CHAPTER ONE

1.0 INTRODUCTION

Lignin is the most structurally complex carbohydrate possessing a high molecular

weight and the most recalcitrant, consisting of various biologically stable linkages
(Perez et al., 2001). The lignocellulose material of plant consists of three main
compounds, namely cellulose, hemicellulose and Lignin. After cellulose, Lignin is
the second most abundant renewable biopolymer in nature. It is most abundant
aromatic polymer in the biosphere. Lignin causes a serious pollution and toxicity
problem in aquatic ecosystem owing to its low biodegradability. Large amounts of
lignocellulosic waste generated through forestry and agricultural practices, paper-
pulp industries, timber industries and many agro-industries pose environmental
pollution problems (Howard et al., 2003). In soil biosphere, a few groups of
organisms are capable of degrading complex Lignin polymers and best examples
are white rot fungi and other fungi such as Phanerochaete chrysoporium,
Streptomyces viridosporus, Pleurotus eryngii, Trametes trogii, Fusarium
proliferatem, Agaricus, Erwenia, Copricus, Mycema and Swterium. However,
commercialization of Lignin degradation by fungi has disadvantages in the form of
problems related to fungal protein expression and genetic manipulations and
showed a lack of stability under practical treatment condition involving high pH,
oxygen limitation and high Lignin concentrations (Crawford and Muralidhara,
2004). For this reason, studies on the bacterial degradation will be more preferable
for Lignin and the production of bacterial Ligninolytic enzymes has seen increased
in recent year. Lignin degrading enzymes are essentially extracellular in nature due
to the large and complex structure of Lignin which cannot enter the cell for
intracellular action (Aarti et al., 2015).

1
 Lignin peroxidase (LiP) is an enzyme first discovered in 1983 was used to
degrade Lignin. Lignin peroxidase is useful in the treatment of colored industrial
effluents and other xenobiotic as it has bioremediation potential to decolorize the
effluents. Decolorization of methylene blue dye was also used as an indicator of
the oxidation ability of Ligninolytic enzyme produced by the potential Lignin
degrading bacterial strains. Lignocellulytic enzymes also have significant potential
application in various industries including chemicals, fuel, food, brewery and wine,
animal feed, textile and laundry, pulp and paper, and agriculture (Howard et al.,
2003). In this study, an investigation was attempted to prepare alkali Lignin
substrate and isolation, degradation and characterization of Lignin degrading
bacteria will be made feasible (Aarti et al., 2015).

Plant biomass is the most abundant renewable biomass on earth and is considered
as an attractive source of bio energy and bio based chemicals. It is mainly
composed of Lignin, cellulose and hemicellulose. The Lignin percentage in
lignocellulosic biomass is around 10–30% and is the second most abundant natural
organic poly-mer. Lignin enables plants to generate rigid structures and provides
protection against hydrolysis of cellulose and hemicellulose. The biotechnological
conversion of lignocellulose into different carbohydrates, including glucose, is the
basis for the production of ethanol, carbohydrates and aromatic products. Such
plant biomass derived products can be used as fuel, polymer precursors, food and
flavor compounds, and pharmaceutical building blocks. For optimizing the use of
plant biomass through biorefining, Lignin degradation has become a key target in
the last few years. Efficient and cost-effective methods for selective Lignin
degradation are in high demand. It is worth noting that, while the recent intensified
efforts in complete valorization of plant biomass, Lignin was already considered as

2
a major industrial by-product in the first half of the previous century (Acebes et al.,
2017).

While cellulose and hemicellulose are built from carbohydrates, Lignin is a highly
cross-linked polymer formed by polymerization of 4-hydroxyphenylpropanoid
monomers (monolignols) through various ether and carbon–carbon bonds. The
phenolic moieties of the monomeric units are p-hydroxyphenyl (H), guaiacyl (G)
and syringyl (S) groups and the percentage of each depends on the plant species
and tissue. The formation of Lignin is triggered by plant per-oxidases and/or
laccases. By oxidizing the phenolic monolignols into their respective phenolic
radical, formation of dimers is catalyzed. Subsequent enzyme-catalyzed single
electron oxidations promote polymerization. Monolignols can couple via various
bonds with a preference of coupling through the -carbon. The most occurring
linkages involve O 4, and 5 bonds (Acebes et al., 2017).

Due to its aromatic nature and highly branched polymer net-work, Lignin is rather
inert towards degradation (Abdel-Hamid et al., 2013). Yet, to complete global
carbon cycling, nature has evolved catabolic pathways since the time that plants
started to produce Lignin (Nelsen et al., 2016). White-rot fungi have developed a
rich collection of extracellular oxidative enzymes to attack and degrade Lignin.
They employ different types of heme-containing peroxidases, which include the
so-called Lignin peroxidases (LiP), manganese peroxidases (MnP), versatile
peroxidases (VP), and dye-decolorizing peroxidases (DyP) (Lambertz et al., 2016).
While some of this peroxidase is capable of attacking Lignin or Lignin fragments,
peroxidases also attack Lignin from a distance. By oxidizing mediators, small
oxidizing agents are generated that can penetrate the branched Lignin polymer to
trigger depolymerization via radical chemistry. Known mediators are Lignin
derived aromatic compounds (e.g. formation of veratryl alcohol cation radical) and

3
manganese ions. For effective peroxidase-based Lignin degradation, also various
fungal oxidases are secreted to produce the required hydrogen peroxide.
Candidates for the extra-cellular production of hydrogen peroxide are aryl alcohol
oxidases, glyoxal oxidases, and various carbohydrate oxidases. Except for
peroxidases, fungi also secrete various copper-containing oxidative laccases that
assist in Lignin degradation. Intriguingly, it seems that the same types of enzymes
used for Lignin synthesis in plants (peroxidases and laccases) are used by fungi to
recycle the aro-matic polymer. Genome sequence analysis of Ligninolytic fungi
has revealed that there is not one defined set of enzymes for Lignin degradation.
The composition of the set of oxidative enzymes being produced depends on the
fungus (Ahmad et al., 2010).

While a wealth of biochemical knowledge has been obtained on fungal degradation

of Lignin, the Ligninolytic capacity of bacteria has been less well studied. While it
appears that white-rot fungi are very well equipped for Lignin degradation,
evidence is growing that also bacteria are capable of delignification. Already in
1930 Phillips et al. reported on a thorough study on Lignin decomposition by “soil
microorganisms”, which presumable will be bacteria (Phillips et al., 1930). While
many claims of bacterial Lignin degradation have been reported since then, only in
the last few decades some bacterial enzymes involved in delignification have been
identified. With this review we aim at providing an overview of the bacterial
enzymes that have been implicated to be involved in degrading Lignin or the
oxidation of Lignin derived degradation products (Ahmad et al., 2010).

4
1.2 BACKGROUND OF THE STUDY

Cow dung (CD) or cow manure is the waste product of bovine animal species that
include domestic cattle (cows, bullock, and buffalo), yak, and water buffalo. Cow
dung is the undigested residue of plant matter which has passed through the
animal’s gut and includes water (80%), undigested residues (14.4%), and
microorganisms (5.6%). The pH of the Cow dung varies from 7.1- 7.4. The fecal
matter in Cow dung is rich in crude fiber (indigestible cellulose, hemicelluloses,
pentosans, Lignin), crude protein, and 24 types of minerals including nitrogen (N),
phosphorus (P), potassium (K), iron (Fe), sulfur (S), magnesium (Mg), calcium
(Ca), cobalt (Co), manganese (Mn), chlorine (Cl) and sloughed of intestinal
epithelium. The portion of fecal matter derived from the rumen of cattle improves
the constituents of Cow dung by enriching with bile pigments (biliverdin),
intestinal bacteria, and mucus (Alfaro et al., 2014).

Cow dung is traditionally used as organic fertilizer in Asian and African

agriculture for ages. In addition to the nutritional contributions to the soil, Cow
dung enhances resistance in plants against pests and diseases, stimulates plant
growth, along with P and S solubilization (Sharma and Singh, 2015). Cow dung
also harbors diverse groups of microorganisms that further enhance soil
biogeochemical processes. In Ayurveda (it is a system of traditional medicine that
has historical roots in the Indian Subcontinent), different processed products
obtained from cattle such as milk, curd, ghee, urine and by-product (dung) are
widely used in medicinal formulations (Sharma and Singh, 2015).

The current status of Cow dung as a bio-resource for sustainable development has
been briefly reviewed by Gupta et al. (2016). In this re-view, the various

5
applications of Cow dung and Cow dung-based microorganisms’ uses in
agriculture, aquaculture, and bioprocesses have been outlined (Cragg et al., 20155).

Mandavgane and Kulkarni (2020) reviewed the valorization of cow urine

and Cow dung in the model bio-refinery. However, several critical aspects such as
microbial diversity, bio-dynamics preparation and uses of Cow dung in agriculture,
underlying mechanisms in bioprocesses, and biotechnological applications (i.e.,
enzymes, bio-methane, and bio-hydrogen) and environmental applications are not
fully discussed in these reviews. In this context, the present review provides a
comprehensive discussion on the underlying mechanisms of Cow dung
microorganisms in agricultural, biotechnological, and environmental applications
in a sustainable circular economy context (Dashtban et al., 2012).

1.3 STATEMENT OF THE PROBLEM

Degration of Lignin from cow dung is the use of naturally-occurring

microorganisms or genetically engineered microorganisms (bacteria and fungi) by
man, to detoxify man made pollutants. Since bioremediation is a microbial process,
it requires the provision of nutrients among other factors or requirements.

Plant cell wall material is composed of three important constituents: cellulose

Lignin, and hemicellulose. Lignin is particularly difficult to biodegrade, and
reduces the bioavailability of the other cell wall constituents. Lignin is the well-
known complex substance covalently bound to side chains of xylans of cell-walls.
It represents an obstacle to microbial digestion of structural carbohydrates both
because it is a physical barrier and because of the depressing effect on microbial
activity, due to the phenolic compounds it contains. Mechanical and chemical
treatment of lignocelluloses in agricultural wastes and poor quality roughages has
been frequently used for improving the quality of such materials, however these

6
methods are either costly or have hazardous impact on ambient environment. In
contrast, biological treatment is a novel trend, which is not only more efficient in
improving the quality of these materials through degradation of their Lignin and
indigestible fiber contents, but also is of less cost and environmental friendly. In
addition, cow dung and other waste manure use waste materials in the production
of chemical pulp, wherein Lignin is degraded and dissolved almost completely (90-
95%) in black liquor. If not removed from the treated wastewater, the Lignin
presents a serious pollution and toxicity problem in aquatic ecosystems, owing to
its low biodegradability and high range of colour. A number of physico-chemical
methods have been developed for the treatment of Lignin from paper mill
wastewater; however, however, these processes are not very effective and costly.
However, this research will help in isolating and identifying of Lignin degrading
biological bacteria from cow dung waste.

1.4 AIMS AND OBJECTIVES

AIM

i. The major aim of this project is to Isolate and analyzed Lignin Degrading
Bacteria From Cow Dung
ii. To isolate potential thermophilic Lignin degrading bacteria from cow
dung compost and examine their Ligninolytic enzyme activities

OBJECTIVES

i. To assess the molecular and physiological characterization of potential

aerobic Ligninolytic bacterial strains, which have been previously
isolated from Sokoto Cow dung.
ii. To identify bacterial species associated with Lignin from cow dung.

7
 iii. To evaluate the anti-bacterial activities of Lignin Degrading Bacteria
From Cow Dung

1.5 SCOPE AND LIMITATION OF THE STUDY

The study is set out to determine and focus on isolation and identification of Lignin
degrading bacteria from cow dung in Sokoto State metropolis. Therefore, this
study will help in determining some selected Cow dung in the areas of Usman Dan
Fodio University Sokoto State.

1.8 SIGNIFICANCE OF THE STUDY

Although several studies have been done to produce Lignin degrading bacteria
from cow dung, this study will help in examining and identifying the process of
degrading bacterias from cow dung. In order to maximize the bacterial Lignin
degradation, and justification to use these Ligninolytic bacterial strains in industrial
and agricultural applications. This study will be useful to the society, hospitals,
clinical; it is a major carbon source and has significant concentration of simple
carbohydrates. It is believed that the findings will help in determining some
chemical constituents of the cell wall components is important to develop a good
mechanism for the conversion of these constituents into more value added products
such as low price chemicals (eg; xylitol, xylose), biofibres, biopulps and ruminant
feed. The conversion of these lignocellulosic materials into value added end
products becomes a hurdle due to lacking of biocatalysts that can overcome the
bio-refining process. The findings will be of benefit to; Specifically, to the
National Agency on Food Drugs Administration and Control (NAFDAC) on the
need to further strengthen their survey and make some research works towards
obtaining Lignin from waste products. Finally, to the general public, scholars and

8
future researchers to be fully aware and able to identify which Lignin can be
degrade from strain of bacteria from the findings of this study.

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 CHAPTER TWO

LITERATURE REVIEW

2.0 INTRODUCTION

Lignin, which is an aromatic heteropolymer that accounts for 15–30% of

lignocellulosic biomass, is the most abundant source of renewable aromatic carbon
in nature. Lignin consists of phenylpropane units combined with different types of
linkages, including -O-4, -O-4, 4-O-5, - , -5, 5-5, and -1. It is a recalcitrant
compound due to its high molecular weight, structural complexity, and relative
insolubility. It can result in many adverse environmental consequences. Waste
water containing Lignin that has been discharged from many industries (e.g., the
paper industry, rice milling businesses, and traditional Chinese medicine
businesses) has led to serious water pollution. Agricultural wastes such as straw
contain stable Lignin, which limits the further application of cellulose and
hemicellulose. These biomass resources are often burned by farmers in China,
which causes serious air pollution (SO2, NO2, CO2, and CO) in autumn and winter
and affects human health. Lignin degradation can help solve the environmental
problems caused by Lignin pollutant emissions or incineration. At the same time,
its degradation can improve the pretreatment efficiency of lignocellulose biomass,
which results in the effective separation of cellulose, hemicellulose, and Lignin and
a mild conversion of biomass to bio-energy as well as other valuable products
Currently, the main Lignin degradation technologies are chemically, physically,
and biologically based. Energy consumption during the physical processes is
normally high and the chemical processes may cause secondary pollution from
potential mismanagement of the chemicals used. On the other hand, the
biodegradation method that uses some fungi and bacteria that have evolved in

10
nature to deconstruct Lignin is environmentally friendly and has relatively lower
energy consumption (Alfaro et al., 2014).

Despite the above-mentioned advantages, the current Lignin biodegradation

approach has several drawbacks. The most active microbes with respect to Lignin
biodegradation that have been identified to date are fungi, such as those belonging
to the white-rot or brown-rot families, which have been widely reported to be
capable of decomposing wood. However, these microorganisms often lack the
capability required for industrial applications, such as fast growth rates. Bacteria
grow faster than fungi, and they have been reported to be able to produce high
value-added products from Lignin (Dashtban et al., 2012).

However, the decomposition and utilization of Lignin by bacteria is limited.

To overcome this, researchers have tried to use protoplast technology to construct
inter-kingdom fusant (the protoplasts of fungi and bacteria will be prepared, and
then the cells will be fused). They successfully prepared fusants of Enterobacter
cloacae and Psathyrella candolleana to degrade alkali Lignin. However, the
degradation efficiency of the fusants was sensitive to the environment, and their
genetic stability was also not clear. In addition to living microorganisms, many
enzymes involved in Lignin degradation have been identified, such as laccase,
Lignin peroxidase (LiP), manganese peroxidase (MnP), dye-decolorizing
peroxidase (DyP), and aryl-alcohol oxidase (AAO). Their mixtures have been used
to degrade Lignin but with relatively high costs and non-universal adaptability for
different substrates (Baldrian, 2006).

The decomposition of Lignin in nature is a result of cooperation among fungi and

bacteria in natural microbial consortia. At present, more attention has been given to
single Lignin-degrading strains rather than to microbial consortia. Some studies

11
have shown that microbial consortia have higher Lignin degradation efficiencies
than single strains and that their ability to adapt to different environments is
greater. For example, when treating different kinds of wastewater, the treatment
efficiency of a microbial consortium is much higher than that of a single
microorganism. Given the potential advantages of microbial consortia, efficient
Lignin degradation by microbial consortia could be obtained by screening water-
preserved wooden antiques from ancient graves. As China has a long history and
culture, large numbers of wooden antiques from various dynasties are unearthed
each year. The hypotheses of our study are that carbohydrates such as cellulose and
hemicellulose will be consumed by microorganisms during long-term burial and
that the remaining Lignin created a good Lignin-rich environment (>60% Lignin
content) for Lignin-degrading microorganisms that could utilize Lignin as a carbon
source. After these wood antiques will be unearthed, they will be soaked in water
for further preservation to avoid dry shrinkage and deformation. During the
process of burial and preservation after excavation, a continuous water-saturated
state is helpful for microorganism survival, while the water-preserved wooden
antiques are further decomposed. Thus, it is feasible to obtain the Lignin-degrading
microbial consortia from samples of water-saturated preserved wood antiques
(Baldrian and Valaskova, 2008).

2.1 HISTORICAL BACKGROUND OF THE STUDY

In recent years, biological method using microorganisms and enzymes has gained
extensive interest in Lignin pretreatment process due its cost effectiveness and
environmental friendliness. Moreover, biological pretreatment process does not
produce toxic compounds as much as physical and chemical process. Lignin
peroxidase (LiP), Manganese peroxidase (MnP) and Laccase (Lac) are the major
enzymes involved in Lignin degradation process. To date, white rot fungi (WRF)

12
such as Trametes versicolor, Trametes hirsuta, P. ostreatus and P. radiata are the
common Lignin degrading microorganisms that are capable of producing all the
three major enzymes. However, Ligninolytic enzymes from fungi have low
stability under high working temperature, high pH condition and high substrate
conditions. In fact, delignification process often involves high temperature up to
70˚C. Moreover, it is proven that bacterial enzymes are capable in degrading
Lignin and address the limitation of fungi (Baldrian and Valaskova, 2008).

Fig2.1 Lignin structure

Sources: (Baldrian, 2006).

2.1.1 LIGNIN PEROXIDASE

Lignin peroxidases will be first discovered in Phanerochaete chrysosporium and

later in Trametes ver-sicolor, Bjerkandera sp., and Phlebia tremellosa, which are
well known white rot fungi. Activity of LiP has previously been detected in some
bacteria, such as Acinetobacter calcoaceticus and Strep-tomyces viridosporus.
LiPs, capable of at-tacking Lignin polymers, are relatively non-specific to their

13
sub-strates. LiPs are known to oxidize different phenolic aromatic compounds and
a variety of non-phenolic Lignin model com-pounds along with many other organic
molecules. These enzymes are usually secreted by given microorganisms as a
family of isozymes whose relative composition and isoelectric points (pI) vary
depending on the growth medium and nutrient conditions. The globular structure
of LiP isolated from P. chrysosporium is composed of eight ma-jor and eight minor
α-helices with limited β components and is organized into two domains that form
an active center cav-ity composed of a heme-chelating single ferric ion. The LiP
contains two glycosylation sites, two Ca2+ binding sites and four disulfide bridges,
all stabilizing the three-dimensional structure of this enzyme. Molecular mass of
LiPs ranges from 35 to 48 kDa and it has a pI between 3.1 and 4.7. (Barcelo et al.,
2004). The high redox potential of LiPs (around 1.2 V at pH 3) makes these
enzymes capable of oxidizing substrates that are not oxi-dized by other
peroxidases. The catalytic cycle of LiP resembles the catalytic mechanism
common to all per-oxidases. In each cycle, the native enzyme is oxidized by H2O2
and generates a compound I intermediate that exists as a ferry oxo porphyrin
radical cation [Fe (IV) = O.+ ]. Next, the enzyme is subjected to two single-
electron reduction steps by the electron donor substrate, such as veratryl alcohol
(VA), leading to a tran-sient formation of compound II [Fe (IV) = O] and a VA
radical cation (VA+ ). The compound II further oxidizes the second VA molecule,
simultaneously returning to its native stage to initi-ate a new catalytic cycle of LiP.
VA, similar to Mn(III), plays the role of being a small molecular weight redox
transfer mediator between the enzyme and its polymeric substrate (Wong 2009).

2.2 BACTERIAL ENZYMES ACTING ON LIGNIN

2.2.1. DyP-type peroxidases

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As described above, white-rot fungi produce several different kinds of heme-
containing peroxidases to trigger Lignin decomposi-tion. However, homologs of
the most common fungal Ligninolytic peroxidases, LiPs MnPs and VPs, have not
been encountered in biochemical studies on Ligninolytic bacteria. Also when
analyzing sequenced genomes or proteomes of Ligninolytic bacteria, no homologs
emerge. It seems that these Lignin-degrading peroxidases, belonging to the super
family of plant peroxidase, are restricted to fungi. Yet, recently it has become clear
that bacteria are relatively rich in another type of peroxidase, the so-called dye-
decolorizing peroxidases. DyPs rep-resent a newly discovered family of heme-
containing peroxidases, which has recently received attention due their ability to
degrade Lignin and other compounds. The first discovered member of this enzyme
family, DyP from Bjerkandera adusta, was isolated and characterized in 1999 (Kim
and Shoda, 1999). Stud-ies on the activity of this enzyme on synthetic
anthraquinone and azo-dyes have served to name this family of peroxidases
(Sugano et al., 2007).

In recent years a large number of bacterial DyPs have been described in

literature which is in line with the observation that putative DyP-encoding genes
are abundantly present in bacterial genomes. In fact, already in 1988 a bacterial
‘Lignin peroxidase’ was described from Streptomyces viridosporus. Unfortunately,
no sequence has ever been deposited for this protein or the respective gene while
several papers have appeared on cloning of the respec-tive gene. Yet, when
analysing the recently sequenced genome of this Streptomyces isolate, a gene
encoding a putative Tat-secreted DyP can be identified. This may well be the
enzyme that was described long before the first fungal DyP was described (Barcelo
et al., 2004).

15
DyPs have a protomer weight of around 40–60 kDa and var-ious oligomeric states
have been observed. They belong to the peroxidase-chlorite dismutase superfamily
of proteins and contain a non-covalently bound heme b cofactor. DyPs show a
dimeric ferredoxin-like fold consisting of a four-stranded anti-parallel -sheet
surrounded by -helices. DyP-type peroxidases contain a highly conserved
GXXDG-motif and a conserved proximal histidine, which acts as the fifth ligand
of the heme iron. Yet, while DyPs are structurally unrelated to the common fungal
peroxidases, they exhibit similar catalytic properties with having similar redox
potentials and reactivates. Furthermore, some of the bacterial DyPs are secreted via
the Tat secretion machinery which adds to the analogy with the secreted fungal
peroxidases (Beeson et al., 2011).

2.3 SPECIES DECOMPOSING LIGNIN

Lignin degradation is caused by certain fungi as well as several bacterial species.

Fungi are more efficient in the breakdown of Lignin than bacteria, in which
delignification is slower and more limited (Bruckmann et al., 2002).

Microbial degradation of Lignin has not been intensively stud-ied in organisms

other than fungi, but there are reports of bacteria that can break down Lignin.
These Lignin-degrading bacteria represent mainly three classes: Actinomycetes, α-
Proteobacteria and γ-Proteobacteria. This ubiquitous group of microbes is widely
dis-tributed in natural ecosystems worldwide and occurs both in terrestrial and
aquatic habitats. Their apical and branching growth, production of aerial or
substrate mycelia, and morphogenetic development resemble filamentous fungi.
Due to the production of secondary metabolites and the use of extracellular
enzymes, these bacterial species are considered major de-composers of
lignocelluloses in soils. In the prokaryotes, one of the most frequently identified

16
and studied Ligninolytic enzymes are laccase. Phylogenic analysis has shown that
representatives of Archaea from four phyla have laccase genes. Yet the greatest
numbers of strains with laccase genes will be identified in Proteobacteria,
Actinobacteria and Firmicutes. Within Cyanobacteria and Bacteroidetes, the
number of strains with identifiable laccase genes was found to be substantially
lower. Showed that Sphingobacterium (phylum Bacteroides) produce manganese
superoxide dismutase, which is able to oxidize Lignin via hydroxyl radical
mechanism (Beeson et al., 2011).

Many strains from the Brucella, Ochrobactrum, Sphin-gobium and Sphingomonas

genera, belonging to the class α-Proteobacteria, decompose Lignin. Within another
group of Ligninolytic bacteria (γ -Proteobacteria) the best known for their
degrading abilities are Pseudomonas fluorescens (producing Lignin peroxidase
LiP), Ps. putida (dye-decolorizing peroxidase DyP (Bruckmann et al., 2002).

2.4 MICROBIAL COMPOSITION OF CATTLE MANURE

Literally, livestock practices vary from one individual to another and from one
geographical location to the other, but eventually, influence the microbial structure
of manure released by the animals. Clearly, manure provides different biological
and physicochemical environment to microorganisms. Specifically, Pachepsky et
al. noted that many manure-based pathogens exist, but the major manure based
zoonotic bacteria, including Salmonella spp., Campylobacter spp., Listeria
monocytogenes, Yersinia enterocolitica, Escherichia coli and protozoa viz.
Cryptosporidium parvum and Giardia lamblia, are present; however others are less
common. Viruses represent another group of pathogens that exist in cattle manure.
Originally, these pathogens inhabit the intestinal tracts of animals and are typically
shed in this habitat asymptomatically. Seemingly, both animals and humans on and

17
off farms are exposed to the potential health risks allied to inadequate management
of manure. Consequently, the fate of these pathogens in manure to pollute,
contaminate and infect the environment and humans, respectively, is based on the
pathogen’s ability to survive in manure following excretion. Nevertheless, the
factors affecting the survival rates of these well documented pathogens excreted in
cattle manure have been enumerated and deliberated on as we proceed through this
paper (Bianchetti et al., 2013).

The dissemination of these pathogens could occur via unplanned and uncontrolled
release by runoff either from livestock facilities or excessive land application of
manure and also through infiltration into soils and groundwater or by the release of
bio-solids and manure residuals upon transportation off farms in circumstances
where land application, marketing or other beneficial uses are appropriate. It is
worth mentioning that vectors, e.g., flies and vermin, may also spread and cause
subsequent infections to other animals with pathogens from stored manure. The
microbes presented below will be chosen based on their probability of
dissemination from cattle manure to humans as well as their endorsed potential
health threats to humans and the environment. Moreover, outbreaks caused by
these organisms have been linked tenuously to cattle in certain instances. Table 2
shows the different pathogen types, their prevalence, storage condition and
survival periods in shed cattle manure after pollution, transportation and infection,
as well as the diseases/symptoms they cause in humans (Boer et al., 2010).

18
Table 2 Pathogen type, prevalence in cattle, and temperature of storage, survival
rates and diseases/symptoms.

Pathogen Type Prevalence Survival Storage Diseases

(%) Rate Temperature
/Symptoms
(Days) (°C)
E. coli O157:H7 16 10 to 5 to 30 Mild to bloody
>100 diarrhea, vomiting,
hemolytic-uremic
syndrome, hemorrhagic
colitis
Salmonella 0 to 13 28 to 196 5 to 30 Salmonellosis
typhimurium,
Salmonella dublin
Campylobacter sp 31.1 7 to 21 5 to 30 Campylobacteriosis,
p. Guillain-Barre
syndrome, reactive
arthritis and
postinfectious irritable
bowel syndrome
Listeria 24.4 168 <20 Listeriosis, flu-like
monocytogenes Symptoms, vomiting,
diarrhea, meningitis,
septicemia,
spontaneous abortions
Yersinia <1 10 to 100 5 to 30 Yersiniosis, diarrhea,
enterocolitica lymphadenitis,

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 pneumonia, abortions
Cyptosporidium 1 to 100 28 to 56 5 to 30 Gastroenteritis and
parvum cyptosporidiosis
Giardia lamblia 10 to 100 7 5 to 30 Giardiasis (diarrhea
and abdominal cramps)
Sources: (Bianchetti et al., 2013).

2.4.1 Classification and Description of the Main Bacterial Pathogens in Cow

Dung

It is quite unrealistic to enumerate all the microbial pathogens present in cattle

manure because of the huge numbers of these pathogens that populate the
gastrointestinal tract and the other systems in the animal. Diverse groups of
microbial pathogens are involved due to the vast array of physical, chemical and
biological constituents contained in cattle manure. However, it is impossible for
every individual pathogen that constitutes the entire bacterial organization in
manure to be isolated, characterized and identified. These challenges may be
linked to the limitations in some of the available methods failing to identify some
of these pathogens owing to the state they may be exhibiting at that particular
instance. In addition, differences in the DNA extraction and purification methods
can affect DNA quality, which in due course can affect the interpretation of
information regarding the microbial communities coexisting in manure samples
(Boer et al., 2010).

However, the possibility of causing an infection depends on the type of bacteria, its
infective dose and the immune status of the individual host. Owing to the variation
in pathogenic/virulence potential across the bacterial domain, the number of cells
necessary to cause infection equally differs from one bacterium to the other. This

20
indicates that as few as 10 cells of a specific bacterium may be sufficient to cause
an infection, whereas a greater number of cells may be required by others to cause
an infection. However, small numbers of some bacteria shed in manure and
introduced into the environment might multiply under favorable conditions
resulting in higher levels, i.e., greater risk, thereby causing contamination of food,
soil and water (Canas and Laccases. 2010).

Escherichia coli is a gram-negative, facultative anaerobic, rod-shaped and motile

bacterium that exists as a normal flora in the intestinal tract of both healthy animals
and humans. It serves as a reliable indicator of fecal contamination and equally
indicates a possibility of the occurrence of enteropathogenic and/or toxigenic
microbes found in food and water, thereby presenting with public health
hazards. E. coli O157:H7 found in cattle manure has been reported as the most
notorious pathogen which produces a potent toxin that can cause serious infection
in humans. This strain can be called verocytotoxic (VTEC) or enterohemorrhagic
(EHEC) or Shigatoxin producing (STEC) E. coli. According to Gerba and Smith,
owing to the pathogen’s low infective dose ascribed to as few as 10 cells and high
pathogenicity, it is incriminated as the causative agent of gastrointestinal diseases
(gastroenteritis). In addition, Karmali highlighted that the bacterium equally causes
attaching and effacing (A/E) lesions by translocation effect or proteins into the host
cells via a type III protein secretion system (Castilho et al., 2010).

Salmonella species belong to the family of Enterobacteriaceae and are Gram

negative, short, plump shaped rods, non-spore forming, and motile, non-
capsulated, aerobic and facultative anaerobic organisms. These pathogens could be
found in a range of animals, including dogs, birds, cats, cattle, pigs and humans
and are responsible for the infection called salmonellosis. Salmonellosis occurs via
ingestion of food or water contaminated with animal feces or by direct contact with

21
animal feces and is characterized by three major symptoms viz. septicemia, acute
enteritis and chronic enteritis. However, the manifestations of salmonellosis vary
depending on the species/strain and the host type (Cazares´-Garcıa et al., 2013).

Campylobacter species are most commonly found in the intestinal tracts of

animals such as cattle, pigs, chickens, wild-living mammals and birds. They are
known to cause a wide variety of disorders in cattle, sheep, and pigs. For example,
Salihu and coworkers mentioned that Campylobacter jejuni and Campylobacter
fetus caused abortion, stillbirths and birth of weak lambs in sheep at some stage in
late pregnancy. Similarly, when transmitted to humans when consuming
undercooked agricultural food products and contaminated water, it causes an
infection called campylobacteriosis, which is a self-limited and sporadic illness. In
addition, C. jejuni and Campylobacter coli are the two most important species
associated with human bacterial gastroenteritis worldwide (Cazares´-Garcıa et al.,
2013).

Listeria monocytogenes is a Gram-positive, facultative, intracellular, rod-shaped

bacterium incriminated as the causative agent of a severe food borne illness in
humans contacted through the fecal-oral route and characterized by localized
cutaneous infections. Dairy cows serve as the main reservoir and high levels of the
pathogen occurs in animals fed with improperly fermented silage contaminated by
growth on manure-fertilized soils. Animal listeriosis is manifested as encephalitis,
meningitis, septicemia and abortions. The organism’s ability to withstand heat,
freezing and refrigeration temperatures have been a call for concern in the food
industries since raw foods, unpasteurized milk, raw vegetables, raw and cooked
poultry and refrigerated foods could serve as vehicles of transmission. This
characteristic is contradictory since it is a non-spore former. Notwithstanding, it

22
can grow over a wide range of environmental conditions (pH and temperature) thus
a longer survival duration in the environment (Choinowski et al., 2015).

Yersinia enterocolitica, a member of the genus Yersinia is a Gram-negative

cocobacillus belonging to the family of Enterobacteriaceae. It is unsporulated,
non-capsulated and lives as an intestinal flora in many species of wild and
domestic animals, including cattle as well as humans. It causes yersiniosis in both
animals and humans manifested as lymphadenitis, acute enterocolitis, nosodum
erythema, septicemia, poliartritis and even death. According to Tirzui and
colleagues, the cases of yersiniosis infections throughout the world are under
reported and the prevalence/incidence varies from country to country owing to the
availability of materials, laboratories and engagement of specialists.
Enterococcus species represent a subgroup of the group D fecal Streptococcus and
as a coccus, they are spherical in shape and occur either singly, in pairs or as short
chains (Choinowski et al., 1999).

They are Gram-positive, facultative anaerobic, lactic-acid producing bacteria that

live as commensal bacteria in the gastrointestinal tract of humans and animals.
Members of this subgroup include Enterococcus faecalis, Enterococccus
faecium, Enterococcusgallinarum or Enterococcus casseliflavus, Enterococcus
mundtii and Enterococcus avium, which exhibit significant differences in the
incidence of virulence factors, antibiotic resistance genes and distribution in fresh
and dry cattle manure (Canas and Laccases. 2010).

Mycobacterium species are acid-fast bacteria reported to be found in cattle

manure. They can survive in store manure/environment for long duration since
they can withstand fluctuations in temperature and pH, dehydration and exposure
to sunlight. This predisposition is ascribed to their acid fast characteristic

23
established by the high lipid and wax content of the mycobacterial cell wall. In
addition, the cell wall components confer upon these bacteria the propensity to
repel or not to absorb water causing them to demonstrate less susceptibility to
some chemical disinfectants (Couturier et al., 2012).

Bacillus and Clostridium species are spore forming bacteria that are commonly

found in cattle manure. They belong to the classes Bacilli and Clostridia
respectively, but both belong to the phylum Firmicutes. They have the tendency to
form spores and persist in that resistant and dormant state when growth conditions
are unfavorable but revert to vegetative cells through germination as growth
conditions becomes favorable. As spores, they are insensitive to heat, desiccation
and disinfectant; consequently, these bacteria will be detected in cattle manure
though in reduced numbers after pasteurization at 70 °C for hours in a study
conducted by Marañón and colleagues (Chen et al., 2015).

Bacillus species identified so far in manure entailed B. anthracis, B. cereus, B.

subtilis, and B. thuringiensis, which are Gram-positive rods, aerobic spore formers
and are mostly harmless and can persist for over years in the soil. The most
peculiar of these species is B. anthracis that causes anthrax (a life threatening and
dreaded disease) especially the pulmonary form through the inhalation of the
bacterial spores. The class Clostridia consists of Gram-positive anaerobic spore-
forming bacteria that are ubiquitous in the gastrointestinal tract and can be divided
into two groups based on their ability to invade and multiply within living tissues
(Couturier et al., 2012).

2.5 MICROBIAL OF COW DUNG

The microbial diversity of Cow dung (coprophilous organisms) has received the
attention of biologists since the last century. The presence of naturally occurring

24
bene-ficial microorganisms, predominately bacteria (bacilli, lactobacilli, and
cocci), and some actinomycetes, fungi, and yeast have been reported in Cow dung.
Cow dung harbors a rich microbial diversity containing almost 60 species of
bacteria (i.e. Bacillus sp., Lactobacillus spp., Corynebacterium spp.), fungi (i.e.
Aspergillus, Tri-choderma), 100 species of protozoa and yeasts (i.e.
Saccharomyces and Candida) (Chen et al., 2015).

2.5.1 Bacteria

Although bacteria and fungi are both important contributors to the composting
process of cow dung, bacteria are more abundant. The general microflora
inhabitant of the cattle gut involves Bacillus, Bifidobacterium, and Lactobacillus.
Velazquez et al. (2004) identified a novel species of xylanolytic, facultatively
anaerobic, motile, gram-variable, sporulated rod bacterium Paenibacillus
flaviporus from fresh and aged Cow dung based on 16S RNA gene sequence
analysis. Investigated microbial analysis of compost using CD as a booster. The
compost supported a high population of bacteria mainly Bacillus pumilus, Bacillus
sphearicus, Bacillus macereans, Bacillus lateosporus, Micrococcus varians, Proteus
mirabilis, and Enterobacter aerogenes. Several bacterial species have been reported
from Cow dung such as Citrobacter koseri, E. aerogenes, Escherichia coli, Kleb-
silla oxytoca, Klebsilla pneumonia, Kluyvera sp., Morgarella morganii, Pas-
teurella spp., Providencia alcaligenes, Providencia stuartii and Pseudomonas spp.
The aerobic heterotrophic bacteria isolated will be Acinetobacter spp., Bacillus sp.,
Serratia sp., Alcaligenes sp., and Pseu-domonas sp (Akinde and Obire, 2008). In a
later study from India, Bacil-lus safensis (PG1), Bacillus cereus (PG2, PG4 PG5),
Bacillus subtilis(BD2) Lysinibacillus xylanilyticus (BD3), and Bacillus
licheniformis (CPP1) will be isolated and identified from CD (Radha and Rao,
2014). The pyrose-quencing of 16S rRNA gene of bacteria obtained from bio-

25
stabilization of CD during vermicomposting was analyzed and Proteobacteria will
be in the highest proportions (Lv et al., 2015).

2.5.2 Actinomycetes

Actinomycetes are members of a heterogenous group of Gram-positive, anaerobic

bacteria accounted for a filamentous and branching growth pattern (Berkowitz,
1994). These actinomycetes are an integral part of CD those have been implicated
in the production of unpleasant flavors, odors, and colors. Of the specific types of
actinomycetes, No-cardia spp. are predominately present among CD microflora.
Moreover, a very high number of nocardioform, Rhadococ-cus coprophillus have
been isolated from the dung of domesticated herbivores. Godden et al. (1983)
reported nine species of actinomycetes in cattle manure; out of these
Micromonospora chalcae and Pseudonocardia thermophila will be cellulose
decomposers. In a recent study, Semwal et al. (2018) isolated 30 actinomycetes
species from fresh CD and all of them belong to Streptomyces spp. based on mor-
phological and chemotaxonomic analysis (16S rDNA sequence) (Castilho et al.,
2010).

2.5.3 Fungi and yeasts

Various authors reported different fungi from Cow dung. For example, Aspergillus
niger, Aspergillus flavus, Aspergillus rapens, Aspergillus fumigatus, Rhizopus
stolonifer, Mucor mucedo, Fusarium spp. and Vericosporium spp. will be reported
in CD (Adegunloye et al., 2007); saprophytic fungi (yeast and molds) such as
Alternaria sp., Aspergillus sp., Cephalosporium sp., Cla-dosporium sp.,
Geotrichum sp., Monilia sp., Mucor sp., Penicillium sp., Rhi-zopus sp.,
Sporotrichum sp., Thamnidum sp., Candida sp.,. Rhodotorula sp., Saccharomyces,
Sporobolomyces, Trichosporon, and Torulopsis sp. will be re-ported by others.

26
Some fungi such as Blastomyces sp., Botryodiplodia theobromae, Fusarium sp.,
Nigrospora sp., Penicillum chrysogenum, Penicillum glabrum, Pleurofrag-mium
sp., and Trichoderma harzianum isolated from Cow dung will be reported as
petroleum oil-degraders in aquatic environments in Nigeria (Cazares´-Garcıa et al.,
2013).

2.5.4 GENOMICS OF COW DUNG MICROFLORA

Several factors determine the microbial community of CD. Diet is the major factor
altering fecal microbial communities, while breed, age, gender and ecological
factors are minor factors that influence fecal microbial communities (Kim et al.,
2014). In most cases, fecal bacteria in cattle have been analyzed using culture-
dependent methods that gave approximately 1% of the actual bacteria present in
the animal gut. A bacterium with a similarity of Clostridium cellulosi was detected
in the fermented CD by 16S rDNA analysis (Couturier et al., 2012).

Dietary components of cattle influence the gastrointestinal microbial ecology and

diversity in CD. Investigated bacterial diversity in CD fed with different diets
(corn-based, forage diet) using metagenomics. Individual fecal samples from 333
cattle will be analyzed for determining the bacterial 16S rRNA gene amplicons. A
total of 2,149,008 gene sequences will be analyzed and two dominated phyla, i.e.
Firmicutes and Bacteroidetes will be found in all fecal samples. Girija et al. (2013)
studied a detailed analysis of CD microbiota based on a culture-independent 16S
rDNA approach. Total community of DNA was extracted from fresh CD and
bacterial 16S rRNA genes will be amplified, cloned and sequenced. This study
detected Acinetobacter, Bacillus, Stenotrop homona and Pseu-domonas that will be
producers of indole acetic acid (IAA) and siderophore. Pooja et al. (2015)
constructed a metagenomic library by cloning CD metagenomic DNA fragments

27
into pGX-1 vec-tor containing green fluorescent protein (GFP). The clones
expressing GPF from the library will be screened on maltose induced fluorescence-
activated cell sorter. One positive clone was isolated and the presence of 2031 bp
open reading frame (ORF) designed as amy 1, encoded for periplasomic -amylase.
Many Acinetobacter and Pseudomonas isolated from CD have been reported to
possess N2- fixing and P solubilizing activity. Several genera of bacteria such as
Bacillus and Pseudomonas will be identified in this study known for antagonistic
properties against bacteria and fungi (Lima-Junior et al., 2016). More recently, the
micro-bial community structure of CD is analyzed through terminal restriction
fragment length polymorphism (Bharti et al., 2016).

Recent technological advances in metagenomics have brought the field closer to

the goal of restoring all genomes within microbial diversity of CD microflora at a
much lower cost. How-ever, there are some new informatics challenges (i.e., high
through-put sequencing of amplified markers/DNA barcodes) that must be ad-
dressed to improve the understating of the complexity of CD microflora (Baldrian
and Valaskova, 2008).

28
 CHAPTER THREE

METHOD AND MATERIAL

3.1 INTRODUCTION

The bacterial lignin degradation have been hindered by a lack of a convenient

assay for lignin breakdown.108, 109 previously there have been a number of
methods in the literature: these have used a mixture of techniques but none have
allowed easy and accurate assessments of a bacteria‟s lignin degrading abilities.
They have also been either inappropriate or unreliable when used on Cow dung
samples, hampering efforts to purify lignin-degrading enzymes. The method and
materials used in this study will be listed below.

3.2 METHODS OF COLLECTION OF SAMPLE

Three Cow dung samples (Indian cow, Jersey and buffalo dung) will be obtain at
certain stages in each of the different location (Kara farm, Gidan Hillani and
UDUS site) First, fresh Cow dung samples (untreated) will be taken after
harmonizing the Cow dung before subjecting them to any management practice.
Secondly, another set of samples will be obtained from Gidan Hillani after
subjecting the cow dung to the three different management practices (already
discussed above). The third set of samples of the Cow dung will be collected at the
time of application and incorporation into the soil (at this stage, the Cow dung
treatments and exposed at the field in storage after the 1 month of
composting/ageing period for different time durations of 12 weeks, 8 weeks, 4
weeks and 0 week). The cow manure are put into plastic containers and taken to
the laboratory. These will be all carefully processed and kept for analysis.

29
3.3 METHOD OF PREPARATION

1000g of Cow dung from Indian Cow commonly known as (Deshi Shanu) will be
obtained and these samples will be oven dried immediately after collection at 65˚C
for 3 days and stored for analysis. The moisture content of the Cow dung is lower
than the other types. The dried cow dung was pounded to be in a powder form. The
powdered material had a net weight of 290g.

1000g of Cow dung from Jersey will be carried out and shadow dried for 5 d. The
moisture content will be high when compared to cow dung from Indian Cow based
on the chemical composition of cow dung for previous experiment (Akpinar and
Urek, 2012). The dried Cow dung will be pounded to obtain a fine powdered
sample. The powdered material had a net weight of 250g.

1000g of buffalo dung will be taken and shadow dried for 5d. The moisture
contents will indicate high when compared to Cow dung from Jersey. The dried
buffalo dung will be pound to be a fine powder. The powdered material had a net
weight of 190g.

All the samples were collected aseptically and preserved in refrigerator at 4°c and
they were tested within 24 hours of collection.

3.4 PREPARATION OF COW DUNG EXTRACTS

100 ml of acetone and ethanol will be added in 10g of powdered different Cow
dung (Indian cow, Jersey and buffalo dung) in a conical flask and it will kept in a
rotary shaker for 3d. The extract will be then filtered using Whatman No 1 filter
paper and stored in a vial for future use.

30
3.5 PREPARATION OF THE DISC CONTAINING COW DUNG EXTRACT

The empty discs will be impregnated with 50μl (2 mg/disc) of acetone extracts of
Cow dung from Indian Cow, Jersey, and buffalo dung separately and dried in the
oven. Similarly, the empty discs will be impregnated with 50μl (2 mg/disc) of
ethanol extracts of cow dung from Indian cow, Jersey, and buffalo dung separately
and dried in the oven. This process is repeated until the disc will completely
saturate with the extract. The disc will be used for the study antimicrobial activity
of cow dung extracts against human pathogens.

3.6 ISOLATION OF BACTERIA

Nutrient agar media will be used for the isolation of Lignin degrading bacteria
from cow dung.

3.6.1 Sample-1st (After 2 days)

Cow dung sample will be streaked on nutrient agar media with the help of
sterilized inoculating loop. Upper surface sample and lower surface sample will be
streaked on nutrient agar media separately. The plates will be incubated at 37⁰C for
24 hours. After incubation, colony characteristics will be studied. The colony will
be transformed to slants for further characterization.

3.6.2 Sample-2nd (After 7 days)

After 7 days, sample will be collected from upper surface and lower surface of
Cow dung separately. Inoculum was made from the upper surface on nutrient agar
medium, after that sterilized the loop again over the flame, till it became hot red.
The plate will turned and streaked over agar surface. Incubation of all the plates
will be done at 37⁰C for 24 hours. Same procedure will be repeated for lower
surface sample.

31
3.6.3 Sample-3rd (after 15 days)

Sample of Cow dung from upper surface and lower surface will be collected after
15 days for isolation of bacteria. By applying the both pour plate method & streak
plate method.

 Streak plate method

The sample will be taken with sterilized inoculating loop and streaked at the
medium of the plates. Then the plates will be incubated at 37⁰ C for 24 hours.

 Pour plate method

In pour plate method the sample was picked from upper surface and lower surface
separately by the help of sterilized inoculating loop and transferred in Petri plates
separately, after that the media (47⁰ C) was poured in the plates. The plates will be
rotated and left for solidification. All plates will be incubated in an incubator at 37⁰
C for 24 hours.

3.7 ISOLATION OF LIGNIN DEGRADING BACTERIA

The Lignin degrading bacteria will be enriched using 1% alkaline Lignin minimum
salt medium (Chandra et al., 2008) and incubated at 120 rpm for 7 days at 37˚C.
Enriched sample of 1ml will be transferred to 99 mL of sterile 0.9% NaCl. The
solution will be stirred vigorously and allowed to settle down. Using 1mL of the
liquid mixture, serial dilutions will be performed. 0.1 ml of serially diluted sample
will be spread on minimal salt medium agar containing alkaline Lignin. The plates
will be incubated at 30˚C for 7 days until colonies developed. The isolated bacteria
will be plated onto fresh MSM-L agar plates repeatedly to obtain pure cultures
(Rahman et al., 2013).

32
3.7.1 Lignolytic Activity

The bacterial isolates will be further screened for lignolytic activity using media
containing methylene blue dye as an indicator. The bacteria possessing lignolytic
enzymes undergo oxidation of indicator dye. The isolated bacteria will be streaked
on LB agar containing 0.25% methylene blue indicator dye. The plates will be
incubated at 30o C for 72 h. The agar plates will be monitored daily for bacterial
growth and decolorization of the methylene blue dyes. The decolorized microbial
colonies will be subjected for identification (Bondounas et al., 2011).

3.7.2 Lignin Peroxidase Enzyme (LiP) Assay

The isolates will be assayed for the presence of Lignin peroxidase enzyme (LiP). A
spectrophotometric assay method for the determination of Lignin peroxidase
activity is based on the demethylation of the methylene blue dye. The method can
be efficiently used for the quantification as its sensitivity is close to veratryl
alcohol assay. The enzyme Lignin peroxidase demethylates methylene blue, the
substrate in the presence of H2O2 (inducer). The final product is a tri-demethylated
methylene blue derivative and Azure C, the reaction occurs at pH-4. Enzyme
activity can be measured as percent decolourization of the methylene blue dye.

All the isolates will be freshly inoculated in 100ml of 0.5% Lignin broth in 500 ml
flask and will be incubated at 30oC at 120rpm for 5 days in a shaker incubator to
obtain a heavy bacterial growth. About 10 ml of the culture broth of the isolates
was taken in cooling centrifuge tubes and the culture broths will be centrifuge at
4˚C at 7000rpm in cooling centrifuge. After centrifugation the tubes will be kept in
ice bath without disturbing it and enzyme assay will be carried out using the
supernatant. Assay protocol-1ml of 50 mM Sodium Potasium Tartarate (pH-4)
buffer, 0.1 ml of 0.1mM H2O2 inducer was added to which 32µM methylene blue

33
as substrate and 10 µl of enzyme solution was added. The solution will be
incubating for 1hr at RT and A650 will be measured. The results will be
interpreted as the percent decolourization of the methylene blue dye by the enzyme
as compared to the control tube calculated as (A650 for control- A650 for test/
A650for control) X100 (Bondounas et al., 2011).

3.8 IDENTIFICATION OF BACTERIA

The Lignin degrading bacterial isolates will be characterized and identified. Colony
characteristics, Gram staining, motility and biochemical tests will be observed to
identify the isolates using the Bergey’s manual of determinative Bacteriology 9th
edition.

Nutrient agar medium with 1% CaCO3 was prepared and sterilized. The media was
allowed to cool to 50-600c and poured into the sterilized Petri plates and allowed
to solidify. Isolated colonies from slant culture was selected and taken carefully
and streaked on the solidified medium in the Petri plates. The Petri plates were
incubated at 370C over-nigh.

3.9 BIOCHEMICAL TESTS

The lignin degrading ability of their isolated bacterial cultures will be identified
using 0.1% Congo-red solution. The Congo-red solution will be flooded on the
plates containing basal medium and kept for 15 mins. After, 15 mins the plates are
washed with NaCl solution. The organism which shows the clearance zone is
confirmed as cellulose degrading bacteria.

The isolated Cow dung sample will be further screened using methylene blue dye
as an indicator for lignolytic activity. The microbes that produce lignolytic
enzymes undergo oxidation of indicator dye. The isolated bacteria were streaked

34
on methylene blue indicator dye (0.25 gL-1) containing LB agar plate. The plates
were incubated at300C for 72 h. the decolorization of methylene dye show the
lignolytic activity of the isolated microbes

3.10 PREPARATION OF PH MEDIA

Media of different pH was prepared by addition of different pH buffer solution

named according to pH as pH-2, pH-4, pH-6, pH-7 & pH-8. Two plates of each pH
buffer will be prepared and the pure culture of isolated bacteria will be transferred
by the help of sterilize inoculating loop aseptically on each nutrient agar media
done by streak plate method. Among the streaked plates, one plate from each pH
will be incubated at 37⁰ C for 24 hours, and second streaked plate of different pH
plates will be incubating at room temperature for 24 hours.

35
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