200 8.810
M6 0.368 0.031 0.08 0.053 0.144 0.066 0.170 0.151 0.394 0.430 6.500 15.110
M7 0.489 0.136 0.278 0.037 0.074 0.022 0.022 0.195 0.390 0.420 3.700 8.800
M8 0.488 0.112 0.229 0.005 0.010 0.013 0.026 0.130 0.265 0.390 3.000 7.690
M9 0.455 0.018 0.015 0.004 0.004 0.000 0.000 0.022 0.019 0.380 3.200 8.400
M10 0.055 0.060 1.140 0.050 0.090 0.000 0.000 0.110 2.040 1.230 21.000 17.000
M11 0.943 0.050 0.055 0.201 0.220 0.112 0.450 0.367 0.725 1.310 15.500 11.920
M12 0.678 0.038 0.051 0.000 0.000 0.007 0.011 0.045 0.062 1.290 21.000 19.300
M13 0.044 0.046 1.020 0.051 1.160 0.000 0.000 0.091 2.180 1.210 23.000 19.000
M14 1.130 0.059 0.040 0.016 0.014 0.020 0.017 0.075 0.071 0.970 15.200 15.670
M15 1.430 0.068 0.044 0.034 0.024 0.090 0.062 0.111 0.130 0.780 8.400 10.700
M16 0.690 0.024 0.033 0.016 0.029 0.000 0.000 0.040 0.062 0.910 10.000 10.980
M17 1.930 0.060 0.042 0.020 0.014 0.280 0.180 0.360 0.236 0.850 13.200 15.500
M18 1.320 0.028 0.002 0.025 0.018 0.038 0.020 0.091 0.058 0.760 11.000 14.470
N1 0.980 0.062 0.080 0.016 0.015 0.000 0.000 0.078 0.095 1.530 24.600# 19.690
N2 0.540 0.062 0.080 0.016 0.013 0.000 0.000 0.222 0.093 1.230 21.000 17.070
N3 0.780 0.046 0.055 0.009 0.080 0.000 0.000 0.055 0.135 0.980 15.100 15.400
N4 1.200 0.037 0.020 0.036 0.027 0.000 0.000 0.073 0.047 0.940 13.000 13.800
N5 1.400 0.048 0.031 0.037 0.022 0.046 0.032 0.131 0.000 0.840 14.200 16.900
N6 0.460 0.026 0.053 0.030 0.068 0.000 0.000 0.056 0.121 0.790 7.800 9.870
N7 0.720 0.071 0.098 0.017 0.020 0.124 0.120 0.212 0.238 0.420 4.300 10.230
N8 0.790 0.034 0.040 0.035 0.041 0.000 0.000 0.069 0.081 0.430 5.200 12.090
N9 0.920 0.020 0.020 0.017 0.018 0.000 0.000 0.037 0.038 0.750 11.300 15.060
* Enzyme activity (IU): µmoles of reducing sugar released/min/ml of enzyme.
**Specific activity: enzyme activity/mg of protein.
# Hyperxylanase producer
Table 4c. Screening of bacterial isolates from the hot springs of Vashisht for hypercellulase and hyperxylanase production
Isolates Protein FPase CMCase β-Glucosidase Total Total Protein Xylanase activity
(mg/ml) *Enzyme **Specific *Enzyme **Specific *Enzyme **Specific cellulase specific (mg/ml) *Enzyme **Specific
activity activity activity activity activity activity activity activity activity
V1 1.290 0.018 0.026 0.026 0.050 0.045 0.033 0.089 0.109 0.620 5.100 8.200
V2 0.790 0.040 0.035 0.102 0.160 0.000 0.000 0.142 0.195 0.970 8.000 8.240
V3 0.780 0.032 0.053 0.006 6.400 0.000 0.000 0.038 0.453 0.430 4.000 9.300
V4 1.070 0.096 0.089 0.090 0.085 0.098 0.080 0.284 0.254 1.260 13.040 10.260
V5 0.842 0.022 0.037 0.123 0.256 0.017 0.020 0.162 0.298 0.730 4.800 6.500
V6 0.820 0.052 0.710 0.012 0.032 0.000 0.000 0.064 0.103 0.740 6.500 13.200
V7 1.100 0.012 0.022 0.030 0.003 0.157 0.140 0.172 0.165 0.680 4.600 9.300
V8 0.930 0.024 0.038 0.066 0.079 0.183 0.190 0.273 0.307 0.490 2.600 5.300
V9 1.290 0.023 0.025 0.164 0.126 0.170 0.147 0.357 0.298 0.950 14.200 14.400
V10 0.920 0.020 0.02 0.034 0.03 0.190 0.20 0.244 0.25 1.280 21.100 16.480
V11 0.820 0.060 0.075 0.098 0.114 0.092 0.130 0.250 0.319 1.020 10.000 9.800

* Enzyme activity (IU): µmoles of reducing sugar released/min/ml of enzyme.

**Specific activity: enzyme activity/mg of protein.
 Due to meager production of cellulase from all thermophilic bacterial
isolates further optimization process of this enzyme was not carried out. However
titers of xylanase enzyme were enhanced significantly by subjecting it to different
favorable environmental parameters.

In similar studies in literature 125 isolates of bacteria were isolated from

northern parts of Thailand. The bacterial isolate were grown on CMC agar at 45,
50 and 550C for 25 h and were examined for their cellulase production by using
congo red test, 32 isolates showed positive results with clear zone around the
culture. Their cellulase activity was evaluated by growing in CMC broth, isolate
CM120-1 exhibited the highest enzyme activity of 22.84 U/ml and specific
activity of 0.15 IU/mg protein, the optimal condition for cellulase production were
450C, pH 7 and 96 h of incubation (Krairithichai and Thongwai, 2000).

Chang et al. (2009) isolated five strains of thermophiles from processed

Brassica waste and the hydrolytic activity on various cellulosic biomass substrate
and their temperature profiles were determined. rrs (16S rRNA) sequencing
identified these strains as Thermoactinomyctes and Bacillus strain. Maximal
cellulase activity corresponded to 2.3 IU/ml of enzyme. However Heck et al.
(2002) isolated 87 bacterial strains among which five strains were selected for
their ability to produce either cellulase or xylanase and were cultivated on
soyabean residue. Bacilllus subtilis BL62 showed the highest specific cellulase
activity, 1.08 IU/mg of protein within 24 h of growth, whereas Bacillus subtilis
BL53 showed the highest specific activity for xylanase, 5.19 IU/ mg of protein
within 72 h of cultivation.

Also a thermoalkalophilic xylanase was isolated from Enterobacter spp.

with significant activity. Maximum xylanase activity was observed in isolate
BGCC#259 (E. cloacae) i.e. 0.056 IU/ml at 24h (Sharma et al., 2009).

4.5 IDENTIFICATION OF SCREENED BACTERIA

First of all, the bacterial isolate N1 were tentatively identified on the basis
of their phenotypic and biochemical characteristics in our research laboratory as
per Aneja (2003) as Bacillus sp. their identification was further confirmed at

90
molecular level by using rrs (16S rRNA) PCR technique and these were identified
as Paenibacillus sp. N1.

Biochemical characterization is being used of identification for

microorganism since long molecular characterization has become more authentic
for identification of microorganism at the global level. Therefore selected strain
was further subjected for their identification at genomic level by using rrs (16S
rRNA) PCR technique as shown in Plate 1. Genomic DNA was isolated from N1
by using protocol of Genei which resulted in decent amount of DNA, good quality
and quantity. The isolated genomic DNA (50-100ng) was used in PCR to amplify
small units of rrs (16S rRNA) using specific primer (16S 1375 U: GCAAGT
CGAGCGGACAGATGGG AGC and 16S 1375 D: AACTCTCGTGGTGTGA
CGGGC GGT) Expected size (in 1375 bp) amplification product in the isolates
was obtained in PCR. The PCR product (1500 bp) was purified from
contaminating product by electroelution of the gel slice containing the excited
desired fragment with Qiaquick gel extraction kit. The elution was carried out in
300 µl of nuclease free water. The purified and amplified gel fraction was used for
sequencing and sequencing was performed in ABI automated sequencer using
16SF primer. The following sequences was obtained after rrs (16S rRNA).

1 tgcctaatac atgcaagtcg agcggacttg aagagaagct tgcttctctg atggttagcg

61 gcggacgggt gagtaacacg taggcaacct gccctcaagc ttgggacaac taccggaaac
121 ggtagctaat accgaatagt tgttttcttc tcctgaagag aactggaaag acggagcaat
181 ctgtcacttg gggatgggcc tgcggcgcat tagctagttg gtggggtaac ggctcaccaa
241 ggcgacgatg cgtagccgac ctgagagggt gatcggccac actgggactg agacacggcc
301 cagactccta cgggaggcag cagtagggaa tcttccgcaa tgggcgaaag cctgacggag
361 caatgccgcg tgagtgatga aggttttcgg atcgtaaagc tctgttgcca gggaagaacg
421 cttgggagag taactgctct caaggtgacg gtacctgaga agaaagcccc ggctaactac
481 gtgccagcag ccgcggtaat acgtaggggg caagcgttgt ccggaattat tgggcgtaaa
541 gcgcgcgcag gcggtcattt aagtctggtg tttaatcccg gggctcaacc ccggatcgca
601 ctggaaactg ggtgacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa
661 atgcgtagat atgtggagga acaccagtgg cgaaggcgac tctctgggct gtaactgacg
721 ctgaggcgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa
781 acgatgagtg ctaggtgtta ggggtttcga tacccttggt gccgaagtta acacattaag

91
841 cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac ggggacccgc
901 acaagcagtg gagtatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga
961 catccctctg atcggtacag agatgtacct ttccttcggg acagaggaga caggtggtgc
1021 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc
1081 ttgatcttag ttgccagcac ttcgggtggg cactctaagg tgactgccgg tgacaaaccg
1141 gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtac
1201 tacaatggcc ggtacaacgg gctgtgaagc cgcgaggtgg aacgaatcct aaaaagccgg
1261 tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag tcggaattgc tagtaatcgc
1321 ggatcagcat gccgcggtga atacgttccc gggtcttgta cacaccgccc gtcacaccac
1381 gagagtttat aacacccgaa gtcggtgggg taaccgcaag gagccagccg ccgaaggtgg
1441 gatagatgat tgggggaagt cgaacaagga gc

After using BLAST tool isolate was identified as Paenibacillus sp. (≅98%
homology) (Plate 2).

Similarly Tan et al. (2001) isolated a xylanolytic thermophilic bacterium

from Gunung Pancar hot springs, rrs (16S rRNA) sequence analysis of xylanolytic
thermophilic bacterium (IT-08) indicated that IT-08 resembled Bacillus
thermoleovorans. Similarly DNA sequencing of the three isolates from Manikaran
thermal springs and phylogenetic analysis revealed that all isolates showed 97 to
100% similarity with Paenibacillus ehemensis, Bacillus cereus and Bacillus
subtilis of H-7, H-9 and R-9 respectively as revealed by Singh et al. (2010).

4.6 SCREENING OF HYPERCELLULOLYTIC AND

HYPERXYLANOLYTIC FUNGI FROM HOT SPRINGS

As is shown in Table 4d, screening of hypercelluloytic and xylanolytic

fungi from hot springs was done and fungal isolate F1 was found the highest
producer of cellulase 0.692 IU/ml i.e. FPase (0.111 IU/ml), CMCase (0.116
IU/ml) and β-glucosidase (0.466 IU/ml) and xylanase activity of (23 IU/ml) as
compared to the other fungus F2 isolated from hot spring of Tattapani having
FPase (0.099 IU/ml), CMCase (0.097 IU/ml), β-glucosidase (0.166 IU/ml) and
xylanase activity of 15 IU/ml.

92
Plate 1. Molecular characterization of hyperxylanolytic bacterial strain N1
using rrs (16S rRNA) technique
 Paenibacillus sp. N1

Plate 2. Hyperxylanolytic bacteria isolated from hot springs

Thermomyces sp. F1

Plate 3. Hypercellulolytic and Hyperxylanolytic fungus isolated from

hot springs
Table 4d. Screening of fungal isolates from the hot springs of Himachal Pradesh for hypercellulase and hyperxylanase production

Isolates Protein FPase CMCase β -glucosidase Total Total Protein Xylanase activity
(mg/ml) *Enzyme **Specific *Enzyme **Specific *Enzyme **Specific cellulase specific (mg/ml) *Enzyme **Specific
activity activity activity activity activity activity activity activity activity
F1 1.200 0.111 0.092 0.116 0.096 0.466 0.38 0.692# 0.568 1.33 23.0## 17.29
F2 0.816 0.099 0.121 0.097 0.118 0.166 0.203 0.362 0.442 1.24 15 12.09

* Enzyme activity (IU): µmoles of reducing sugar released/min/ml of enzyme.

**Specific activity: enzyme activity/mg of protein.

# Hypercellulase producer
## Hyperxylanase producer
 Due to meager production of cellulase from all thermophilic bacterial
isolates further optimization process of this enzyme from bacteria was not carried
out. However titers of xylanase enzyme were enhanced significantly by subjecting
it to different favorable environmental parameters.

Twenty strains of thermophilic fungi were screened for their production of

xylanolytic and cellulolytic enzymes in liquid medium containing birchwood
xylan as substrates (Mendoza et al., 2006). Among them a thermophilic fungus
was identified as Thermomyces lanuginosus had optimum temperature and pH for
growth at 550C. Maximum enzyme activities of 5,846 and 840 IU/ml were
obtained in liquid Mandel’s and Reese medium with 3% corn cob at pH 6.0 after
7 days.

Boonlue et al. (2003) had found Scytalidium thermophilum AF101-3 strain

as the best isolate produced highest xylanase and corncob decomposing activity
than other strains isolated from the Japanese’s soil at temperature of 500C

4.7 IDENTIFICATION OF SCREENED FUNGI

F1 strain based upon its short and septate mycelium and brownish color
was identified as Thermomyces sp.F1 and based upon higher enzyme units, it was
selected for the further studies (Plate 3).

4.8 PRODUCTION AND OPTIMIZATION OF XYLANASE FROM

HYPERXYLANOLYTIC BACTERIAL STRAIN Paenibacillus sp. N1
UNDER SUBMERGED FERMENTATION.

Standardization of various cultural conditions i.e. medium, substrates,

incubation time, inoculum size, pH, temperature, amino acid, nitrogen source and
additives was done as these were essential parameters to maximize the
extracellular enzyme production.

4.8.1 Effect of different media

The effect of different media on xylanase production has been enlisted in

Table 5 and fig 4 and it was found that the highest xylanase activity was in Basal
salt medium (BSM) i.e. 27.200 IU/ml while the least enzyme production 12.011

94
IU/ml was observed in Nakamura medium followed by TGY (26.900 IU/ml) and
Xylan medium (26.221 IU/ml). Statistically xylanase produced in BSM was
significantly higher than others. 10.569 % increase in xylanase activity depict in
Basal salt medium in comparison to control.

Nutrient concentration in the medium had influenced production of

xylanase by the bacterial isolate. The culture showed the maximum growth in the
Basal medium containing ammonium sulphate (0.6%), ammonium chloride
(0.1%), sodium chloride (0.05%), yeast extract (0.5%), potassium hydrogen
phosphate (0.3%), magnesium chloride and calcium chloride as the growth
supplements in the medium and seemed to have promoted extracellular xylanase
production. Sodium chloride present in the medium probably helped in
maintaining the osmotic balance of the medium while magnesium sulfate was a
cofactor for a variety of metabolic reactions. Basal medium containing (NH4)2SO4
and NH4Cl as its major ingredients and both being rich source of nitrogen (Briggs
et al., 2005) had caused higher xylanase production from bacteria. Xylanase
secretion and its extracellular performance are known to depend directly on the
type of ions present in solution. In the present study, Reese medium contained a
high concentration of the metal ions in the medium, as a result enzyme production
was low, which could be due to the blockage of the secretion of protein into
external medium (Kaupinen et al., 1997).

Basal medium also contained yeast extract which was the most important
source of nitrogen in the medium in terms of the accessibility and composition to
get the higher yield of the xylanase (Sharma and Bajaj, 2005). Higher xylanase
production using defined medium indicated that the presence of the simple sugars
and compounds could be utilized easily by the bacterium and enhanced the cell
ability to produce the xylanase enzyme (Basar et al., 2010).

Dhillion et al. (2000) isolated a Bacillus circulans AB16 from garbage

dump, the highest xylanase activity was in basal medium of 55 IU/ml. B. pumilus
showed a 3.4-fold increase in the xylanase production was achieved using the
optimized culture medium consisting of (g/l): K2HPO4, MgSO4.7H2O,

95
Table 5. Effect of media on xylanase production from Paenibacillus sp. N1

Protein conc. Xylanase activity % increase/

Media (mg/ml) (IU) decrease in
xylanase activity
Basal’s Medium 1.100 *27.200 + 10.569
**(24.720)
Nakamura Medium 0.571 12.011 - 50.979
(21.000)
Emerson Medium 0.630 15.511 - 36.731
(24.600)
TGY medium 0.941 26.900 + 9.795
(28.621)
Xylan Medium 0.910 26.221 + 6.900
(28.700)
Reese Medium 0.902 24.600 -
(27.222)
CD 0.05 0.177 0.153
S.E. (difference of mean) 0.081 0.069

* IU: µmoles of reducing sugar released / min / ml of enzyme.

** Value in parentheses depict specific activity i.e. enzyme activity/ mg of protein.
+ indicates an increase in xylanase activity over the control
- indicates an decrease in xylanase activity over the control

Table 6. Effect of incubation time on xylanase production from

Paenibacillus sp. N1

Protein conc. Xylanase activity % increase or

Incubation Time (Days) (mg/ml) (IU) decrease in
xylanase activity
1 0.960 *25.521 -6.250
**(26.516)
2 1.211 27.441 + 4.411
(23.470)
3 1.340 29.461 + 8.312
(21.980)
4 1.222 28.453 + 4.595
(23.320)
5 1.100 27.200 -
(24.720)
6 0.390 15.312 - 43.750
(39.230)
CD 0.05 0.167 0.177
S.E. ( difference of mean) 0.079 0.081
*
**
Same as in Table 5
+
-

CaCl2.2H2O, NaCl, peptone, yeast extract and wheat bran. Similarly a

thermoalkalophilic Arthrobacter sp. MTCC 5214 produced optimal extracellular

96
xylanase in medium supplemented with modified basal salt medium of pH 9
supplemented with 0.5% birch wood was isolated locally from a sediment sample
collected from sediment sample collected from the Mandovi estuary, west coast of
India (Khandeparker and Bhosle, 2006).

4.8.2 Effect of incubation time

Effect of incubation time on xylanase production has been evaluated in

Table 6 and fig 5a. Enzyme activity was measured at regular intervals up to
period of 6 days viz. 1, 2, 3 ,4, 5, 6 . Highest xylanase activity was noticed on 3rd
day (29.461 IU/ml) from Paenibacillus sp. N1 followed by a gradual decline with
increase in incubation time. Least enzyme production was noticed on 6 day
(15.312 IU/ml) of fermentation. Statistically enzyme produced on 3 day was
found significantly higher than others. An 8.312 % increase in xylanase activity
has been noticed on 3 day of fermentation in comparison to control.

When growth of Paenibacillus sp. N1 was studied, it showed exponential

phase extending from 12 to 48 h and then entering into stationary phase at 72h.
Enzyme production (fig 5b) when compared with growth profile, peak in yield
was noted on 3rd day i.e. in stationary phase. Similar reports were shown in
literature where highest yield of enzyme was found in exponential/ stationary
phase. A decline in enzyme activity afterwards can be because of the optimum
period was probably due to proteolysis or due to depletion of nutrient available to
microorganisms, causing a stressed microbial physiology resulting in inactivation
of enzyme (Flores et al., 1997).

Maximum production of xylanase was observed in culture incubated at

500C, pH 7 for 12 h with cell density of 1.78 × 109 by Bacillus sp. AQ-1 under
submerged fermentation (Wahyuntri et al., 2009). Similarly maximum xylanase
production 125 IU/ml was recorded in the stationary phase (36h) of culture by
Bacilllus subtilis isolated from estuarine environment by optimization of culture
conditions (Annamalai et al., 2009).

97
 Maximum titers of xylanase (2600 IU/l) was obtained by Streptomyces
sp.-SU9 in the medium containing oat spelt as a substrate with pH of 9 in shake
flask condition after 96 h of fermentation (Bajaj et al., 2010). Optimum
production of both enzymes was achieved after 5 days incubation on a rotary
shaker (200 rpm) at 35 °C and initial pH 7.0 by Streptomyces galbus NR (Amany
et al., 2004).

4.8.3 Effect of pH

Table 7 and fig 6 depict the effect of pH on the medium for xylanase
production by Paenibacillus sp. N1. Xylanase production was estimated at 4.0,
5.0, 6.0, 7.0, 8.0, 9.0, 10.0 pH for hyperxylanolytic bacteria. Xylanase production
from Paenibacillus sp. N1 strain ranged between 24.000 to 31.860 IU/ml at
different levels of pH which were significantly at par to each other. The results in
present study revealed an alkalophilic nature of the bacterial isolate which was an
accessible attribute for further use. An increase of 8.319% in xylanase activity
was noticed in comparison to control.

Since the pH of the medium influences the growth of microorganisms as

well as enzyme production. If cultivation of the organisms is carried at an
unfavorable pH, it may limit the growth and xylanase production by substrate in
accessibility. The use of alkaline xylanase has special advantage in the industry as
it allows direct enzymatic treatment of the alkaline pulp and avoids the cost of
incurring and time consuming steps of pH re-adjustment thus alkaline xylanase
that are stable at high temperature are more beneficial because of saving in
cooling cost and time, alkaline active xylanase may also find a potential
application in addition to pulp bleaching (Srinivasan et al., 1999 & Jain 1995).
Some industries such as laundry detergents, leather and paper require alkaline and
thermostable enzymes (George et al., 2001). Industrially desirable characteristics
like thermostability and alkalophilic nature make them a potentially commercial
important enzyme in industries. The pH of the medium strongly affects many
enzymatic processes and transport of various components across the cell
membrane (Moon and Parulekar, 1991).

98
 As reported, many alkalophilus had already been explored for enzyme
production because of their special significance in the industry. Anuradha et al.
(2007) reported three xylanolytic bacteria isolated from the sugarcane field and
identified as the Bacillus sp. and had optimal pH 9.0 for strain Sta and strain Stb
and 8.0 for strain Stc. These properties qualify the enzyme to be novel and thus
exhibits favorable potential for application to bleaching in paper and pulp
industries and other relevant.

Nakamura et al. (1995) isolated Thermoalkalophilic Bacillus sp. Strain

TAR-1 from soil producing an extracellular xylanase. The enzyme was most
active over the range of pH 5.0 to 10.0 at 500C, the optimum temperature for
activity were 750C at pH 7.0 and pH 9.0. Xylanase R was stable up to 650C at pH
9.0 for 30 min in the presence of xylan. Cultural condition for higher xylanase
production by an alkalophilic Bacillus pumilus strain MK001 were optimized
under submerged fermentation by Kapoor et al. (2008), maximum yield of
xylanase was obtained at pH 9 i.e. 610.0±40.0.

4.8.4 Effect of temperature

Table 8 and fig 7 showed the data on effect of temperature on xylanase

production from hyperxylanolytic bacteria. The bacterial isolates had shown the
capability to grow as well as produce xylanase at temperature ranging from 30 to
500C. Maximum xylanase titres have been produced at 500C from Paenibacillus
sp. N1 strain (31.860 IU/ml) which were significantly higher than others
statistically. Minimum xylanase production for Paenibacillus sp. N1 was observed
at 300C (6.320 IU/ml). Optimum temperature (500C) range obtained for potential
enzyme production in the present study has reflected an inclination towards being
thermophilic in nature and thus could become an asset for commercial use in
industries. Since Paenibacillus sp. N1 was isolated from hot water springs with a
possibility to isolate thermophiles having hyperenzyme producing potential thus
fulfilling one of objective of present study. This temperature is also referred by
other authors as being the best temperature for many of the microbial xylanase
processes.

99
 Thermophiles can tolerate higher temperature by using increased
interaction than non - thermotolerant organisms, because of the presence of

Table 7. Effect of pH on xylanase production from Paenibacillus sp. N1

pH Protein Xylanase activity % increase or

conc.(mg/ml) (IU) decrease in
xylanase activity
4 0.981 *20.312 -31.093
**(24.750)
5 1.200 24.00 (20.081) -18.532
6 1.311 25.502 - 13.441
(19.771)
7 1.340 29.461 -
(21.980)
8 1.480 30.311 + 2.881
(14.440)
9 1.670 31.860 + 8.139
(19.390)
10 0.970 24.501 - 16.830
(25.56)
CD 0.05 0.162 NS -
S.E.(difference of mean) 0.078 3.171 -
*
**
Same as in Table 5
+
-

Table 8. Effect of temperature on xylanase production from Paenibacillus

sp. N1

Temperature(0C) Protein Xylanase activity % increase or

conc.(mg/ml) (IU) decrease in
xylanase activity
30 0.560 *6.320 -80.225
**(11.251)
35 0.640 9.331 - 70.804
(14.530)
40 0.931 15.302 - 119.34
(16.452)
45 1.119 24.711 - 22.681
(22.071)
50 1.670 31.860 -
(19.390)
55 1.257 25.000 - 21.777
(19.881)
CD0.05 0.171 1.030 -
S.E. ( difference of mean) 0.078 0.476 -
*
**
Same as in Table 5
+
-

100
hydrophobic, electrostatic and disulphide interaction (Kumar and Nussinov,
2001). Temperature variation was a special feature because it can penetrate
physical barrier and can have dramatic effects on the structure of macromolecules
and also affects all levels of biological adaptation (Hickey and Singer, 2004).

Thermostable microorganisms are the potential sources of the

thermostable enzymes. An increase in Gibbs free energy change of hydration
(Gromiha et al., 1999) and increase in number of salt bridges, side chain-side
chain interactions (Kumar et al., 2000), aromatic clustera (Saelensminde et al.,
2009), contact between the residues of hydrogen bonds (Saraboji et al., 2005), ion
pairs (Maugini et al., 2009), electrostatic interaction of charged residues (Yao et
al., 2002), amino acid coupling patterns, hydrophobic free energy and
hydrophobic residues in thermophilic protein have shown enhance protein
stability (Saraboji et al., 2005). Thermal stability of xylanase is an important
property due to its potential application in several industrial processes, use of such
enzymes has been expected to greatly reduce the need for pH and temperature
adjustments before the enzyme addition.

Sharma and Bajaj (2005) has reported the maximum xylanase production
by an alkalophilic isolate Streptomyces sp. CD3 with optimum enzyme activity at
pH 8 and temperature 500C. Organism efficiently uses the wheat bran and
bagasses as a substrate under submerged fermentation and produced 2.211 and
1.896 IU/ml of xylanase. Sa-Pereira et al. (2002) showed the synergestic effect of
Bacillus subtilis using a culture medium with oat spelts xylan as a xylanase
inducer. At 500C xylanase productivity obtained after 11h of shake flasks, 96,000
U/ l/ h and in reactor 104,000 U/ l/ h. Optimal xylanase production of about 12
U/ml was achieved at pH 6.0 and 500C within 18 h fermentation.

Optimum temperature of the isolated cellulosome type enzyme from

thermophilic Bacteroide sp. strain P-1 was performed by Ponpium et al. (2000)
for cellulase and xylanase at pH 7.0, enzyme has optimum temperature of 600C
for cellulases and 500C for xylanase activity.

101
4.8.5 Effect of inoculum size

Effect of inoculum size on xylanase production is evaluate in Table 9 and

fig 8. Different inoculum size used were 2.5%, 5%, 7.5%, 10%, 12.5 and 15%
(v/v). Optimum inoculum size was found to be 12.5% for xylanase production
(35.850 IU/ml) from Paenibacillus sp. N1 while the least xylanase production was
observed at 2.5% i.e. 11.361 IU/ml. An inoculums size of 12.5% showed an
11.126% increase in xylanase activity over the control.

In the present experiment, inoculum size ranging from 2.5% to 7.5%

probably was very low in turn resulting in lower number of cells in production
medium thus causing lesser production of enzyme. A further increase in the
amount of inoculum i.e. 10 - 12.5% had improved titers of enzyme reaching to
maximum @12.5% showing an equilibrium between the number of cells and
substrate concentration at this level thus enhanced xylanase production beyond
that (15%) enzyme activity had dipped low because of overload in number of cells
causing nutrient starvation. It seems that an inoculum size of 2.5% to 10% in
BSM medium did not cause an overload of cell facing nutrient limitation whereas
inoculum size of 15% was so high that the nutrients were consumed faster and
overall resulting in less cell growth and leading to lower enzyme yield whereas at
inoculum size of 12.5% (v/v), a balance between the proliferating biomass and
available nutrient was seen. Enzyme activity was maximum at optimal level
because at this point equilibrium was maintained between the inoculum size and
availability of the substrate while decline in enzyme yield at the larger inoculum
size might be due to the formation of the thick suspension and improper mixing of
the substrates in shake flask (Omsojasola et al., 2008)

As is also shown in literature that an increase in the amount of inoculum

improves the growth of the microorganisms up to a certain point resulting in
higher enzyme units however beyond this point, there can be a reduction in the
microbial activity due to nutrient limitations whereas a lower amount of
inoculation causes lower number of cells in the production medium thus
producing lesser enzyme (Nagar et al., 2010).

102
 Kulkarni and Rao (1996) reported that 10% inoculums size was optimum
for xylanase production by Bacillus sp. NCIM 59. Sindhu et al. (2006) reported
that 10% inoculums size was found to be optimum for production of xylanase by
B. megaterium.
Similarly Kapoor et al. (2008) the maximum xylanase production from an
alkalophilic Bacillus pumilus strain MK001 at an inoculums size of 1.25% (v/v)
under submerged fermentation.

4.8.6 Effect of amino acids

Table 10 and fig 9 reveal data on effect of amino acid on xylanase
production by isolate Paenibacillus sp. N1 was studied by adding different amino
acids i.e phenlyalanine, alanine, cysteine, tryptophan, arginine and glutamic acid
at concentration of 10 µg/ml in to Basal salt medium. Maximum xylanase titers
i.e. 40.601 IU/ml were obtained, when tryptophan was added as nitrogen source in
the medium while minimum enzyme activity (13.00 IU/ml) was recorded with
alanine. Other amino acids have lead to lesser production of xylanase ranging
from 30.611 IU/ml with arginine, 23.111 IU/ml with phenylalanine, 25.501 IU/ml
with cystiene, 20.612 IU/ml with glutamic acid respectively. Statistically xylanase
produced from tryptophan was significantly higher than others. When tryptophan
was added into the medium an 13.22% increase in xylanase activity was observed
over the control when there was no addition of tryptophan.

As is well known that amino acids are fundamental structural units of

proteins. Enzymes seem to be modulated by interaction of cations with amino acid
residue involved into the active site. Amino acid enhanced the growth rate as well
as improve protein synthesis they may directly or indirectly involved absorbed by
the cell. However specificity of amino acids affects the overall rate of enzyme
production. In case of isolate Paenibacillus sp. N1 tryptophan has been found to
be a most prominent amino acid to increase xylanase production. The
enhancement in the xylanase yield in the presence of tryptophan could be due to
the presence of indole functional group and the oxidation dissimilation of
tryptophan is catalysed by special sequence of enzymes, the physiological role of
which is to convert this heterocyclic substrate to aliphatic fragments that can enter
the central pathways of cellular intermediatory metabolism.

103
Table 9. Effect of inoculum size on xylanase production from Paenibacillus
sp. N1

Inoculum size (%) Protein Xylanase activity % increase or

conc.(mg/ml) (IU) decrease in
enzyme activity
2.5 0.810 *11.361 -68.268
**(14.062)
5 0.821 14.000 -55.555
(15.000)
7.5 0.890 14.162 -68.691
(15.911)
10 1.670 31.860 -
(19.390)
12.5 2.241 35.850 +11.126
(15.981)
15 0.820 12.162 -61.396
(14.821)
CD 0.05 0.177 1.037 -
S.E.(difference of mean) 0.081 0.476 -

*
**
Same as in Table 5
+
-

Table 10. Effect of different amino acids on xylanase production from

Paenibacillus sp. N1

Amino acid Protein Xylanase activity % increase or

conc.(mg/ml) (IU) decrease in
xylanase activity
Phenylalanine 1.000 *23.111 - 42.250
**(23.160)
Alanine 0.830 13.000 - 67.501
(12.042)
Cysteine 1.201 25.501 - 36.251
(21.251)
Tryptophan 2.460 40.601 +13.222
(16.500)
Arganine 1.461 30.611 - 23.511
(20.951)
Glutamic acid 0.930 20.612 - 48.512
(22.151)
C.D.0.05 0.177 1.460 -
S.E.( difference of mean) 0.086 0.666 -

*
**
Same as in Table 5
+
-

104
 The role of amino acid in promoting the enzyme titers is well cited in
literature varying from organism versus aminoacid,β-phenylalanine had enhanced
xylanase yield in case of Bacillus pumilus strain after 40 h of incubation at 370C
closely followed by leucine and tryptophan (Kapoor et al., 2008). Other
aminoacid used in the study visually alanine, methionine, threionine, cystiene
have exerted a strong impression in xylanase synthesis thus promoting that need
of aminoacid to enhanced the xylanase syntheisis in organism depended.

4.8.7 Effect of carbon sources

Table 11 and fig 10 show the data on effect of different substrate i.e.
arabinose, mannose, sucrose, xylose, dextrose, lactose, rabinose, fructose for
hyperxylanase producing bacteria Paenibacillus sp. N1. Optimal level of xylanase
production was observed when xylose was used as a carbon source for the enzyme
production (40.601 IU/ml) which was significantly higher than others. Lowest
enzyme production was observed in the medium containing 1% treated rice straw
(4.000 IU/ml) as a source of carbon while rest of carbon sources (arabinose,
mannose, sucrose, dextrose, lactose, rabinose, fructose) showed xylanase activity
between 38.702 to 6.411 IU/ml.

The diverse pattern found in enzyme production along with different

carbon sources used in the present experiment is directly linked to the specific
carbon used in the present study. This clearly indicates that one of the major
factors that affects the enzyme production is carbon source used in the production
medium (Seyis and Aksoz, 2005). Some of the carbon source used in the medium
supports the good growth as well as good enzyme synthesis, while other supports
the good growth as well with reduced enzyme synthesis (Satyanarayanan, 2007).
Carbon sources are also essential elements for microorganism during the period of
growth and metabolism (Nagar et al., 2010).

Sugars serve as nutrient for microbial growth. Xylan containing residue

may serve as the inducer for promoting the xylanase production (Beg et al.,
2000). It appears that low molecular mass fragments of xylan play a key role in
the regulation of xylanase biosynthesis that include xylose, xylobiose,

105
xylooligosaccharides, heterodisaccharides of xylose and glucose and their
positional isomers. Xylose induced the production of the enzyme as compared to
other sugars.

Table 11. Effect of different carbon sources on xylanase production from

Paenibacillus sp. N1

Carbon source Protein conc. Xylanase %increase or

(mg/ml) activity decrease in
(IU) enzyme activity
Arabinose 1.621 *38.702 - 32.522
**(23.800)
Mannose 1.231 25.301 - 36.750
(20.811)
Sucrose 0.310 5.111 - 87.251
(19.222)
Dextrose 0.430 8.600 -125.622
(18.601)
Xylose 2.460 40.601 -
(16.500)
Lactose 0.980 25.302 -36.751
(25.811)
Rabinose 0.711 14.509 - 63.756
(20.050)
Fructose 0.233 6.411 - 8.423
(27.800)
1% Rice straw 0.180 4.000 -90.000
(19.161)
C.D 0.05 0.171 0.820 -
S.E.(difference of 0.081 0.392 -
mean)
*
**
Same as in Table 5
+
-

Kapoor et al. (2008) reported the maximum production of xylanase by

Bacillus pumilus strain MK001 when xylose was added as the carbon sources, at
370C under the shaking condition 200 rpm, pH 9 showed the enzyme production
of 1190 IU/ml on birch wood xylan and 1150 IU/ml on oat spelt xylan was
observed. Similarly a xylanase producing alkalophilic Bacillus NT-9 obtained on
the selective medium were studied by Xiao-Fang et al. (2004). The medium
composed of xylose 1. 5%, (NH4)2 SO4 0.25%, K2HPO4 0.1%, MgSO4 · 7H2O
0.02%, with the initial pH of 10, was suggested to be optimal for the enzyme

106
production. When cultivated at 37°C for 72 h, the enzyme activity elaborated by
the strain may reach as high as 10.5 U/ml.

Effects of xylose on xylanase production by a thermophilic Bacillus sp.

showed diverse patterns on corn cob (CC) and wheat bran (WB) under SmF and
SSF by Gupta and Kar (2008). In SmF, enzyme synthesis was stimulated by
xylose on supplementation with both MSS and YEP by 41.38% and 27.47%.
Under SSF xylose stimulated xylanase synthesis by 44.01%, on wheat bran
supplemented with MSS, it decreased the enzyme activity by 12.89% with YEP
supplementation.

4.8.8 Effect of nitrogen sources

Table 12 and fig 11 depict the effect of nitrogen on xylanase production

by supplementing the medium with different organic and inorganic nitrogen
sources like yeast extract, beef extract, peptone, casein, tryptone, ammonium
nitrate, sodium nitrate, sodium nitrite, ammonium carbonate and di-ammonium
hydrogen phosphate at a concentration of 0.5% in Basal salt medium. Among all,
maximum enzyme activity was obtained in the medium supplemented with the di-
ammonium hydrogen phosphate (48.000 IU/ml) while minimum enzyme
production (20.612 IU/ml) recorded when casein was added as a nitrogen source
in to the medium. In comparison to control, 15.870 % increase in xylanase
activity was observed when (NH4)2HPO4 was added as a source of nitrogen into
the medium.

Effect of different organic and inorganic nitrogen source have shown a

variable effect on xylanase production from Paenibacillus sp. N1. Among all
nitrogen tested in the present study (NH4)2HPO4 closely followed by beef extract
have shown a notable higher production of xylanase. Inorganic nitrogen i.e.
(NH4)2HPO4 consists of ammonium ions and phosphoric acid ammonium salt has
enhanced the growth rate as well as improved the protein expression by mediating
ammonium assimilating enzymes (Wang et al., 2009). Phosphoric acids probably
containing inorganic phosphate are important constituents of cellular
biomolecules such as cAMP, nucleic acid and coenzyme which play a regulatory

107
role in the synthesis of both primary and secondary metabolites (Nagar et al.,
2010).

Table 12. Effect of different nitrogen sources on xylanase production from

Paenibacillus sp. N1

Nitrogen source Protein conc. Xylanase %

(mg/ml) activity increase/decrease
(IU) in xylanase
activity
Beef extract 2.420 *46.900 +17.074
**(19.380)
Peptone 1.230 29.900 -25.361
(24.301)
Yeast extract 2.460 40.601 -
(16.500)
Casein 0.872 20.612 -48.547
(23.051)
Tryptone 2.372 45.411 + 13.357
(19.162)
NH4NO3 0.932 21.900 - 45.332
(23.492)
NaNO3 2.340 44.810 + 11.587
(19.144)
NaNO2 2.350 45.000 + 12.331
(19.141)
NH4CO3 1.340 31.411 - 21.590
(26.082)
(NH4)2H2PO4 1.320 21.601 - 46.078
(16.364)
(NH4)2HPO4 2.760 48.000 + 15.870
(17.391)
C.D 0.05 0.151 1.081 -
S.E.( difference of mean) 0.076 0.522 -

*
**
Same as in Table 5
+
-

The optimization of NH4 ions have been shown to stimulate the growth as
well as at the same time increase the enzyme yield because of protein inhibiting
nature at low temperature. Among organic sources had stimulated the xylanase
production. Since it is a balance source of protein in terms of composition and
accessibility. It could also be possible that organic nitrogen provided by beef
extract contains that amino acid required by Paenibacillus sp. N1 strain and these
amino acid are absorbed directly by these isolates.

108
 Cultural variables resulted in a marked enhancement in the secretion of
cellulase and alkali thermostable xylanase by extreme thermophiles Geobacillus
thermoleovarans. When tryptone was added into the medium, peak in enzyme
production was attained within 42 h in a fermenter as compared to 72 h in shake
flask. Optimization of the cultural condition was result in the 7.72 fold increase in
the enzyme activity (Sharma et al., 2007). Thus it is clear that ability of cell to
utilize a particular compound is complex affair, depending upon genetic ability of
the organism.

A xylanase producer Bacillus mojavensis strain, called AG137, isolated

from cotton farm (Kashan-Iran) produced 249.3308 IU/ml of xylanase at pH 8
and temperature 37oC, within 48 h in submerged fermentation in enzyme
production medium supplemented with a mixture of 1% (w/v) yeast extract and
1% (w/v) tryptone used as an optimum nitrogen sources and led to an increase in
xylanase yield from 194.68 IU/ml under non-optimized fermentation condition to
302.466 IU/ml in optimized (Sepathy et al., 2011).

A thermophilic Bacillus circulans AB 16 from the garbage dump, capable

of producing a novel extracellular thermo stable xylanase in basal medium
containing rice straw under shake condition, an enhanced xylanase yield was
observed when tryptone was added i.e. 21.6 to 47 IU/ml (Dhillon et al., 2000).

4.8.9 Effect of additives

The effect of different additive on xylanase production evaluate in Table

13 and fig 12. Different surfactants used were Tween 20, Tween 80, SDS, PEG
2000. Maximum xylanase activity i.e. 52.300 IU/ml was found in Tween 20
which was statistically found significantly higher than others. Minimum
production of xylanase i.e. 7.500 IU/ml when SDS was used as an additive. An
8.958% increases in enzyme activity was observed when Tween 20 was added
into the medium in comparison to control which do not contains Tween 20 as a
additive into the medium.

109
Table 13. Effect of additives on xylanase production from Paenibacillus sp.
N1

Protein Xylanase %
conc. activity increase/
Additive (mg/ml) (IU) decrease in
xylanase
activity
Polyethylene Glycol 2000 (PEG 2.590 *46.440 - 3.250
2000) **(17.930)
Tween 20 2.984 52.300 +8.958
(17.550)
Tween 80 2.321 43.291 - 9.812
(18.650)
Sodiun Dodecyl Sulphate (SDS) 0.780 7.500 - 84.500
(10.000)
C.D.0.05 0.108 0.188 -
S.E ( difference of mean) 0.086 0.081 -

*
**
Same as in Table 5
+
-

Addition of Tween 20 has increases the xylanase yield which might be

caused due to multiple reasons such as its favorable effect on cellmembrane
permeability (Uma Maheshwar Rao and Satyanarayana, 2003), addition of this
surfactant positively affected the enzyme substrate interaction leading to a more
effective conservation of substrates (Erikson et al., 2002). Tween 20 a surfactant
disrupts non specific binding of enzyme to substrate and thus exerts a positive
effect on the desorption and recycling of xylanase. It is also shown to increase
enzyme stability and prevention of enzyme denaturation leading to 8.911%
increase over control i.e. without Tween 20.

A considerable decrease in xylanase activity (7.5 IU/ml) upon adding ionic

surfactant SDS has almost completely inhibited enzyme production which might
be due to its denaturation by conformational changes in tertiary and secondary
structure of protein or by binding of surfactant to the active site of enzyme or
change in nature of substrate by decreasing the availability by reaction sites
(Kuhad et al., 1998).

110
 Addition of Tween 80 that can increase the xylanase production 1.5 fold
by Bacillus subtilis using cheap agro-residue i.e. wheat bran as a substrate and
enhanced the enzyme production to 2995.20 ± 200.00IU/ml. Optimum
temperature for the enzyme production was 500C. Enzyme was 100% stable over
the pH ranges from 5 to 11 (Nagar et al., 2010).

Enzyme secretion by thermophilic Geobacillus thermoleovorans was

enhanced when the basal salt medium was supplemented with xylan (0.015%) and
Tween-80 (0.1% v/v). Optimization of the culture condition resulted in a 7.72 fold
enhancement in enzyme production. The cellulase free xylanase was optimally
active at pH 8.5 and 800C, and useful in the bioleaching of paper pulps (Sharma et
al., 2007).

The significant increase in extracellular xylanase activity from

Paenibacillus sp. N1 is a main highlight of submerged fermentation, thus fulfilling
one of the main objectives of our present study. The stepwise increase in xylanase
titers starting from 24.60 IU/ml to 52.300 IU/ml and escalating yield of the
xylanase upto 113.38% (fig 13), which is an outcome of optimizing the various
process parameters i.e. nutrients, incubation time, pH, temperature, incubation
time, etc. The optimization of these fermentation factors particularly physical and
chemical parameters are of primary importance in the development of any
fermentation process owing to their impact on the yield practicability and
economy of the process (Wenster-Botz, 2000). The optimal conditions derived for
Paenibacillus sp. N1 for xylanase production designate this isolate as an
extremophile which has been found hyperxylanolytic as well as thermophilic and
alkalophilic strain This combination of these rare attributes along with the
standardized parameters from Table 5 to 13, designate it as a highly cherishable
strain and thus can be recommended for commercial application at industrial
scale.

111
4.9 PRODUCTION AND OPTIMIZATION OF CELLULASE FROM
HYPERCELLULOLYTIC FUNGUS Thermomyces sp. F1 UNDER
SOLID STATE FERMENTATION

Result in Table 14 and fig 14 revealed the production of extracellular

cellulase by Thermomyces sp. F1 during biodegradation of untreated and
pretreated rice straw (Oryza sativa) using Vogel’s medium, pH 5.5 with inoculum
size of 10% for 8 days. In microwave treated biomass, higher cellulase enzyme
activity of 34.430 U/gds i.e. FPase (11.311 U/gds), CMCase (9.630 U/gds) and β-
glucosidase (13.500 U/gds) was obtained, whereas enzyme activity in untreated
biomass was 16.750 U/gds i.e. FPase (6.211 U/gds), CMCase (4.511 U/gds) and
β-glucosidase (6.060 U/gds). It was also observed that enzyme activity was higher
in treated biomass as compared to untreated biomass (Plate 4).

Table 14. Cellulase production from Thermomyces sp. F1 using untreated

and microwave treated rice straw (Oryza sativa) under SSF

Untreated Treated biomass t cal

biomass
Protein (mg/g) 14.000 17.222 + 2.415
FPase *6.211 11.311 + 3.825
**(0.443) (0.656)
CMCase 4.511 9.630 + 3.839
(0.900) (0.559)
β-glucosidase 6.060 13.500 + 5.58
(0.432) (0.738)
Total cellulase (U/gds) 16.750 34.430 + 13.26
(1.196) (1.999)

* U/gds: µmoles of reducing sugar released / min / g of enzyme.

** Value in parentheses depict specific activity i.e. enzyme activity/ g of protein.
+ indicates an increase in cellulase activity over the control.
- indicates a decrease in cellulase activity over the control.

SSF systems, which are closer to natural habitat of microbes, prove more
efficient in producing certain enzymes and metabolites (Babu and Satyanarayan
1995). The choice of appropriate substrates is of great importance for the
successful production of cellulase. The substrate not only serves as the source of
carbon but also produce the necessary inducing compounds for the organism
(Haltrich et al., 1996).

112
 In the present study, rice straw being rich source of cellulose (equals to
60%) was selected as a carbon substrate for cellulase production. Rice straw an
important agricultural waste generated in bulk, inexpensive in nature and
abundantly available, inturn could lead to cost effective production of otherwise a
costly enzyme. Lesser production of enzyme, is due to the reason that native
lignocellulosic biomass i.e. cellulose present in it is generally in bound form i.e.
shielded by lignin and hemicelluloses around it. Moreover complex and
crystalline form of cellulose render it as an inaccessible form of substrate for
enzyme production (Park et al., 2002). It has been explored that pretreatment of
the lignocellulosic biomass increases the accessibility of cellulose and
hemicelluloses to the action of hydrolytic enzymes substantially producing more
titers of enzymes. Pretreatment alter or remove structural and compositional
impediments to hydrolysis and subsequent degradation processes in order to
enhance digestibility, improve the rate of enzyme hydrolysis and increase yields
of intended products (Moiser et al., 2005). Therefore suitable pretreatment given
to the rice straw became a prerequisite for increasing the availability of substrate
i.e. cellulose for enhanced production of cellulase. Microwave irradiated
pretreatment was chosen in solid state fermentation study because of the reasons
viz highly effective, physical method and pollution free approach as no discharge/
toxic byproducts are generated.

Similar studies pertaining to solid state fermentation for cellulase

production using lignocellulosic biomass has been enlisted by many authors.
microwave irradiation of lignocellulosic biomass is the latest pretreatment method
recently has been applied for production of enzymes and fuels. Microwave
technology is supposed to simply three structural polymers along with extraneous
components. When the crystalline region is placed between electromagnetic field
it acts polarized generating a charge on crystalline interphase.

Kumarkura and Kaetsu (1978) studied the effect of irradiation for

pretreatment of bagasse prior to its enzymatic hydrolysis. The pretreated bagasse
resulted in double yield of glucose by the hydrolysis compared to untreated one.
Akhtar et al. (2001) produced cellulases by Bacillus subtilis used for

113
saccharification of wheat straw, rice straw and bagasse. Wheat straw, rice straw
and bagasse were separately pretreated with (1-4% w/v) NaOH for 4 h. It has been
seen that saccharification level has been increased to 33 in comparison to 4%
sachharification of untreated wheat straw when the substrate was pretreated with
2% NaOH for 4 h at room temperature. In the related study by Dahoot and
Noomrio (1996) the rate of β-glucosidase and CM-cellulase production by
Aspergillus fumigatus in H2SO4 and HCI pretreated wheat straw mineral medium
reached maximum. Ojumu et al. (2003) reported high cellulase activity from 3%
pretreated saw dust, bagasse and corn cob as substrate respectively.

Another important factor responsible for higher cellulase production is

solid state fermentation carried out using nutrient rich medium as fermentation
liquor. It has an additional advantage over simpler water for releasing higher titers
of enzyme because nutrient rich supplementation would help to establish as well
as facilitate the growth of fungus thus leading to proper utilization of substrate
eventually increasing the production of cellulase.

In literature pretreated lignocellulosic biomass has been used successfully

for cellulose production in solid state fermentation alkaline pretreated rice straw
was being used as the carbon source by Trichoderma reesei F-418 for cellulase
production i.e. 16.2 U/gds under the optimum culture condition of 75 % (v/w)
moisture, pH 4.8 for 5 days incubation at 28±2ºC (Fatma et al., 2010). In addition
the Vogel’s medium supplemented with the various nitrogen sources ammonium
sulphate and ammonium nitrate, yeast extract, peptone and tri sodium citrate
served as an important source of nutrient for the cellulase production and was
used as the moistening agent.

The solid waste of sago industry i.e. cassava waste containing 13.4% of
cellulose, 2.9% protein by dry weight was fermented by Aspergillus niger,
Aspergillus terreus and Rhizophus stolonifer under solid state fermentation in
nutrient rich medium. Highest cellulase activity was observed on 10th day, by R.
stolonifer mediated fermentation (Pothiraj et al., 2006).

Cellulase production was carried out in solid state fermentation using

waste from the vinegar industry for Trichoderma koningii AS3.4262. Optimal

114
FPase of 6.90 IU/ml and CMCase activity of 23.76 U/gds were obtained after 84h
of incubation with media containing vinegar waste, having optimal moisture
content of 50%, pH 5.0 and incubation temperature of 300C (Lui and Yang, 2007).

4.9.1 Effect of moisture level

Table 15 and fig 15 reveal the production of cellulase during degradation
of untreated and pretreated lignocellulosic biomass i.e. rice straw as a substrate
using vogel’s medium of pH 5.5 with inoculum size of 10%. The optimization
was done at different moisture level i.e. 1:2, 1:4, 1:6 and 1:8. Among the different
moisture level 1:6 was found to be the best for cellulase production by
Thermomyces sp. F1 producing total cellulase of 43.012 U/gds i.e. FPase (12.363
U/gds), CMCase (10.400 U/gds) and β-glucosidase (20.153 U/gds) in microwave
treated biomass. However, in untreated biomass 33.826 U/gds of cellulase i.e.
FPase (10.010 U/gds), CMCase (7.566 U/gds) and β-glucosidase (16.250 U/gds)
was noticed at this moisture level. Minimum cellulase activity was observed at
moisture level of 1:2 of 6.932 IU/ml i.e. FPase (2.410 U/gds), CMCase (1.900
U/gds) and β-glucosidase (2.622 U/gds) in untreated residue while 8.407 IU/ml
i.e. FPase (3.063 U/gds), CMCase (1.733 U/gds) and β-glucosidase (3.611 U/gds)
in treated residue. 29.100% (in treated biomass) and 9.788% (in untreated
biomass) increase in cellulase activity was observed at moisture level of 1:6 in
comparison to control i.e. at moisture level of 1:4 (Plate 5).

The moisture level plays a very crucial factor in SSF that determines the
success of the process. A higher moisture level than the optimal level causes a
decrease in porosity, lower oxygen transfer and alteration in rice bran particle
whereas a lower optimum moisture level leads to reduction in solubility and
swelling of solid substrates (Narahara et al., 1982). Optimum moisture level of
60% attained in the present experiment is supported by the fact that high moisture
enhances fungal growth and cellulase production when lignocellulosic substrates
are the carbon sources in the SSF (Panagiootou et al., 2003). Moreover
Thermomyces sp. F1 being thermophilic in nature and required high incubation
temperature for growth, that may leads to evaporation of moisture present in the
substrate, as fungal growth is directly related to moisture level, for supporting the
fungal growth a higher moisture i.e. 60% is required.

115
Table 15. Cellulase production at different moisture level from Thermomyces sp. F1 using untreated and microwave treated rice
straw (Oryza sativa) under SSF

Untreated biomass Treated biomass

Moisture Protein FPase CMCase β- Total % increase Protein FPase CMCase β- Total %
level conc. glucosidase cellulase or decrease conc. glucosidase cellulase increase
(mg/g) (U/gds) in cellulase (mg/g) (U/gds) or
activity decrease
in
cellulase
activity
1:2 7.400 *2.410 1.900 2.622 6.932 -58.811 15.000 3.063 1.733 3.611 8.407 -73.111
**(0.325) (0.256) (0.354) (0.933) 0.204) (0.115) (0.240) (0.579)
1:4 15.000 6.221 4.522 6.063 16.820 - 17.122 11.200 9.053 12.872 33.125 -
(0.414) (0.301) (0.404) (1.148) (0.700) (0.528) (0.751) (1.934)
1:6 20.111 10.010 7.566 16.250 33.826 +9.788 31.200 12.363 10.400 20.150 43.012 +29.100
(0.497) (0.375) (0.808) (1.681) (0.396) (0.333) (0.641) (1.370)
1:8 14.000 4.711 4.411 6.532 15.654 - 15.222 19.611 6.382 5.382 11.540 24.321 - 26.578
(0.336) (0.315) (0.466) (1.114) (0.325) (0.274) (0.588) (12.401)
C.D0.05 1.338 0.979 0.188 0.163 0.174 - 1.338 0.163 0.163 0.188 0.163 -
S.E. 0.580 0.424 0.816 0.070 0.039 - 0.580 0.070 0.070 0.008 0.070 -
(difference
of mean)
*
**
Same as in Table 14
+
-
 Untreated biomass Treated biomass

Thermomyces sp. F1

Plate 4. Production of cellulase enzyme from Thermomyces sp. F1

using untreated and treated rice straw in SSF
 Untreated biomass Treated biomass

Substrate: medium 1:4

Untreated biomass Treated biomass

Substrate: medium 1:6

Untreated biomass Treated biomass

Substrate: medium 1:8

Plate 5. Effect of moisture level on cellulase production from
Thermomyces sp. F1
 As revealed in many studies, moisture level may vary ranging from 20%
(Sanghi et al., 2007), 40, 60 to 80 % depending upon the substrate used and
temperature of incubation. In a similar study a higher cellulase activity was
recorded by Gao et al. (2008) using Aspergillus terreus M11 on lignocellulosic
material i.e. corn stover having 80% of moisture in corn stover for enzyme
activity up to 581 U/gds.

4.9.2 Effect of temperature

Table 16 and fig 16 exhibit secretion of cellulase at different temperature

30, 35, 49, 45, 50, 550C from Thermomyces sp. F1 using untreated and microwave
irradiated rice straw . There was a consistent increase in enzyme activity along
with temperature ranging from 30-500C reaching maximum at 500C i.e. 43.041
U/gds (FPase (12.463 U/gds), CMCase (10.337 U/gds) and β-glucosidase (20.041
U/gds)) in treated rice straw and 33.240 IU/ml (FPase (10.400 U/gds), CMCase
(7.422 U/gds) and β-glucosidase (16.400) U/gds) in untreated biomass was
noticed. Further increase in temperature resulted in remarkably low activity
enzyme activity of 5.220 U/gds (2.600 U/gds of FPase, 1.986 U/gds of CMCas
and 1.880 U/gds of β-glucosidase)) and 5.282 U/gds i.e. 3.220 U/gds of FPase,
1.011 U/gds of CMCase and 1.051 U/gds of β-glucosidase in untreated and
treated biomass respectively as shown in Plate 6.

The results so obtained prove thermophilic nature of Thermomyces sp. F1

0
(50 C) isolated from hot spring of Manikaran. Thermophilic fungi grow at high
temperature exceeding 450C and their special advantage is that enzyme produced
by them has been found to be more heat stable than the corresponding enzymes
produced by mesophlilic fungi (Maheshwari et al., 2000). Fungi provide an
attractive enzyme option to industries as compared to enzymes from bacterial
system. Thermophilic fungi are capable of secreting high level of enzymes and
also of finding novel enzyme variants with high temperature optima and a long
"shelf-life," and these desirable characteristics for commercial applications.
Utilization of enzymes in industrial processes often encounters the problem of
thermal inactivation of the enzyme. A considerable amount of research has been
focused on the search for novel

117
Table 16. Cellulase production at different temperature from Thermomyces sp. F1 using untreated and microwave treated rice
straw (Oryza sativa) under SSF

Untreated Biomass Treated Biomass

Temperature Protein FPase CMCase β- Total %increase Protein FPase CMCase β- Total %
0
( C) conc. glucosidase cellulase or conc. glucosidase cellulase increase
(mg/g) (U/gds) decrease (mg/g ) (U/gds) or
in cellulase decrease
activity in
cellulase
activity
30 15.611 *2.900 3.811 8.40 0 15.111 -54.600 22.811 4.40 0 3.860 12.400 20.662 - 51.993
**(0.128) (0.244) (0.538) (0.192) 0.169 0.543) 0.905)
35 19.600 4.460 3.872 10.261 18.593 -43.018 25.240 6.500 6.400 11.533 24.000 -44.276
(0.227) (0.197) (0.523) (0.948) (0.257) (0.253) (0.602) (0.967)
40 21.000 6.262 4.411 13.400 24.073 --26.034 27.422 6.411 7.400 15.600 29.411 - 31.523
(0.298) (0.210) (0.638) (1.145) (0.233) (0.269) (0.568) (1.072)
45 23.421 6.503 7.400 14.800 28.703 -13.710 29.433 8.511 8.600 18.062 35.173 -18.409
(0.277) (0.315) (0.631) (1.225) (0.298) (0.292) (0.613) (1.194)
50 20.111 10.400 7.422 16.400 33.240 - 31.200 12.463 10.337 20.241 43.041 -
(0.517) (0.415) (0.770) (1.653) (0.416 ) (0.330) (0.642) (1.371)
55 2.888 2.600 1.986 1.880 5.220 -84.30 3.022 3.220 1.011 1.051 5.282 - 86.510
(0.901) (0.687 ) (0.652) (0.265) (1.06) (0.323) (0.347) (1.923)
C.D0.05 1.290 1.687 0.199 1.034 0.740 - 11.406 0.162 0.162 0.126 0.740 -
S.E.(difference 0.592 0.774 0.089 0.474 0.339 - 5.234 0.074 0.074 0.058 0.339 -
of mean)

*
**
Same as in Table 14
+
-
microbial isolates having the ability to produce thermotolerant and alkalophilic
cellulase. Thermophilic bacteria (including thermophilic actinomycetes) and
thermophilic and/or thermotolerant fungi have been found particularly suitable for
this task (Beg et al., 2000). High temperature and low pH tolerant enzymes are
preferred for the hydrolysis due to the fact that most current pretreatment
strategies relay on acid and heat treatment. In addition, thermostable enzymes
have several advantages including higher specific activity and higher stability
which improve the overall hydrolytic performance (Viikari et al., 2007). Enzyme
produced by them at high temperature ±500C found to have many industrial
applications.

An alkaline cellulase producing fungus i.e. Chaetomium sp. (NIOCC 36)

was isolated from mangrove leave and wood, capable of growing in a wide range
of temperature 500C. Maximum enzyme production at high temperature was
obtained when cotton seed was used as a substrate (Ravindran et al., 2010).
Similarly an extracellular enzyme having thermostablility up to 500C was
secreated by Penicillium notatum NO-923 having highest CMCase of 67.49±1.2
U/gds and FPase of 67.49±1.33 U/gds activity at 300C (Das and Ghosh, 2009).

4.9.3 Effect of incubation time

Effect of incubation time i.e. 4, 5, 6, 7, 8, 9 and 10 days on cellulase

production by Thermomyces sp. F1 shows the data in Table 17 and fig 17. There
was a consistent increase in enzyme in enzyme from initial day of incubation upto
8 days. Highest cellulase i.e. 43.070 U/gds (FPase 12.460 U/gds, CMCase 10.030
U/gds and β-glucosidase 20.580 U/gds) in microwave treated cellulosic biomass
and in untreated biomass 33.540 U/gds (FPase 10.020 U/gds, CMCase 7.423
U/gds and β-glucosidase 16.197 U/gds) in untreated biomass was marked on 8th
day of fermentation. Afterwards a decline in cellulase production was recorded on
9th and 10th day i.e. 21.833 and 18.710 U/gds in untreated biomass and 32.145 and
20.835 U/gds in pretreated residue respectively (Plate 7).

119
 Untreated biomass Treated biomass

40oC

Untreated biomass Treated biomass

45oC

Untreated biomass Treated biomass

50oC

Plate 6. Effect of temperature on cellulase production from

Thermomyces sp. F1
 Untreated biomass Treated biomass

7th day

Untreated biomass Treated biomass

8th day

Untreated biomass Treated biomass

9th day
Plate 7. Effect of incubation time on cellulase production from
Thermomyces sp. F1
Table 17. Cellulase production at different incubation time from Thermomyces sp. F1 using untreated and microwave treated rice
straw (Oryza sativa) under SSF
Untreated Biomass Treated Biomass
Incubation time Protein FPase CMCase β- Total %increase Protein FPase CMCase β- Total % increase
(Days) conc. glucosidase cellulase or conc. glucosidase cellulase or decrease
(mg/g) (U/gds) decrease (mg/g ) (U/gds) in cellulase
in cellulase activity
activity
4 8.772 *4.902 2.3000 11.021 18.223 -47.246 9.670 5.022 3.200 15.121 23.343 -84.965
**(0.558) (0.262) (1.256) (2.107) (0.519) (0.330) (1.563) (2.413)
5 9.782 6.091 4.110 12.142 22.341 - 33.389 13.880 7.170 5.492 15.111 27.773 - 35.521
(0.622) (0.419) (1.354) (2.279) (0.516) (0.398) (1.088) (2.817)
6 17.550 6.501 5.110 11.461 24.072 - 28.228 18.331 6.082 4.051 18.454 28.587 - 33.638
(0.374) (0.419) (0.652) (1.317) (0.331) (0.291) (1.006) (1.556)
7 18.222 6.311 6.201 15.051 27.593 -17.740 25.980 9.170 8.033 19.562 36.765 -14.766
(0.363) (0.310) (0.734) (1.572) (0.352) (0.309) (0.752) (1.415)
8 20.111 10.020 7.423 16.197 33.540 - 31.200 12.460 10.030 20.580 43.070 -14.608
(0.507) (0.241) (0.709) (1.717) (0.399) (0.321) (0.641) (1.380)
9 17.881 7.321 2.460 (12.052 21.833 -21.019 20.881 11.021 6.492 14.632 32.145 -25.368
(0.409) (0.137) (0.673) (1.481) (0.527) (0.312) (0.700) (1.539)
10 11.781 4.200 2.460 12.051 18.710 -44.215 12.771 6.192 3.493 11.150 20.835 -51.074
(0.356) (0.208) (1.022) (1.588) (0.484) (0.273) (0.873) (1.631)
C.D0.05 0.948 0.947 0.945 0.393 1.152 - 0.175 0.132 0.132 0.682 0.945 -
S.E.(difference 0.442 0.441 0.441 0.183 0.537 - 0.081 0.061 0.061 0.318 0.440 -
of mean)
*
**
Same as in Table 14
+
-
 Studying the incubation time revealed that the maximum cellulase
production was obtained when fungi was cultivated for 8 days using rice straw as
a substrate. With longer cultivation time than 8 days, a trend of decreased
cellulase was obtained in the present study. Melo et al. (2007) reported that the
enzyme level declined with prolonged incubation, this could be due to the loss of
moisture or denaturation of the enzyme resulting from variation in pH during
fermentation. Singh et al. (2009) reported that the decrease of enzyme activity
may be due to the accumulation of cellobiose. The time of highest cellulase
activity was depending upon the substrate and fungus used in the fermentation
process (Ojuma et al., 2003 & Alam et al.,1994).

The ability of Aspergillus terreus for the production of cellulolytic enzyme

and reduction of lignocellulosic contents of rice straw in solid state fermentation
was investigated by Jahromi et al. (2011). Enzyme activity was maximum i.e.
FPase (410.76 U/gds), CMCase (351.96 U/gds) and β-glucosidase (16.37 U/gds)
on 8 day of fermentation.

When cultivatiuon of Trichoderma viride NCIM1051 on solid substrate

coconut pith was used for the production of cellulase under SSF (Munsiwaran and
Charaylun,1994). Maximum cellobiose activity obtained on 8 day of fermentation
i.e. 1.8 U/gds.

Quiroz-Castanedo et al. (2009) studied cellulolytic properties of two white

rot fungi Bjerkandera adusta and Pycnoporus sanguineus, cultivated on wheat
straw agar medium. Highest cellulase was synthesized on the 6th day of culture
and CMCase activity was noticed 1.6 times higher on the 8 day of fermentation.
Similarly Jayant et al. (2011) revealed that after 8 days of incubation, newspaper
as a substrate having pH 5 and 400C was the best source of carbon for the
enhanced production of cellulase in the compatible mixed culture under Solid
state fermentation.

Percent increase in cellulase activity in Thermomyces sp. F1 from

microwave treated rice straw after optimization of different parameters viz
pretreatment, moisture, temperature, incubation time under SSF has been shown

121
in fig 16 where an increase of 157.13% was shown over unoptimized conditions
which was significantly higher statistically. After optimization an increase in
cellulase titers from 16.750 U/gds (in untreated biomass) to 43.070 U/gds (in
pretreated biomass) is a remarkable achievement of the present study. Higher
yield of cellulase along with thermophilic and acidophilic nature of Thermomyces
sp. F1 render it as a desirable strain for enzyme producing industry thus finally
meeting an important objectives of our study (Fig 18).

4.10 PRODUCTION AND OPTIMIZATION OF XYLANASE FROM

HYPERXYLANOLYTIC FUNGUS Thermomyces sp. F1 UNDER
SOLID STATE FREMENTATION

Table 18 and fig 19 shows the extracellular xylanase synthesis during

biodegradation of untreated and microwave treated rice straw (Oryza sativa) by
Thermomyces sp. F1 using Vogel’s medium as substrate with inoculum size of
10%. Maximum enzyme activity (260.800 U/gds) was obtained in treated biomass
as compared to untreated biomass with less enzyme activity of 240.000 U/gds
(Plate 8).

Xylanases have expanded their use in many processing industries, such as

pulp and paper, food and textile. Therefore an attempt has been made to isolate
thermophilic fungi to produce enzymes from cheap biomass. The use of purified
xylan as a substrate for enhanced xylanase production was uneconomical (Xia and
Cen, 1999). To reduce the cost of xylanase production using lignocellulosic
material as a substrates is an attractive approach rather than opting for the
expensive pure xylan (Haltrich et al., 1996; Beg et al., 2000; Senthikumar et al.,
2005). Using abundantly available and low cost agricultural byproducts as
substrate for the production of xylanase is one of the ways to substantially reduce
the enzyme production cost (Srinivasan and Rele, 1999).

Solid state fermentation is an attractive method for xylanase production

especially for fungal cultivations, because it presents many advantages such as the
highest productivity per reactor volume as well as the lower capital cost
(Purkarthofer et al., 1993 & Pandey et al., 1999). The physical support and the
energy required for a fungus to grow and produce the desired metabolite is

122
primarily provided by the substrate and also for reducing the cost of enzyme,
selection of cheap and easily available substrate appears to be essential. Therefore
it’s important to select a desirable substrate for solid state fermentation (Pandey et
al., 2001). Rice straw important agricultural byproducts are produced worldwide
in enormous quantities. It is an excellent cost effective substrate for large scale
production of xylanase in solid state fermentation (Sanghi et al., 2008), The
suitability of using rice straw as a substrate for xylanase production is that it
contains hemicelluloses fermenting into considerable amount of soluble sugars
required for the growth of the microorganism and is able to remain loose even in
moist condition, therefore providing large surface area for the fungal growth, main
advantage of using it as a substrate is it costs almost nothing and its use also
reduces the emission of heat and carbon dioxide (Ali et al., 2011).

Table 18. Xylanase production from Thermomyces sp. F1 using untreated

and microwave treated rice straw (Oryza sativa) under SSF
Untreated Treated t cal
biomass biomass
Protein conc.(mg/g) 20.600 27.000 + 4.5030
Xylanase *240.000 260.800 + 1.56
activity(U/gds) **(11.651) (9.659)
* U/gds: µmoles of reducing sugar released / min / g of enzyme.
** Value in parentheses depict specific activity i.e. enzyme activity/g of protein.
+ indicates an increase in xylanase activity over the control.
- indicates a decrease in xylanase activity over the control.

Several structural and compositional factors affect fermentation ability of

lignocellulosic materials for xylanase extraction, hemicellulose sheathing and
degree of hemicelluloses acetylating etc. These barriers differ in relative
significance, depending on the substrate material. Different pretreatments of
lignocellulosic substrates such as physical thermal or chemical treatment are
reported in the literature to make the substrates more conducive to SSF. The
alteration of the substrate using pretreatment techniques leads to a change in
partial depolymerization of hemicelluloses physical nature of lignin, increases in
the available surface area, increases in pore sizes, and deacetylation of
hemicelluloses, which enhance availability of the substrate and decrystallization

123
of cellulose (Shah and Datta, 2005). The latest pretreatment method applied to
lignocellulosic biomass for production of enzymes and fuels is the microwave
treatment. Microwave technology is supposed to simplify three structural
polymers along with extraneous components. This physical pretreatment increases
the amorphous region of hemicellulose and thus has a dramatic effect on
decomposition because of lignocellulose softening.

Zhu et al. (2006) found that xylose could be recovered as crystalline

xylose during the microwave/acid/alkali and microwave/acid/alkali/H2O2
pretreatment. The enzymatic hydrolysis of pretreated rice straw showed that the
pretreatment by microwave/acid/alkali/H2O2 had the highest hydrolysis rate and
glucose content in the hydrolyzate.

Sherief et al. (2010) investigated bioprocessing of lignocellulosic biomass

for biodegradation and production of bioethanol using thermotolerant Aspergillus
fumigatus under solid state fermentation conditions. Alkali pretreatment was
given to rice straw twenty grams of milled dried rice straw was suspended in 160
ml of 1% (w/v) NaOH and pretreated in an autoclave at 121 °C for 1 h. The
residues were collected and washed extensively with water until washings turned
neutral. The residues were dried at 65°C for two days. Dried biomass is grinded to
ensure more uniform particle size distribution.

Several fungal strains were isolated from soils collected from Southern
Kerala, India and screened for the xylanase production using Czapek’s agar
medium (Nair et al., 2008). Total 69 strains were isolated from Pitchavaram
mangroves by Muthezhilan et al. (2007) on the basis of secondary screening
Penicillium oxalicum was selected and optimized for xylanase production in SSF
using cheaper source like wheat bran (3.89 U/gds at 450C temperature and pH 8).

Similarly Taneja et al. (2002) reported an alkalophilic Aspergillus

nidulans KK-99 produced from an alkaline thermostable xylanase (40 IU/ml) in
basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15%) pH
10.0 and 370C.

124
4.10.1 Effect of moisture level

Table 19 and fig 20 reveal the effect of different moisture levels i.e. 1:2,
1:4, 1:6 and 1:8 on xylanase production under SSF. Thermomyces sp. F1 produced
maximum xylanase of 281.000 U/gds with microwave treated biomass rice straw
at moisture level of 1:6 and in untreated biomass lesser xylanase production i.e.
250.000 U/gds was obtained. These values were significantly higher than xylanase
production at other moisture levels of 1:2, 1:4, 1:8. 7.692% increase in pretreated
biomass and 4.166% increase in untreated biomass were observed at moisture
level of 1:6 in comparison to control i.e. at moisture level of 1:4 (Plate 9).

Moisture content of the substrate is one of the crucial factors influencing

the outcome of SSF. Lower moisture content causes a reduction in solubility of
nutrients provided to organisms by SSF. Increase in moisture contents greatly
enhances the enzyme activity thus attributes to the faster growth of fungus at high
moisture and in turn the subsequent early initiation of enzyme production It has
also been cited in literature that high moisture level enhances fungal growth and
xylanase production when lignocellulosic substrates were the carbon sources in
SSF (Purkarthofer et al., 1993; Alam et al., 1994 & Bakri et al., 2003). It’s
concluded that the degree of hydrate of substrate plays an crucial role on the
growth of fungi and enzyme production. A large number of fungi are known to
grow well on moist substrates in the absence of free flowing water, whereas many
bacteria are unable to grow under these conditions, as a result the majority of
studies involving in SSF are conducted using fungi. As in the present study the
thermophilic fungal isolate had been was growing at a higher temperature of 500C,
it required up to 60% of the moisture content that could provide favorable
moisture conditions for the growth of fungus consequently synthesizing more of
xylanase.

In literature the highest xylanase activity (13230 U/gds) was obtained with
the initial moisture contents of 83% by and extracellular xylanase producing
thermophilic fungus Paecilomyces thermophila J18 at 500C on the lignocellulosic
material when wheat straw was used as the substrate.

125
 Untreated biomass Treated biomass

Plate 8. Production of xylanase enzyme from Thermyces sp. F1 using

untreated and treated rice straw in SSF
 Untreated biomass Treated biomass

Substrate : medium 1:4

Untreated biomass Treated biomass

Substrate : medium 1:6

Untreated biomass Treated biomass

Substrate : medium 1:8

Plate 9. Effect of moisture level on xylanase production from
Thermomyces sp. F1
Table 19. Xylanase production at different moisture level from Thermomyces sp. F1 using untreated and microwave treated rice
straw (Oryza sativa) under SSF

Untreated biomass Treated biomass

Moisture level Protein conc. Xylanase activity % increase or Protein Xylanase % increase or
(mg/g) (U/gds) decrease in conc. activity (U/gds) decrease in
xylanase activity (mg/g) xylanase activity
1:2 15.111 *140.411 -41.531 23.600 178.232 -36.313
**(9.292) (7.552)
1:4 20.623 240.011 - 27.012 260.832 -
(11.639) (9.656)
1:6 26.600 250.000 +4.166 30.011 281.000 +7.692
(9.398) (9.333)
1:8 19.623 130.000 - 45.832 22.400 201.802 - 46.703
(6.625) (9.008)
C.D0.05 0.188 1.334 - 0.946 0.955 -
S.E.(difference 0.081 0.578 - 0.410 0.414 -
of mean)
*
**
Same as in Table 18
+
-
 Similarly Thermomyces lanuginosus isolate yielded xylanase activities of
over 5,000 IU/gds when solid substrate fermentation (SSF) was evaluated for
microbial production of xylanase from Eucalyptus grandis as a carbon source by
Szendefy et al. (2006).
Optimum conditions for xylanase production by T. lanuginosus TUB F-
980 were determined at an optimal moisture content (80%), pH (7.0) and time
course (≥5 days). Also the production of xylanase by Thermomyces lanuginosus
strains was examined on bagasse pulp under solid state fermentation (Christopher
et al., 2005). The optimum pH for xylanase production was 7.0 at 80% moisture
level.
4.10.2 Effect of temperature
Table 20 and fig 21 exhibit xylanase activity at temperature ranging from
30, 35, 40, 45, 500C. Thermomyces sp. F1 showed highest xylanase production at
500C i.e. 250.000 U/gds in untreated biomass and 281.071 U/gds in treated
biomass which was found significantly higher than xylanase production at other
temperature range. Statistically lowest enzyme production was obtained at 300C
i.e. 100.700 U/gds in untreated biomass and 144.00 U/gds in treated biomass
(Plate 10).

Temperature and pH are important environmental parameters that

determine growth rate of microorganism/ fungus and significantly affect the
production of xylanase. According to Aiba et al. (1973) at lower temperature, the
transport of substrate across the cells is decreased and lower yield of product is
obtained. Due to the promising thermostability and acidic tolerance of
thermophilic fungal enzyme, they have good potential to be used for hydrolysis of
lignocellulosic residues at industrial scale.

Similar reports referring to bacterial thermophilic species have also been

furnished by various author for xylanase production using lignocellulosic
substrate in SSF. Bacillus subtilis isolated from the compost by Saleem et al.
(2002) produced a significant amount of xylanase when grown in different
pretreated carbon sources such as rice straw, wheat straw, bagasse, wheat bran and
kraft pul producing a significant amount of xylanase activity i.e. 3.2 and 3.5 U/ml
after 14 h of fermentation at 500C.

127
Table 20. Xylanase production at different temperature from Thermomyces sp. F1 using untreated and microwave treated rice
straw (Oryza sativa) under SSF

Untreated biomass Treated biomass

Temperature Protein conc. Xylanase activity % increase or Protein Xylanase % increase or
(0C) (mg/g) (U/gds) decrease in conc. (mg/g) activity(U/gds) decrease in
xylanase activity xylanase activity
30 18.802 *100.700 -60.000 20.611 144.000 - 52.333
**(5.356) (6.986)
35 19.503 152.000 -54.432 23.422 180.041 -43.872
(7.794) (7.687)
40 20.402 196.000 - 35.465 24.400 240.051 -23.544
(9.607) (9.838)
45 23.401 220.000 -12.000 27.400 270.000 - 3.940
(9.401) (9.854)
50 26.620 250 .000 - 30.022 281.071 -
(9.433) (9.362)
55 5.011 19.022 - 92.391 9.001 23.110 - 91.778
(3.800) (2.555)
C.D. 0.05 0.181 11.564 - 0.162 8.176 -
S.E. (difference of 0.081 5.184 - 0.073 3.669 -
mean)

*
**
Same as in Table 18
+
-
 Penicillium sclerotrium had shown highest enzyme activity at 500C after 5
days on wheat straw under SSF. The temperature for optimum activity was 50ºC
and optimum pH 4.5 (Knob and Carmona, 2008). Similarly optimization of the
different fermentation parameters i.e. fermentation time, incubation and
inoculums volume leads to 21.7% increase in extracellular enzyme activity at
450C by Thermomyces lanuginosus 195 under solid state fermentation (Gaffney
et al., 2009).

A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied

for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid
state fermentation by Shah and Datta (2005). 500C was the optimal temperature
for the xylanase activity i.e. 3065 U/gds on untreated corncob moistened with
modified Mandels and Strenberg medium, pH 5.0, with 1:5 moisture levels at
300C in 4 days of cultivation.

Yang et al. (2006) reported the high level of xylanase production by

thermophilic fungus Paecilomyces thermophila J18, strain grown at 500C and
producing a higher xylanase activity using the lignocellulosic material under the
solid state fermentation especially wheat straw, under the optimized conditions the
xylanase yield was high as 18580 U/gds.

4.10.3 Effect of incubation time

Table 21 and fig 22 describe the enzyme activity measured at regular
intervals of 7 days viz 2, 3, 4, 5, 6 and 7 to study the effect of incubation time on
xylanase production under SSF. Xylanase production was increased with the
passage of time ranging from 2 day onwards and highest xylanase activity was
recorded on 5 i.e. 250.042 U/gds. Afterwards xylanase production recorded
consistently decreases on 6 and 7 day reaching upto 112.033 U/gds and 100.021
U/gds respectively (Plate 11).

The duration needed for incubation might depend on the growth rate of
microorganism and its enzyme production pattern (Topkas et al., 2003). The
enzyme production of the fungal grown on the 5 day in the present study indicates
that the maximum enzyme might have been produced either in the exponential
growth phase, as during these phase growth elements such as amino

129
Table 21. Xylanase production at different incubation time from Thermomyces sp. F1 using untreated and microwave treated rice
straw (Oryza sativa) under SSF

Untreated Biomass Treated Biomass

Incubation time Protein conc. Xylanase activity % increase or Protein Xylanase % increase or
(Days) (mg/g) (U/gds) decrease in conc. (mg/g) activity decrease in
xylanase activity (U/gds) xylanase activity
2 13.812 *200.001 -.18.455 17.200 240.031 - 16.512
**(14.482) (13.955)
3 18.600 230.003 - 8.333 21.831 256.000 -11.993
(12.362) (11.726)
4 22.200 239.000 4.400 25.822 265.200 - 5.693
(11.300) (10.271)
5 26.520 250.042 - 30.040 281.052 -
(9.464) (9.365)
6 19.611 112.033 -55.200 23.622 126.000 - 55.123
(5.712) (5.334)
7 12.200 100.021 - 59.321 17.400 122.000 - 63.075
(8.198) (7.011)
C.D0.05 0.177 26.793 - 0.162 7.406 -
S.E. 0.081 12.299 - 0.074 3.399 -
(difference of mean)
*
**
Same as in Table 18
+
-
 Untreated biomass Treated biomass

40oC

Untreated biomass Treated biomass

45oC

Untreated biomass Treated biomass

50oC

Plate 10. Effect of temperature on xylanase production from

Thermomyces sp. F1
 Untreated biomass Treated biomass

4th day

Untreated biomass Treated biomass

5th day

Untreated biomass Treated biomass

6th day
Plate 11. Effect of incubation time on xylanase production from
Thermomyces sp. F1
acids, nitrogen, vitamins, protein and enzymes required for essential metabolic
processes or stationary phase are being synthesized by the fungus.

The other studies quoted by the authors with different fungal spp. have
shown enzyme secretion optima matching with their growth profiles. The initial
moisture content, cultivation time, inoculum size and concentration of basal
medium were optimized in solid state fermentation (SSF) for the production of
xylanase by an Aspergillus niger. The activity and productivity of xylanase
obtained after 5 days of fermentation were 5,071 IU/gds of rice straw and 14,790
IU l–1 h–1. The xylanase activity predicted by a polynomial model was 5,484
IU/gds of rice straw (Park et al., 2002).

Five types of agricultural wastes were used for the production of

xylanolytic enzyme by Aspergillus flavus K-03 by Kim (2005). Maximum
production of xylanase was achieved in basal liquid medium containing rice bran
as carbon source after 5 days of incubation at pH 6.5 and temperature of 25oC.
Maximum xylanase activity obtained at pH 5.5~10.5 and more than 70% of the
original activity was retained at pH 6.5 and 7.0. Highest production of xylanolytic
enzyme obtained when 0.5% of rice bran supplemented with basal liquid medium.

Okafor et al. (2007) reported a xylanase producing fungal strain

Penicillium chrysogenum PCL501 isolated from wood-wastes containing different
carbon sources (oat spelts xylan, wheat bran, sawdust and sugarcane pulp. Media
containing sugarcane pulp gave a peak value of 1.39 U/ml at 144 h of
fermentation obtained using sugarcane as a substrate.

In solid state fermentation of Thermomyces sp. F1 using pretreated rice

straw for xylanase production after optimizing important environmental
conditions viz pretreatment, moisture, temperature and incubation time has lead to
an increase of 17.099% in extracellular xylanase activity (fig 23). An impressive
yield of 281.052 U/gds of xylanase from thermophilic and acidophilic
Thermomyces sp. F1 is a main attraction of the conducted experiments thus
recommending its scope for commercial utilization. Overall the optimization
study of cellulase and xylanase enzyme was confined to three hypercellulolytic

131
and xylanolytic bacteria and fungus isolated from a least explored hot and humid
niche i.e. thermal springs of Himachal Pradesh. The search in this area has ended
in highly encouraging achievements which are not only of academic interest but
after refinement of the study virtually can ignite the interest of enzyme oriented
industry.

132
 Chapter-5

SUMMARY AND CONCLUSION

In the present study entitled “Optimization of hydrolytic enzymes
produced from thermophilic microorganisms for degradation of cellulosic
biomass”, an attempt has been made for the isolation of most efficient cellulolytic
and xylanolytic thermophilic bacteria and fungi from hot springs of Himachal
Pradesh, their screening to select hyperenzyme producing strain and optimization
of various growth and nutritional parameters which could further enhance the
yield of extracellular cellulase and xylanase from potential isolates.

In total, 67 isolates in which 65 were bacteria (27 isolates from

Manikaran, 27 from Tattapani and 11 from Vashisht) and 2 were fungi (F1 from
Manikaran and F2 from Tattapani) had been isolated from the hot springs of
Himachal Pradesh. Different physiochemical and biochemical tests were
performed with these bacterial isolates for their tentative identification and
eventually confirming identification at molecular level using rrs (16S rRNA)
technique for the best selected isolates based upon their highest enzyme
synthesizing capacity. An extracellular xylanase producing bacterial stain N1 was
identified as Paenibacillus sp. N1 while hypercellulolytic and xylanolytic fungus
was identified as Thermomyces sp. F1 and these isolates were selected for further
enzyme optimization study.

After the qualitative analysis of enzyme produced from different bacterial

thermophiles isolated from hot springs of Himachal Pradesh, Paenibacillus sp. N1
showed the maximum extracellular xylanase production on Reese’s media i.e.
24.60 IU/ml and minimum production of extracellular xylanase was observed in
T11 i.e. 2.10 IU/ml. The isolate Paenibacillus sp. N1 on the basis of its maximum
xylanase activity was selected for the further course of study under SmF.

Optimization of different factors i.e. media, incubation time, pH,

temperature, inoculum size, amino acids, carbon sources, nitrogen sources and
additives for the production of extracellular xylanase from hyperxylanolytic
bacterial strain Paenobacillus sp. N1 was carried out under the submerged
fermentation. Similarly the different parameters viz. microwave irradiation
pretreatment, moisture level, temperature and incubation time with selected
agricultural residue - rice straw were optimized for hypercellulolytic and
xylanolytic fungal strain Thermomyces sp. F1 under solid state fermentation.

The best yield of extracellular xylanase production by Paenobacillus sp.

N1 i.e. 52.300 IU/ml was obtained in the Basal salt medium after 3 days at pH 9,
temperature 500C, inoculum size 12.5%, amino acid- tryptophan carbon source -
xylose, nitrogen source- (NH4)2HPO4 and additive- Tween 20. After optimization
of different process parameters, xylanase activity from the hyperxylanolytic
bacteria was enhanced from 24.600 to 52.3000 IU/ml and percent increase in the
enzyme activity had gone upto 113.38% which was recordable significantly
higher statistically.

An isolation of extremophilic bacteria Paenobacillus sp. N1 from hot

water spring of Manikaran (H.P.) is a major contribution of the present study.
Thermoalkalophilic nature of Paenobacillus sp. N1 is a highly desirable attribute
which advocates its use for commercial application.

Lignocellulosic substrate i.e. rice straw (Oryza sativa) was selected as a

substrates for the production of cellulase and xylanase from fungal isolate
Thermomyces sp. F1 under SSF. Optimization of different environmental factors
i.e. microwave pretreatment, moisture level of 1:6 temperature at 500C and
incubation time of 8 days for the cellulase production under SSF had yielded
cellulase titers of 43.07 U/gds from 16.75 U/gds resulting in an impressive
increase of 157.13 % in cellulase activity.

As far as xylanase production was concerned, an increase in the enzyme

activity i.e. 281.000 U/gds by thermophilic fungus Thermomyces sp. F1 was
obtained after standardizing different environmental conditions i.e. microwave
pretreatment of biomass, moisture level of 1:6, Temperature at 500C and
Incubation time of 5 days causing high increase of 17.099 % in xylanase activity.

134
 The isolation of Thermomyces sp. F1 with a rare combination of being
hypercellulolytic as well as xylanolytic alongwith thermophilic and acidophilic
attribute is one of the main highlights of the present assignment.

Biotechnological processes are based on crude enzymes thus it becomes

very important to increase their activity in the culture supernatants by selecting
the best carbon source as well as growth parameters. The above conducted study
showed the successful attempt of optimization of different parameters for
enhancement of level of enzyme i.e. cellulase and xylanase from extremophilic
bacterial and fungal isolates with a cost effective approach of using cheap and
abundant lignocellulosic waste as a carbon source for their production.

135
 Chapter-6

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 Dr. Y. S. Parmar University of Horticulture and Forestry,
Nauni, Solan (HP)
Department of Basic Sciences
Title of Thesis : “Optimization of hydrolytic enzymes produced from
thermophilic microorganisms for degradation of cellulosic
biomass”
Name of the Student : Shruti Pathania
Admission Number : F-2009-09-M
Major Advisor : Dr. (Mrs.) Nivedita Sharma
Major Field : Microbiology
Minor Field(s) : i) Biochemistry
Degree Awarded : M.Sc. (Microbiology)
Year of Award of Degree : 2011
No. of Pages in Thesis : 159 + V
No. of words in Abstract : 270
ABSTRACT
The present study was carried out to isolate cellulolytic and xylanolytic microorganisms
from the hot springs of Himachal Pradesh (i.e. Tattapani, Manikaran and Vashisht), their
screening and optimization of cellulase and xylanase production under submerged and solid state
fermentation. Among all the bacterial isolates, bacterial strain N1 and Fungal strain F1 were
selected for enzyme production studies depending upon their higher cellulase (FPase, CMcase
and β-glucosidase) and xylanase producing capability. The selected bacterial isolates N1 and F1
were identified as Paenibacillus sp. and Thermomyces sp. respectively by physiological,
biological and rrs (16S rRNA) PCR technique. Under the submerged fermentation different
parameters (i.e. medium, incubation time, pH, temperature, inoculums size, amino acid, carbon
source, nitrogen and additives) were optimized for the maximum production of extracellular
xylanase by bacterial strain Paenibacillus sp. N1. 113.38% increase in xylanase activity was
noticed after optimization of different parameters under the submerged fermentation. Similarly
production of cellulase and xylanase was carried out under the solid state fermentation from
hyperenzyme producing fungal isolate Thermomyces sp. F1, using untreated and pretreated
lignocellulosic biomass i.e. Rice straw which was subjected to microwave irradiation
pretreatment. An enhanced production of extracellular cellulase and xylanase was observed by
optimization of different parameters i.e. moisture level, temperature and incubation time. A
significant increase in cellulase and xylanase was observed under the solid state fermentation
over the untreated biomass statistically. The present study strongly proves the success of
optimization of different parameters for enhancement in level of enzyme i.e. cellulase and
xylanase with a cost-effective approach of enzyme production using a cheap and abundant
lignocellulosic agricultural waste i.e. rice straw as a carbon source.

Signature of Major Advisor Signature of the Student

Countersigned

Professor and Head

Department of Basic Sciences
Dr. Y. S. Parmar University of Horticulture and Forestry,
Nauni, Solan-173 230 (HP)

159
ANOVA for Table 5
Xylanase activity (IU/ml)

Source df SS MS F
Treatment 5 650.08 130.02 1796.90
Error 12 0.086 0.00723
Total 17 650.17

Protein concentration (mg/ml)

Source df SS MS F
Treatment 5 0.609 0.121 12.18
Error 12 0.120 0.010
Total 17

ANOVA for Table 6

Xylanase activity (IU/ml)

Source df SS MS F
Treatment 5 404.55 80.910 8092.01
Error 12 0.120 0.010
Total 17 404.67

Protein concentration (mg/ml)

Source df SS MS F
Treatment 5 1.646 0.324 36.55
Error 12 0.166 0.0088
Total 17 1.7313

ANOVA for Table7

Xylanase activity (IU/ml)

Source df SS MS F
Treatment 6 217 36.167 2.38
Error 14 212.35 15.168
Total 20 429.35

Protein concentration (mg/ml)

Source df SS MS F
Treatment 6 1,123 0.1868 20.06
Error 14 0.1304 0.00931
Total 20 1.2517
ANOVA for Table 8
Xylanase activity (IU/ml)

Source df SS MS F
Treatment 5 1585.9 317.19 932.91
Error 12 4.0800 0.3400
Total 17 6443.4

Protein concentration (mg/ml)

Source df SS MS F
Treatment 5 2.589 0.517 55.65
Error 12 0.116 0.0093
Total 17 2.701

ANOVA for Table 9

Xylanase activity (IU/ml)

Source df SS MS F
Treatment 5 1801.3 360.26 1059.60
Error 12 4.080 0.3400
Total 17 1805.4

Protein Concentration (mg/ml)

Source df SS MS F
Treatment 5 5.5172 1.1034 110.34
Error 12 0.1200 0.0100
Total 17 5.6732

ANOVA for Table 10

Xylanase activity (IU/ml)

Source df SS MS F
Treatment 5 1320.0 264.01 394.04
Error 12 8.040 0.6700
Total 17 1328.1

Protein concentration (mg/ml)

Source df SS MS F
Treatment 5 5.483 1.096 109.86
Error 12 0.1200 0.00100
Total 17 5.603
ANOVA for Table 11
Xylanase activity (IU/ml)

Source df SS MS F
Treatment 8 4896.8 612.10 2661.32
Error 18 4.140 0.2300
Total 26 4901.00

Protein Concentration (mg/ml)

Source df SS MS F
Treatment 8 13.918 1.739 173.98
Error 18 0.1800 0.0100
Total 26 14.098

ANOVA for Table 12

Xylanase activity (IU/ml)

Source df SS MS F
Treatment 10 3649.2 364.92 891.01
Error 22 9.0104 0.4095
Total 32 3658.2

Protein Concentration (mg/ml)

Source df SS MS F
Treatment 10 14.651 1.4651 168.93
Error 22 0.1908 0.00867
Total 32 14.842

ANOVA for Table 13

Xylanase activity (IU/ml)

Source df SS MS F
Treatment 3 3691.9 1230.6 123062.85
Error 8 0.00800 0.0100
Total 11 3692.0
Protein Concentration (mg/ml)

Source df SS MS F
Treatment 3 7.896 2.6321 263.21
Error 8 0.0800 0.0100
Total 11 55.084
ANOVA for Table 19

Sources of df Mean sum of squares

variance Untreated Treated
Protein Xylanase Protein Xylanase
conc. activity conc. Activity
Inoculum 3 67.035 3.649 35.594 7025.9
Error 8 0.0800 0.502 0.252 0.257

ANOVA for Table 20

Sources of df Mean sum of squares

variance Untreated Treated
Protein Xylanase Protein Xylanase
conc. activity conc. Activity
Inoculums 4 31.5000 40.042 39.711 20.200
Error 10 0.02 1.0319 0.00802 1.0382

ANOVA for Table 21

Sources of df Mean sum of squares

variance Untreated Treated
Protein Xylanase Protein conc. Xylanase
conc. activity Activity
inoculum 5 84.176 226.83 74.133 17.333
Error 12 0.0100 1.4534 0.0835 1.5456
ANNOVA for Table 15

Source of df Mean Sum of Squares

Variance Protein CMCase FPase β- Total Protein CMcase FPase β- Total
glucosidase enzyme conc glucosidase enzyme
activity activity
Moisture 4 81.839 16.129 30.529 102.87 1658.00 156.72 45.621 56.014 137.76 646.54
Error 10 0.5055 0.0100 0.270 0.00752 0.00145 0.5055 0.00752 0.00753 0.0100 0.00750

ANNOVA for Table 16

Source of df Mean Sum of Squares

Variance Protein CMCase FPase β- Total Protein CMcase FPase β- Total
glucosidase enzyme conc glucosidase enzyme
activity activity
Temperature 5 165.80 14.196 24.759 83.831 304.67 44.888 34.037 32.239 140.80 508.53
Error 12 0.5261 0.019 0.9002 0.338 0.1733 41.107 0.00835 0.00833 0.00505 0.1733

ANNOVA for Table 17

Source of df Mean Sum of Squares

Variance Protein CMCase FPase β- Total Protein CMcase FPase β- Total
glucosidase enzyme conc glucosidase enzyme
activity activity
Incubation 6 63.845 16.525 11.026 12.961 98.185 177.17 19.294 23.357 32.835 177.79
time
Error 14 0.2933 0.2917 0.2922 0.0505 0.432 0.001 0.00576 0.00576 0.1571 0.291
 CURRICULUM VITAE

Name : Shruti Pathania

Father’s name : Sh. Dilbag Singh Pathania

Date of birth : 15 Aug 1989

Sex : Female

Marital status : Unmarried

Nationality : Indian

Educational qualifications

Certificate/degree Class/grade Board/University Year

Matric First C.B.S.E 2004
10+2 First C.B.S.E 2006
B.Sc. First G.N.D.U 2009

Whether sponsored by some state/ : Nil

Central Govt./Univ./SAARC

Scholarship/Stipend/Fellowship, any : Nil

Other financial assistance received
During the study period

( Shruti Pathania )