Wolkite University

College Of Natural and Computational Science

Departiment of Biotechnology
Proposal Title On: Isolation and Screening of Potential Cellulose
Degrading Bacteria from Food Wastes
Proposal Prepared By:
1. Daniel Mulu……………...Ncsr/176/07
2. Debebe Landina……....…Ncsr/181/07
3. Lidia Ashenafi….….….…Ncsr/408/07
4. Mustefa Sema………...…. Ncsr/494/07
5. Rehima Kedir…...………. Ncsr/520/07
6. Salyate Dawud…………... Ncsr/529/07
7. Tasfaye Debelo………....... Ncsr/594/07
8. Tigist Nigatu……………... Ncsr/630/07
Advisor: Mr. Getaw Abera (Msc.)
Proposal Submitted To: Department Of Biotechnology for Partial Fulfillment
of the Requirement for the Degree of Bachelor Science in Biotechnology

Wolkite, Ethiopia

April, 2018
Contents…………………………………………………………………………………………...Page
Abbreviations..............................................................................................................................................ii
1. INTRODUCTION.............................................................................................................................1
1.1. BACKGROUND OF THE STUDY..........................................................................................1
1.2. The statement of the problem...................................................................................................2
1.3. Objective of the study................................................................................................................2
1.3.1. General Objective..............................................................................................................2
1.3.2. Specific Objective...............................................................................................................2
1.4. Significance of the study............................................................................................................3
2. LITERATURE REVIEW.................................................................................................................3
2.1.1. Cellulolytic Fungi...............................................................................................................3
2.1.2. Cellulolytic Bacteria...........................................................................................................4
2.2. Cellulolytic Enzyme...................................................................................................................4
3. MATERIALS AND METHODS......................................................................................................8
3.1. Description of the Study Area...................................................................................................8
3.4.2. Screening of Cellulose Degrading Bacterial strains............................................................10
3.5. Characterization of Cellulolytic Bacteria...................................................................................10
3.5.1. Morphological Characterization...........................................................................................10
3.5.2. Biochemical Characterization of Isolates.............................................................................10
3.6.Heavy metal tolerance assay.........................................................................................................11
3.7.1. Antibacterial Activity of Cellulolytic Bacteria.....................................................................12
3.7.2.Antifungal Activity of Cellulolytic Bacteria..........................................................................12
3.9. Statistical analysis.........................................................................................................................13
4. EXPECTED OUTPUTS..................................................................................................................13
5. BENEFICIARIES:..........................................................................................................................13
6. WORK PLAN..................................................................................................................................14
Table: 1......................................................................................................................................................14
Table: 2......................................................................................................................................................15
8. REFERENCE..................................................................................................................................16

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Abbreviations
 ANOVA: Analysis of Variance
 CBD: Cellulose Binding Domain
 CBH: Cellobiohydrolase
 CBM: Cellulose Binding Module
 CI: Cellulolytic Index
 CMC: Carboxyl Methyl Cellulose
 PDA: Potato Dextrose Agar
 SPSS: Statistical Package for the Social Sciences

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1. INTRODUCTION
1.1. BACKGROUND OF THE STUDY

Food waste is an unwanted part of raw or cooked food discarded during or after food preparation
to the environment. It isno longer fit for consumption and forms a significant part of domestic
waste. Large variety of microorganisms can exist in food waste such as bacteria, fungi, protozoa
and viruses (Kaur and Arora, 2012). Cellulose is commonly found in plant cell walls and
abundantly exists on the earth (Ram et al., 2014). This cellulosic biomass is a renewable and
abundant resource with great potential for bioconversion to value-added bio-products (Sadhu and
Kanti, 2013). Wastes from agricultural and industrial sources accumulated in the environment
present a huge reservoir of cellulose.Cellulose has crystalline structure made from long polymers
of β 1-4, linked glucose units (Ram et al., 2014). Those microorganisms are used this waste as
their source of food and utilize it by breaking it into simple molecules. Food wastes contain
different macromolecules and microorganisms secret extracellular enzymes to degrade those
molecules down and make it easily usable. Microorganisms able to hydrolyze cellulose molecule
are called cellulolytic microorganisms.

Among many different microorganisms existing bacteria are the major leading cellulose
degraders. Bacteria have high growth rate compared to fungi and have good potential to be used
in industries. Bacteria are present everywhere in diverse ecological habitats. They are considered
highly valuable as they are used in fermentation processes, much as brewing, baking, cheese and
butter manufacturing, chemical manufacturing as well as other diverse biological activities (Saha
et al., 2014).

Studies have been done for long years up on isolation and characterization of cellulose degrading
bacteria from different varieties of sources such as soil, municipal wastes, feces of ruminants,
composts and from different ecological niches (Wilson, 2011). That cellulose degrading bacteria
secret extracellular enzyme called callulase. Some papers tell that cellulases production by
various bacteria belonging to the genera of Bacillus,Cytophaga,Cellvibrio,
Cellvibrio,Cellulomonas, Pseudomonas and Micrococcus(Anita et al., 2017).Those bacteria have
an important role in the biosphere to reduce complex polymer cellulose into valuable products
such as monomer sugar, microbial biomass proteins, compost, antibiotics and other material to
everyday use for man (Kaurand Arora, 2012).

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For successful bioconversion of cellulosic materials, theimportant parameters are the nature of
cellulose and optimal conditions for catalytic activity and production of enzymes. Cellulose
quality, temperature, aeration, carbon sources, incubation period, medium additives, pH of the
medium and presence of inducers are important factors to be considered for the optimized
production of cellulase enzymes. The purpose of this work will be basically to examine the
possible utilization of cellulose degrading bacteria in food waste from cafeteria for highest
cellulase activity and bacterial growth at optimum working conditions such as pH and
temperature.

This activity will be achieved through different steps; isolation of cellulose bacterial strains from
food wastes of cafeteria; selection of the most potent isolate producing cellulase activity and
optimization of working conditions affecting glucose biosynthesis by the preselected
isolate.Therefore, this research paper or study mainly will have a focus on cellulose degrading
bacteria in food waste.

1.2. The statement of the problem

Most foods are mainly from plant or vegetable sources such as fruits, seeds, roots and from
different parts of them. Those vegetables consist of cellulose which composed of lignin,
hemicelluloses and lignocelluloses. Therefore, there waste disposal to the environment cause a
serious problem of waste treatment and pollution control since they are not easily biodegradable.
Microorganisms have capacity to hydrolyze cellulose substance in to simple molecules of
biodegradable and convert it to other valuable products are important in conversion by using
their extracellular enzyme.Therefore, we are initiated to find out and isolate potential
microorganism particularly bacteria from food wastes to use it for waste treatment process and
other various industrial process using cellulose containing raw material.

1.3. Objective of the study

1.3.1. General Objective

The general objective of this study is isolation and screening of potential cellulose degrading
bacteria from food waste samples

1.3.2. Specific Objective

 To isolate cellulose degrading bacteria.
 To determine cellulose degrading activity of the bacteria
 To determine growth ability of bacteria on the different range of temperature and pH
 To examine the growth of cellulose degrading bacteria on heavy metals
 To determine their antimicrobial activities and their potential.

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1.4. Significance of the study

The significance of this study will be isolation and identification of cellulose degrading bacteria
from food wastes. This study will have a remarkable role in industrial process utilizing
microorganisms having cellulosic activity. In starch industry and leather industry cellulose is
highly needed. To satisfy the demand of this enzyme this study contributes its side by isolating
effectivecellulose producing bacteria.Cellulose degrading bacteria also important in breakdown
of complex cellulotic compounds to simple sugars in wastes for easy treatment. Therefore, the
study will have great role in waste degrading and control of contamination.

2. LITERATURE REVIEW

2.1. Cellulose Degrading Microorganisms

Cellulolytic microorganisms are primarily carbohydrate degraders and are unable to use proteins
or lipids as energy source for growth. But, anaerobic cellulolytic species have a restricted
carbohydrate range, limited to cellulose and/or its hydrolytic products (Sukumaran et al., 2005).
The best studied cellulolytic environment is the rumen where plant cell walls degrading process
done in animals’ intestine by a very dense andcomplex mixture of anaerobic
microorganisms.Some cellulose degrading microorganisms found in animal stomach like
protozoa, fungi and bacteria arethe major cellulose degraders (Wilson et al., 2011).Cellulolytic
microorganisms have evolved two strategies for utilizing their cellulases: discrete noncomplexed
cellulases and complexed cellulases (Sukumaran et al., 2005).

2.1.1. Cellulolytic Fungi

Fungi are eukaryotic organism and most of the individuals are saprophytic which are the main
producers of extracellular cellulase and are efficient in degradation of major polymers such as
cellulose and lignin. Most commonly studied fungal include speciessuch as genus Trichoderma,
Aspergillus, Humicola, Penicillium and others (Sari et al., 2017). Most of the cellulolytic fungi
belong to Ascomycota and Basidiomycota phylum. Several fungi can metabolize cellulose as an
energy source, only few strains are capable of secreting a complex of cellulase enzymes, which
could have practical application in the enzymatic hydrolysis of cellulose.

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 In addition to T. reesei, other fungi like Humicola, Penicillium and Aspergillus have the ability
to yield high levels of extracellular cellulases (Sukumaran et al., 2005). Filamentous fungi are
used in many industrial processes for the production of enzymes and metabolites. Some of
advantages employing fungi for enzyme production are low-cost material with high productivity,
faster production, and amenable modified enzymes. Furthermore, the extracellular enzymes
which is normally secreted outside cells can be easily recoverable from the culture media
(Vishwanatha et al. 2010; de Souza et al. 2015).

2.1.2. Cellulolytic Bacteria

The cellulolytic bacteria have been isolated by various researchers from different samples .
Rastogi et al., 2009, have isolated cellulose-degrading bacteria belonging to the genera of
Brevibacillus, Paenibacillus, Bacillus and Geobacillus. Gupta et al., 2012, isolated eight
cellulose hydrolyzing bacteria from invertebrates showing hydrolytic capacity in the range of 4.0
to 9.0 and found that isolates produced CMCase in the range of 0.162 to 0.400 IU/ml.
Khianngam et al., 2014, isolated cellulolytic bacteria belonging to the genera ofBacillus,
Paenibacillus and Lysinibacillus from oil palmmeal samples. Bacteria are interesting microbial
group for the faster production of cellulase because of their high growth rate and easy handling and
adaptability to various genetic manipulations (Saha et al., 2014).

2.2. Cellulolytic Enzyme

Cellulases are a group of enzymes which play an important role in the hydrolysis process of β-1,
4-glycosidic linkage in cellulose. The complex of cellulolytic enzymes consists of endo-cleaving
(endoglucanases), exocleaving (cellobiohydrolases) and β-glucosidases. A complete hydrolysis
of crystalline cellulose involves synergistic actions of these cellulolytic enzymes (Lynd et al.,
2002; Sari et al., 2016). Cellulases are the third most significant commercial enzyme in the world
market. Cellulases, solely or in a mixture with other enzymes, are involved in several industries
including biofuel, food, feed, beverages, paper, textile, pharmaceutical, agricultural etc. (Kuhad
et al. 2011).

Recently, researches on cellulolytic enzymes have been done intensively due to their important
role as the lignocellulosic material source in the process of bioethanol production (Okeke et al.,
2015). Cellulase plays important role in the process of fermentable sugars production from
cellulose, which is the primary polysaccharides in lignocellulosics. Cellulase production is the
key phase of the enzymatic cellulose hydrolysis process.
Isolation and characterization of cellulolytic microbes provide a good starting point for the
discovery of such beneficial enzymes. Therefore, much research is aimed to obtain new
microorganisms producing cellulase with higher specific activities (Rathnan et al., 2012).

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2.3.Cellulase Enzyme Cellulolytic Mechanisms

Microorganisms produced extracellular cellulases that are either free or cell associated to
hydrolyze and metabolize insoluble cellulose. Those have different catalytic mechanisms based
on glycosidic bonds.Glycoside hydrolases cleave glucosidic bonds by using acid–base catalysis.
The hydrolysis is performed by twocatalytic residues of the enzyme: a general acid (proton
donor) and a nucleophile/base (Zhang and Percival, 2013). Depending on the spatial position of
these catalytic residues, hydrolysis occurs via retention or inversion of the anomeric
configuration. The complex of cellulolytic enzymes consists three main hydrolyzing mechanisms
or activities like endo-cleaving (endoglucanases), exo-cleaving (cellobiohydrolases), and β-
glucosidases.

2.3.1. Endoglucanases (Endo-1, 4-β-D-Glucan Glucanohydrolases)

Endoglucanases randomly cut β-1,4-bonds of cellulose chains at internal amorphous sites and
generate new oligosaccharides chains of different length and new ends. (Sadhu and Kanti,
2013).Differentendoglucanases are produced by archaea, bacteria,fungi, plants, andanimals with
different catalyticmodules. Fungal endoglucanases in general possess a catalyticmodule with or
without a cellulose binding module (CBM), while bacterial endoglucanases may possess multiple
catalytic modules,cellulose binding modules, and other modules with unknown function(Zhang
and Percival, 2013).

2.3.2. Exoglucanase (1, 4-β-D-Glucan Cellobiohydrolases)

Exoglucanases act in a possessive manner on the reducing or non-reducing ends of cellulose

polysaccharide chains and release either glucose or cellobiose as major products. These enzymes
are active against crystalline substrate such as Avicel, amorphous celluloses and the one called
acellooligosaccharides. However, they are inactive against cellobiose or substituted soluble
celluloses such as CMC (Sadhu and Kanti, 2013).

2.3.3. β -Glucosidases (β-D-Glucoside Glucohydrolases)

β-Glucosidases that do not contain a cellulose binding module hydrolyze soluble cellodextrins
and cellobiose to glucose. The activity of β-glucosidases on insoluble cellulose is negligible and
inactive against crystalline or amorphous cellulose. It degrades cellobiose, which is a known
inhibitor of CBH and endoglucanase (Sadhu and Kanti, 2013).

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2.4. Cellulolytic mechanism of cellulose in bacterial system

Researchers focused on mode of cellulase action in bacterial system based on four structures
believed to be important in adhesion to cellulose (Sadhu and Kanti, 2013) including large
multicomponent complexes cellulosomes, fimbriae or piliadhesions, Carbohydrate epitopes of
bacterial glycocalyx layer and enzymebinding domains.

2.4.1. Adhesion via Celluloses like Complexes

Cellulosomes are large, stable, multi-enzyme complexes specialized in the adhesion to

anddegradation of cellulose that reside with protuberances visible on the cell surface.
Thecellulosome complex is composed of a central catalytic subunit termed scaffoldinwhich
contains a cellulose binding domain (CBD) and a number of attachment sites calledcohesins,
which serve to bind the enzymatic submits.

2.4.2. Adhesion via Fimbriae or Pili

Fimbriae or pili, which have been implicated in bacterial adhesion which are surfaceappendages.
It is found in gram-negativebacteria. Structural subunits of fimbriae are responsible for the
adhesions. InRuminococcus albus, a novel forms of cellulose-binding protein have
beenrecognized that belongs to the pil protein and most similar to the fimbrial proteins ofgram-
negative, pathogenic bacteria (Sadhu and Kanti, 2013).

2.4.3. Adhesion via Carbohydrates Epitopes of Bacterial Glycocalyx

Most of the evidence about adhesion via carbohydrate epitopes has been found fromelectron
microscopy observation (Zhou et al., 2001). Ifglycocalyx carbohydrate was removed by
periodate oxidation with the protease anddextranase treatment, the adhesionto cellulosehas been
decreased significantly.

2.4.4. Adhesion via Cellulose- Binding Domains of Cellulolytic Enzymes

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It has been revealed that two functional domains are found in cellulase structure viz. theactive
catalytic domain responsible for the hydrolytic cleavage of the glycosidic bonds andthe binding
domain that binds the bacterial enzymes to its substrate (Zhang and Percival, 2013). The
cellulose bindingdomain (CBD) is linked to the catalytic core by linkers rich in hydroxyl amino
acids. Because of the conserved aromatic residues, it was thought that CBD attached tocellulose
either by hydrogen bonding or hydrophobic interaction.

2.5. Applicationof Cellulases in VariousIndustries

Microbial cellulases find applications in various industries. Cellulases were initially investigated
several decades back for the bioconversion of biomass which gave way to research in the
industrial applications of the enzyme in animal feed, textiles and detergents and in the paper
industry. With the shortage of fossil fuels and the arising need to find alternative source for
renewable energy and fuels, there is a renewal of interest in the bioconversion of lignocellulosic
biomass using cellulases and other enzymes (Sukumaran et al., 2005).

2.5.1. Pulp and Paper Industry

In the pulp and paper industries cellulase and hemicellulose have been used for biochemical
pulping processes. Interestingly, cellulases employed inbio-modification of fiber, hand sheet
strength properties, de-inking of recycled fibers and for improving drainage and run ability of
paper mills (Ramesh et al., 2011).In contrast to chemical processing,
biomechanicalpulpingusingcellulases resulted in substantial energy savings (20–40%) during
refining and improvements in hand-sheet strength properties. (Rajeev et al., 2005).
Endoglucanases have the ability to decrease the pulp viscosity with a lower degree of hydrolysis
and cellulases have also been reported to enhance the bleaching ability of softwood Kraft pulp
producing a final brightness score comparable to that of xylanases treatment (Ramesh et al.,
2011).

2.5.2. Textile Industry

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Cellulases are the most successful enzymes used in textile wet processing, especially finishing of
cellulose-based textiles. The major application of cellulases in the textile industry are for bio-
polishing of fabrics and producing stonewashed look of denims, as well as for improving fabric
softness and brightness (Sadhu and Kanti, 2013).Traditional stonewashing of jeans involves
amylase-mediated removal of starch coating and treatment of jeans with pumice stone in large
washing machines. Cellulases have been successfully used for the bio-stoning of jeans and bio-
polishing of cotton and other cellulosic fabrics. During the bio-stoning process, cellulases act on
the cotton fabric and break off the small fiber ends on the yarn surface, thereby loosening the
dye, which is easily removed by mechanical abrasion in the wash cycle. The advantages in the
replacement of pumice stones by a cellulose-based treatment include less damage of fibers,
increased productivity of the machines, and less work-intensive and environment pollution
reduction (Ramesh et al., 2011).

2.5.4. Food Processing Industry

Cellulases have a wide range of potential applications in food biotechnology as well. The
production of fruit and vegetable juices requires improved methods for extraction, clarification,
and stabilization. Cellulases also have an important application as a part of macerating enzymes
complex (cellulases, xylanases, and pectinases) used for extraction and clarification of fruit and
vegetable juices to increase the yield of juices (Ramesh et al., 2011).The macerating enzymes are
used to improve cloud stability and texture and decrease viscosity of the nectars and purees from
tropical fruits such as mango, peach, papaya, plum, apricot, and pear. Enzyme mixtures
containing pectinases, cellulases, and hemicellulases are also used for improved extraction of
olive oil (de Carvalho et al., 2008).

2.5.5. Biofuel

The most important application currently being investigated actively is in the utilization of
lignocellulosic biomass for the production of biofuel. They are the most abundant and renewable
resource available for mankind but their use is limited only due to lack of effective technologies.
The potential application of cellulase is the conversion of cellulosic materials to glucose and
other fermentable sugars, which in turn can be used as microbial substances for the production of
a variety of fermentation products like ethanol (Rajeev et al., 2005).

2.5.6. Waste Management

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The wastes generated from forests, agricultural fields, and agro industries contain a large amount
of unutilized or underutilized cellulose, causing environmental pollution. Nowadays, removal of
those wastes by bioremediation technique is the major focus of environmentalists and other
organization. This uses the involvement of enzymes dominantly cellulase (Gupta et al., 2011).

3. MATERIALS AND METHODS

3.1. Description of the Study Area

The study will be conducted from March, 2018 to June, 2018 in Wolkite University,
Biotechnology laboratory at the Department of Biotechnology in Wolkite, Gurage zone in SNNP
of Ethiopia. Wolkite town is situated at distance of 337km from Hawassa (capital city of SNNP
regional state) and 158km away from south west of Addis Ababa. The geographical location of
the town is approximately 8˚ 33̍ N latitude and 37˚ 59̍ longitude E.The average elevation of the
town is about 1870m above sea level.

3.2.Sample Collection
Samples will be collected directly from three places including student cafeteria, lounges and
from a place it dumped from the environment. Pre-sterilized screw cap glass vials or bottles will
be used for the sample collection purpose. Randomly selected one cm 2 blocks of waste patch
were scrapped with scapula and collected in separate vials. Each sample will be kept in clean
sterile sample bottles sealed and transferred to the biotechnology laboratory for isolation of
cellulose degrading bacteria and stored at 40C.

3.3. Experimental Design

3.3.1. Nutrient Agar Medium
Nutrient agar medium will be used for growth of bacterial culture from the samples (Kaur and
Arora, 2012). It has main composition of 0.5 % Peptone; 0.3 % beef extract/yeast extract; 1.5 %
agar; 0.5% Sodium chloride in distilled water. The medium will be prepared by dissolving 2gm
of nutrient agar powder in round bottom flask on hot plate. Then it will be sterilized in autoclave
for 15min at 121 0C and after carefully takeout from autoclave. After that, it will be poured to pre
sterile Petri dish plates under sterile condition. Finally, it will allowto cool and solidified.

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3.3.2. EnrichmentMedium

An enrichment technique will be used for the isolation of cellulose degrading bacteria as of
(Saini et al.,2017). Enrichment of cellulolytic bacteria will be achieved in mineral salt solution of
(in g/l): NaCl,6.0;(NH4)2SO4,1.0; KH2PO4,0.5; K2HPO4,0.5; MgSO4,0.1; CaCl2,0.1and 0.1%
CMC (Sainiet al., 2017).The media will be prepared and used for screening of bacterial isolates
which able to utilize cellulose.

3.4. Isolation and screening cellulose degrading bacteria strains

3.4.1. Isolation of Bacterial strains

The food waste samples will be suspended in water by vigorous vortexing and serial dilutions
will be made up to 10-6 using sterile distilled water in test tubes. 0.1 ml of appropriate dilution
will be spread growth media already prepared before plate at pH 6.8 and incubated at 37 0C for 48
hours for bacteria culture growth (Kaur and Arora, 2012). Streaking plate method will be used for
obtaining pure bacterial strain. The purified colonies will be preservedat 4 oC for further
identification and screening. After, the one with cellulose degrading ability will be determined
using Enrichment technique.

3.4.2. Screening of Cellulose Degrading Bacterial strains

Pure cultures of bacterial isolates obtained will be individually transferred (Vinotha and
Maheswari, 2014)and will be grown on the minimalagar medium supplemented with 1% CMC
(carboxymethylcellulose) of adjusted pH 7.0 at 30 0C for 4-5 days.The plates will beflooded with
0.1% Congo red dye solution for 15 minutesand will be distained with 1M NaCl solution (Kaur
and Arora, 2012; Sari et al., 2017). The appearance of azone of hydrolysis or clear zone around
the colonies indicates synthesis ofextracellular cellulases by the microbes (Subodh et al., 2012).
The cellulolyticpotential of the positive isolates will be evaluated by cellulolytic index (CI), that
is ratio of diameter of zone ofhydrolysis to the diameter of colony.

3.5. Characterization of Cellulolytic Bacteria

Identification of cellulolytic bacteria will be carried out based on their morphological and
biochemical characterization tests (Lokhande and Musaddiq, 2015).

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3.5.1. Morphological Characterization
Morphological of potential isolate will be characterized by colony shape, size, elevation, color,
margin and configuration by observation under microscope (Vimal et al., 2016). Bacterial
isolates will be stained by methylene blue on the glass slide and will be observed under light
microscope for morphology identification.

Gram staining: Gram stain will be performed to observe the cellular morphology and gram
nature of the bacteria based its absorption of dye (Saha and Santra, 2014). This will be carried
out by using standard techniques with a step-wise application of Crystal Violet Solution, iodine
solution, ethanol (95%) and Safranin solution as described in(Das et al.,2010).

Iodine staining:Potential isolates with cellulase production were identified by Grams iodine dye
staining method as method used by (Vimal et al.,2016).0.133 grams of Potassium iodide and
0.067 grams of Iodine will be dissolved in 20 ml distilled water and it will be prepared for iodine
staining of bacterial isolates.

Motility test: Bacterial motility will be observed directly by microscope. A drop of bacterial
suspension will be placed in to glass slide and put cover slip in the center (Ivanen et al.,2009).

3.5.2. Biochemical Characterization of Isolates

Potential isolates will be characterized by different biochemical methods catalase test, citrate
utilization test, starch hydrolysis, Urease and methyl blue (Das et al., 2010; Morris et al., 2008).

Catalase test: Catalase test will be carried out by using 3% hydrogen peroxide. The cells from a
culture will be mixed with a drop of hydrogen peroxide on a clean slide using the inoculating
loop. The presence and absence of bubbles will be recorded as positive and negative
respectively, (Das et al., 2010).

Starch hydrolysis: This test will be carried out by dividing starch agar plate into two equal
sectors using a marker. After labeling the organism’s name, the test organisms will be spot
inoculated and incubated for 24hours(Das et al., 2010).Zone of hydrolysis of starch will be
detected as a brownish clear zone in a blue black background after flooding the starch agar plate
with iodine solution. The presence of zone of hydrolysis on the plate indicated the ability of the
test organism to metabolize starch.

Urease taste: Urease test will be carried out by preparing urea agar containing phenol red as pH
indicator. After inoculating the agar with the test isolate and incubating the culture for 24 h, color

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change of the agar from red to pink will be observed and recorded as a positive result for urease
test (Das et al., 2010; Zaved et al., 2008).

3.6.Heavy metal tolerance assay

Heavy metals such as Arsenic, Zinc, Mercury and others will be used for determination of the
growth of the bacterial strains. The sterilized agar medium will be prepared and poured on plates
for bacterial growth. The plates will be inoculated with bacterial suspension through spread plate
method. Various concentrations of each metal compounds will be added on the media. For each
metal concentration separate Petri plate with pre-inoculated bacteria will be used (Saha and
Santra, 2014). All the plates will be incubated at 37±2 °C for 48 hours. The zone of inhibition
each colony will be measured against each concentration of the heavy metal. Plates without any
metal concentration will be treated as a control plates.

.7 . Detection of Antimicrobial Activity

Antimicrobial activity of these cellulolytic isolates will be determined by well diffusion method
against different bacterial and fungal human pathogens This is to examine to observe their ability
to work against other microbial or their competition with other microorganisms. For this work all
the isolates will be screened for antibacterialand antifungal activity by cross streak method. In
the crossstreak method, the new isolates will be streaked on nutrient agar by one side of petri
plate and the plates will be incubated overnight at either roomtemperature or 37 °C.

After incubation, the test human bacterialpathogens and fungal pathogens will be streaked on the
other side corner of the bacterial isolates (Saha and Santra, 2014). The zone of inhibitionagainst
human bacterial pathogens and fungal pathogens wereobserved after 48 hour of incubation.
Plates with the same mediumwithout inoculation of bacterial isolates but with
simultaneousstreaking of test organisms were maintained for controls.

3.7.1. Antibacterial Activity of Cellulolytic Bacteria

Antimicrobial activity of these isolates will be determined against different known laboratory
available bacterial human pathogens such as Staphylococcus aureus, Escherichia coli,

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Pseudomonas aeruginosa, Enterococcus and others on fresh Nutrient agar media at 37°C against
a positive control (Streptomycin 1mg/mL). Wells of 0.5 cm will be made with the help of a
sterile borer on the media and 100µl of the supernatant and streptomycin will be poured to the
respective wells while for negative control sterile distilled water will be used (Ahmadet al.,
2013). Then zones of inhibition for the supernatant and positive control will be measured.

3.7.2.Antifungal Activity of Cellulolytic Bacteria

The antifungal activity of these isolates will be determined against pathogenic fungi such as
Candida albicans and Aspergillus niger on fresh Potato Dextrose Agar (PDA) plates and they
will be incubated at 25-27 °C for 5 days. A sterile cotton swab will be used to uniformly spread
the fungal culture on PDA plates. The plates will be allowed to dry for 15 minutes. Wells of 0.5
cm will be made with the help of a sterile borer on the media and 100 µl of the supernatant and
Fluconazole (Concentration 1mg/ml) will be poured to the respective wells while for negative
control sterile distilled water will used (Ahmadet al., 2013). Then zones of inhibition for the
supernatant and positive control will be measured.

3.8. Determination of optimal pH andTemperature

Pre-selected isolate will be dispensed in test tubes containing mineral salt broth medium with
0.1% cellulose and adjusted from pH 5 to 8. To determine their growth potential in different
range of pH level. It will be incubated at 37°C for 96 h. Following the incubation, growth of the
cultures will be measured by observation of the optical density at 560 nm(Kaur and Arora,
2012). Same procedure will be followed for determining bacterial growth at varying temperature.
Tubes will be incubated at different temperatures ranging from 30 to 65 0C for 96 hoursand high
temperature resistant isolates will be identified.

3.9. Statistical analysis

One-wayanalysis of variance (ANOVA) will be done using Statistical Package for the Social
Sciences (SPSS) for the determination of significant differences within different conditions.
Three replicates will be determined for each condition. A significant difference will be found
when p< 0.05.

4. EXPECTED OUTPUTS

By this research expected results and its main scope will be isolation of bacterial strains having
high cellulolytic activity from the samples and which are having potential to grow in high
temperature, pHand resist to heavy metals. Those isolates will be used for different purposes.

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5. BENEFICIARIES:

At the end of this research work many sectors of organizations will be benefited. Among those
many industries such as pulp and paper industry, leather industry, textile industry, wine and
brewery industry, food processing industry and waste management are the major beneficiaries of
the output. This is since microorganisms and enzymes have greater role in any biological
processes in industries for safe and good quality product production.

6. WORK PLAN
The total estimated duration time for this research will be three months including the
fromproposal preparation to final report submission date.

Table: 1

Activities wil be done Months (March-June/ 05/03/2018-05/06/2018)

March A p r i l M a y J u n e

Title selection
Literature review reading 1 5 - 3 0
Preparation of proposal 2 0 - 3 0
Proposal presentation 2 9 - 3 0
Sample collection 02-04
Laboratory work 05-17
Media preparation 05-06
Sample inoculation 0 6
Colony isolation identification 07-17
Report writing 05-20 20-30
Submis ion of inal report and presentation 01-01

7. WORK BUDGET

Table: 2.

Activities Cost requirement in BIRR

Sample collection 8 0 0
Chemicals purchase 2 0 0 0
Transportation 2 0 0

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 Materials (equipment) 1 5 0 0
Paper and pen 1 0 0
Personal labor 1 5 0 0
For communication 5 0 0
Report writing and printing 3 0 0
Data connection 1 0 0
Contingency 6 0 0
Total cost 7 6 0 0

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