Asian Journal of Microbiology and Biotechnology

7(1): 17-27, 2022

ISSN: 2456-8341

ISOLATION OF Bacillus subtilis CAPABLE OF DEGRADING

AGRICULTURAL WASTES

IZEGAEGBE DANIEL OMOIKHOJE a*, KAMAL NEPAL a AND GEORGE KARANI a

a
Department of Biotechnology, Faculty of Life Science, Jain University, Bengaluru, India.

AUTHORS’ CONTRIBUTIONS
This work was carried out in collaboration among all authors. All authors read and approved the final
manuscript.

Received: 06 March 2022

Accepted: 11 May 2022
Published: 14 May 2022 Original Research Article
__________________________________________________________________________________

ABSTRACT

Epe district is a town located on the north side of Lekki, Lagos state. Epe’s temperature ranges from about 40 to
70 degrees centigrade. Considering humidity, temperature peaks high most of the time. The months with the
highest temperatures are February, March, and April. And the rainy seasons start from June to September. The
sample solid waste materials considered for this study were chopped and weighed approximately 3.0 to 3.5 Kgs,
were mostly taken from the Epe district and then moved to a garden where it was let to decompose for 4 weeks.
The soil test sample that was used weighed approximately 2.5 to 3.0kgs and was taken to the laboratory for
further investigation for the presence of bacteria. Objective: The goal of the study includes the extraction of the
Solid waste sample and isolation of B. subtilis bacteria. Other bacillus species like Pseudomonas spp, B. cereus,
Bacillus subtilis, E. coli, and B. thuringiensis. were found in the sample. According to previous research, B.
subtilis has been found to produce cellulase enzymes that degrade cellulose into sugars. Agriculture wastes like
rice straw and husk, maize, wheat, woodchips, and other agricultural waste are rich in cellulose. Other enzymes
like pectinase and lignin peroxidase are found in bacteria that degrade complex components like cellulose,
hemicellulose, and lignin in agricultural waste. The B. subtilis was bacteria of interest whereby isolation was
done by screening microorganisms producing cellulase using CMC agar. Then identification of the bacterial
isolate was done through morphological and physiological characterization and identifying the genus level
according to Bergey’s Manual of Determination Bacteriology (8 th edition). API strips (20E kit) were used in
identifying gram-positive bacteria.

Keywords: Solid waste; enzyme; bacteria; fungi; cellulose.

TERMINOLOGIES 1. INTRODUCTION

BNF: Biological nitrogen Fixation In Solid Waste (SW), it is the type of waste that
NA: Nutrient Agar incorporates transcendentally domestic waste
NH3: Ammonia (household waste) with at times, the expansion of
N2: Nitrogen industrial waste that is gathered by a district in a given
P: Phosphorus region. For the most part, solid waste is either made
TOC: Total Organic Carbon Content up of either semi-solid or solid forms of waste where
IPM: Integrated Pest Program that prohibit high risk in industrial wastes or domestic
_____________________________________________________________________________________________________

*Corresponding author: Email: izegaegbedaniel371@gmail.com;


waste [1,2]. The disposal of the solid waste industry
isolates solid waste into four significant classes which
rely upon the state wherein, they are deployed. For
instance, General solid Waste or MWS Material,
Industrial Solid Waste, Residual Solid Waste, and
Irresistible Solid Waste is also known as infectious
solid waste [3]. The microorganism that abides in this
waste is assembled under solid Waste Microbiota
(SWM) [4]. The most well-known organism that is
found in strong waste are microbes and growths are
involved in the degradation process also including
Algae and protozoa like Pseudomonas takes part in
the degradation process.
Microorganisms, as their name proposes, are tiny
organic entities that are found around us and inside
our bodies and typically require a magnifying
instrument to notice them. These microorganisms are
being arranged into a wide scope of classes that
incorporates microorganisms, infections, parasites,
archaea, protozoa, and algae. A few microscopic
organisms and growths are notable for the interaction
of corruption. The term "microbial corruption" here
refers to the microbial difference in normal mixtures,
consistently those that have an opposite effect on
human prosperity, [5] to less harmful or more
accommodating constructions, in the earth or the
experiment place. This technique collects data on the
qualities, impetuses, and paths that help sustain
things, engineer remediation of dirty conditions, and
anticipate the fate of fabricated materials around the
world.
Lagos state is grappling with a major waste disposal
issue. Markets, buildings, canals, streets, roads, and
land all around Lagos are littered with decaying piles
of rubbish. Astonishingly, the quantity of solid waste
in Nigeria is growing exponentially [6,7]. The present
attempts to get rid of them are not going far enough.
These problems include inadequate budget, fast-
growing populations, inaccuracy in planning and
enforcement of current standards as well as
uncontrolled urban development. Despite Lagos
State's reputation as a model for ecological
sustainability, the area nonetheless has a large amount
of untreated solid waste [8]. It has accumulated as a
result of the local population's poor waste disposal
practices and the negligence of waste processing
enterprises.
2. LITERATURE REVIEW
2.1 Microorganism in Soil
B. subtilis is a ubiquitous, gram-positive, and rod-
shaped bacteria that form the spore during
unfavorable conditions, thus resisting heat. B. subtilis
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Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
is a perfect multifunctional probiotic. For the
manufacture of vitamins, acetoin, hyaluronan and
other compounds, B. subtilis is extensively utilized as
an industrial cell factory. It is profoundly found in the
soil and non-pathogenic places. Biocontrol
mechanisms found in Bacillus subtilis allow it to
combat diseases caused by pathogens in both a direct
and indirect manner. Many secondary metabolites,
hormones, cell wall-degrading enzymes, and
antioxidants are synthesized by the plant as part of its
defensive system directly in response to pathogen
attacks as part of this mechanism [9, 10, 11]. The
indirect mechanism includes the promotion of plant
growth and the development of acquired systemic
resistance. Bacillus subtilis is also capable of
solubilizing soil pH, increasing nitrogen fixation, and
producing siderophores that promote its development
and inhibit the growth of pathogens.
B. subtilis is known to produce commercially
important products like amylase and proteases etc. It
is catalase-positive, which is the most important
characteristic for the identification of this organism.
In terms of both limiting the development of harmful
bacteria and improving nutrient absorption. Other
Microorganisms that can also be found in the soil are
Pseudomonas spp, B. cereus, E. coli, B. thuringiensis
etc.
2.2 Biodegradable Waste
We can concur that innovation has upgraded personal
satisfaction and has brought forth development in
different fields. This unpredictably affects
humankind, climate change, and the ecosystem.
Concerning the plastic water bottles that are
extremely advantageous to utilize, however, this may
take thousands of years to disintegrate yet its
debasement is a test of nature. Presently there are
huge amount of wastes that can be disintegrated or
non-degraded [12]. Also, regardless of whether it is
biodegradable or non-biodegradable, all trash is
hurtful to human existence and a misfortune to the
climate.
Materials and substances can be named as
biodegradable in case they are handily decayed by
microbes and other regular creatures and don't add to
contamination. Biodegradable waste is handily found
in civil solid waste like kitchen waste, agricultural
waste, food waste, and paper waste, [13] which are as
a rule debased by organisms (microorganisms,
growths), abiotic segments like temperature, UV,
oxygen, and so on. They are separated into carbon
dioxide, methane, water, and other essential regular
blends by different cycles like soil fertilization, high-
impact absorption, anaerobic handling, or relative

ways. Biodegradable waste influences the climate just
when they are available in abundance. They can create
are a huge amount of microbial populace around the
waste which can cause numerous transferable
illnesses to people, and animals [13]. It can produce
an awful odor, discharge certain gas on the interaction
of burning, and dumping grounds can go about as a
breeding place for specific vectors or transporters like
mosquitoes and rodents which eventually can
different hurtful illnesses. Furthermore, the negative
impact of microbiota on human health may cause
allergy, inflammatory bowel disease (IBD), obesity,
diabetes, and even cancer. For instance, the
composition of gut bacteria can indicate the risk of
diseases in each person.
2.3 Microorganisms Degradation
Composting blends including bulking agents such as
lignocellulosic wastes or by-products will generally
take longer to decompose than those containing other
biological wastes, such as animal wastes. The
presence of phenolic compounds in lignocellulosic
materials might also hinder some of the bacteria
present in the compost. Mathur (1998), on the other
hand, pointed out that the ammonia generated by
combining these materials with wastes that generate
ammonia would neutralize and auto-oxidize the
phenolics compound.
2.4 Dynamics of Biodegradation Phases
Biodegradation is a significant reduction in the
component of solid waste soil. When biodegradation
happens, it limits the contamination movement and
diminishes microbiota mass in the subsurface of the
solid waste. The degradable wastes are more degraded
than the non-degradable waste. The process of
biodegradation is a biochemical process caused by the
action of microorganisms [14, 7, 15]. This happens
when a compound loses an electron and gains a
positive charge and has a negative charge by gaining
electrons. During this process, electron acceptor
oxygen goes about under aerobic conditions or
harmful natural conditions. The oxidation of the
natural conditions is coupled to decrease atomic
oxygen which is referred to as aerobic respiration.
2.5 Factors Affecting Soil Biodegradation
2.5.1 Physiological factors
2.5.1.1 Rates of soil degradation
Soil state and process and also the presence of catalyst
are key factors that lead to soil degradation.
The number of "catalysts" present under these
conditions represents the product of microbial
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Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
organisms ready to metabolize the soil. Similarly, the
number of enzymes possessed by each cell has a
similar effect. Consequently, every element that
impacts soil essence consolidation is determined by
the number of microorganisms present in the cell's
enzymes [16]. This can either accelerate or slow the
pace of soil disintegration. However, this emphasized
that the rate of biodegradation is not always stable and
is not that variable for soil concentration; rather, it
slows as the concentration of the soil falls.
2.5.2 Extent of soil degradation
The amount to which a specific enzyme is
metabolized in soil essence where it involves the
affinity and the availability of the soil essence. This
affinity is an intrinsic property of an enzyme where it
is evaluated by its structure. The affinity differs
among various enzymes and each enzyme performs
the same functions performed by various
microorganisms in populations.
2.5.3 Microbial physiology and their general
indicators
There are usually 50:14:3 proportions of C, N, and P
in microorganisms. In order for infinite microbial
development to occur, adequate amounts of these
supplements should be available in a useable form and
to legal extents. For example, the quantities of
phosphorus that bacteria may use for growth may be
unavailable, either naturally or synthetically [17].
2.5.4 Availability of nutrient
Organic carbon mixes are analyzed based on their
approximate total organic carbon content (TOC). A
sample's TOC is calculated by taking into account
compounds that cannot be digested or that are not
easily metabolized by organic molecules. This method
will overstate the amount of carbon [18] that is
accessible to microbes. Also, since the surface soils
contain more microorganisms, this makes them have a
greater microbial population density than deep soils,
which have a more prominent density in
soil compared to water bodies.
Soil degradation can be slowed or accelerated
depending on the quantity of N2 available to
microorganisms in the form of organic N2 (NH4+),
nitrate (NO3–), and/or nitrite (NO2–). A nitrogen
ratio of less than 40, indicates that there is enough
nitrogen available.
Microorganisms may utilize both inorganic
(orthophosphate) and organic types of phosphorus,
depending on the solvent used. This happens when the
ratio of carbon to phosphorus is more than 120:1 and

carbon to nitrogen to phosphorus ratio (C/N/P) of
100:10:1 is increased calcium (Ca) and magnesium
(Mg) concentrations might cause phosphates to be
released more quickly, reducing the quantity available
for microorganism. Different nutrients present in soils
like sulfur (S), Ca, Mg, and potassium (K), including
trace numbers of minerals, are regularly found in
sufficient amounts for metabolic needs.
2.5.5 Electron terminal acceptors
Due to the fact that the microbes' digestion of organic
soil essences frequently includes the component being
oxidized, it is necessary to use the electrons obtained
from these cycles in order for them to degrade various
combinations. These bacteria can receive electrons
from an array of organic substances of carbon and
inorganic materials (Table 1). The amount of energy
obtained by microorganisms from these metabolic
cycles varies. Aerobic respiration produces more
energy than methanogenesis (the cycles are recorded
in Table 1 from the most energy conserved to the
least). The rate of soil deterioration is usually related
to the quantity of energy absorbed at first [19].
O2 is the terminal electrophile in the aerobic process.
The accessibility of O2, where it is achieved through
the dispersion from the environment, or can also
disperse through water. Whereby when it comes in
contact with water it is easily dissolvable and this
makes it move faster [17]. When this happens, it
typically slows down the rate of soil deterioration. So,
therefore, it is said to be slow when the soil does not
have enough availability of O2 diffuse from the air
surface of the earth. However, this makes the water
content in the soil below and will not make the limited
Oxygen dissolve properly in the soil. As a result, O 2
may be used quicker than it is replaced, and the
technique may become anaerobic.
2.6 Respirometry of Soil Degradation
Respirometry of soil determines the percentage of
oxygen consumption or carbon dioxide (CO2)
composition in soils, which indicates the amount of
microbial activity on site. This perception is by
observing the amount of O2 needed through soil
deterioration and comparing it to the rate recorded in
a nearby location that is not affected. The increased
oxygen intake assessed low amounts of O2; this is
taken as a sign of higher respiration action, which is
conceivable as a result of soil degradation activities.
Nonetheless, there are various issues with using
respirometry to demonstrate soil deterioration. The
most significant aspect of this method is that it
monitors the oxygen usage that occurs throughout the
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Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
metabolism of all carbon sources. If the sample
provided contains a tiny proportion of the total
organic carbon, the increased oxygen consumption
due to soil activities might rather be increase a bit in
the aggregate. Furthermore, the approach is only
useful for detecting cellular respiration and does not
account for Fermentation or other cycles involved
with soil degradation.
2.6.1 Temperature
The temperature also has an unpredictably large
influence on biodegradation paces, by influencing the
paces of enzyme-catalyzed reactions. For every 10oC
reduction in temperature, the pace of biodegradation
is cut into sections and these Biodegradation paces are
generally extremely slow at 0oC. High soil
temperatures also result in increased soil microbial
performance and a faster pace of biodegradation up to
a limit of around 65oC. Therefore, the pace of
biodegradation may change occasionally and the
microbial metabolic performance can itself increase
soil temperature.
2.6.2 Moisture
Water impacts the pace of microbial metabolism since
it impacts the measure of dissolvable Water impacts
the pace of microbial metabolism since it impacts the
measure of water-insoluble materials available. Also,
the hydrostatic stress and concentration of PH of
aquatic and terrestrial habitats, as previously stated,
the moisture in the Porosity of soil influences the
exchange of oxygen. Under severe water stress, O2
could be lost rapidly than it could be replaced in the
soil pore, resulting in anaerobic soil. It could slow
down the degradation process and induce significant
alterations in microbial metabolic activity. However,
the soil moisture content should be in the range of 25
to 85 in the percentage of the moisture content, with a
range of 50–80 percent being optimal for the
degradation process.
2.6.3 PH
The pH of the soil is proportional to the acidification
or measure of the acidity in water content. However,
degradation rate may occur in a wide pH range, with a
flow rate of 6.5 - 8.5 being usually ideal for biological
degradation in terrestrial and marine systems, with pH
values ranging from 5 to 9 been deemed acceptable.
In this scenario, a slight change in soil pH impacted
nutrient uptake, such as dissolvable properties of (P).
When an essential mineral in an organic system is at a
threshold pH of 6.5 and decreases with pH values
greater or lesser than this value.

Table 1. Electron terminal acceptors
Electron acceptor Chemical symbol
Oxygen O2
Nitrate and nitrite NO3 & NO2
Ferric iron Fe+3
Manganese Mn+4 & Mn+2
Sulfate SO4–2, S2O3–2 & SO3–2
Organic compounds Many
Carbon dioxide CO2
Source: Hayat et al. 2010.
2.7 Modes of Microbiota and its Impact on
Biodegradation
2.7.1 Bacteria in the soil
Bacterial organisms are the most numerous microbes
in the soil. These bacterial species in Soil are
unicellular organic substances with a considerable
number of bacteria in a single weight unit of
Soil depending on Soil characteristics. A few days
after a rainfall, changes in soil moisture, and
variations in soil temperature and carbon substrate
may cause the bacteria population to grow [15,20,21].
This has the potential to have an impact on the soil's
nutritional qualities. Among the many different kinds
of bacteria, only a few microbe species are
particularly sensitive. Whereby, any changes in the
climatic impact on the soil have the potential to easily
kill those microbes. While some microbes are highly
intense, they can endure severe situations such as high
temperatures, low temperatures, or high humidity.
Only a few bacteria are affected by certain varieties of
plants. It is often impossible to understand the
microbial behaviors and relationships in an
environmental setting, [22] or which kind of bacteria
organisms are generally prevalent in distinct soil
types, based on their widespread dispersion and
metabolic flexibility.
2.7.2 Function of soil bacteria
For decades, soil microorganisms had always been
employed in crop production. Bacteria's primary roles
[21] are: (1) Supplying and increasing the availability
of nutrients present in the soil for plant intake; (2)
Promoting plant development, e.g. by the synthesis of
phytochemicals; (3) regulating or suppressing the
action of plant diseases; (4) enhancing soil physical
properties; (5) bioremediation or microbial leaching
of inorganics. Bacteria have lately been employed in
the soil to mineralize organic contaminants, i.e,
bioremediation of contaminated soils [23]. In
agricultural development, when it comes to
maximizing plant genetic potential. In plants,
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Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
Symbol process
Aerobic respiration
Aerobic respiration (denitirification)
Anaerobic respiration
Anaerobic respiration
Anaerobic respiration (sulfate reduction)
Fermentation
Methanogenesis
microbial interactions are important because the root
microbiome is important. This assists in the
biotransformation, mobilization, absorption, and
alteration of the nutritional requirements for the
development of plants. Biological methods are
becoming more popular as a supplement to chemical
fertilizers for increasing crop production in an IPM
system.
2.7.3 The impact of microbiota on biodegradation
Bacteria, or microbes living beings, are generally
utilized in immense scale mechanical operations.
These microorganisms are necessary for the synthesis
of many Catabolism. Like bioethanol, alcohols,
lactate, and lactoflavin, which include the change of
biochemical compounds that help in the identification
and elimination of toxins. Biofertilizers and metal
toxicity can both be reduced by using organisms.
Insulin is an example of a non-microbial compound
that can be made using microorganisms. The
microbiota has the following roles during
biodegradation:
2.7.3.1 Production of metabolite
Ethanol is widely used as a dissolvable, extractant,
and radiator fluid by living organisms. Some stains,
greases, disinfectants, insecticides, pyrotechnics, and
polymerizers. Polymerized fibers are also based on
this material, as well as plasticizers [24]. Plasticizers,
braking fluids, extractants, and petroleum compounds
all benefit from the creation of N-butanol, which is
also produced by microbes. Glycerol, mannitol, and
butanol are all commonly used in the
pharmaceutical and food industries, respectively,
whereas glycerol is used in both medicine and food
manufacturing.
2.7.3.2 Bio-fertilizers
Microbes are added to soil to improve plant growth by
increasing the number of nutrients available for plant
growth. There are a number of regularly used bio-

fertilizers that make phosphates available to plants in
order to boost crop development and yield. Biological
conditions in which plants naturally occur depend on
parasites called mycorrhizae for adequate nutrient
absorption and long-term survival of the plants.
Nitrogen fixation is a process by which Azospirillum
bacteria increase plant growth and aid in the
production of food [25, 26].
2.7.3.3 Production of insulin using microbes
People with type-2 diabetes have been treated for
many years with insulin generated from the pancreas
of animals killed for their meat. As a result, patients
are less likely to have hypersensitivity to the insulin
produced by genetically engineered bacteria.
Researchers use a new technology called recombinant
DNA to insert an insulin-producing human gene into
bacteria's DNA [27]. It's in these large, specially
treated steel fermentation tanks that the gene-modified
bacteria work their magic, producing a large amount
of insulin. As soon as the yeast has fermented,
researchers extract and purify insulin for use by
diabetes patients. Microbes cannot spread if the
equipment is not always kept sterile.
2.7.3.4 N2 fixation symbiosis
It is essential for plant growth and food and feeds
production since N2 is required for the cell production
of enzymes, proteins, photosynthesis, Chromosomes,
and RNA. To supply legumes with nitrogen, rhizobia
bacteria use nitrogenase, an enzyme that converts N2
from the atmosphere into nitrate [28,29]. The
physiological nitrification in (BNF) process is
responsible for 65 percent of the nitrogen now used in
agriculture and will continue to play important role in
future sustainable crop production systems. The
symbiotic relationship between legumes and N2-
fixing bacteria, which converts atmospheric N2 to
NH3 in legumes, is the primary source of important
metabolic processes in BNF [30].
3. RESEARCH METHODOLOGY
The sample waste was collected; the isolation and
identification of microbiota were done.
In this study, soil test sample was collected from
decomposed agricultural waste. The agricultural
wastes that were used are cabbage waste, rice straw,
and maize waste. The soil test sample was examined
Table 2. The raw material concentrations
Material Rice straw
[DM (Dry matter, g·kg−1) 864.7 ± 1.3
The mean ± standard deviation of the three samples is shown in the table.
Source: Du, G, et al, 2021
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Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
and also the analysis of biodegradation was done by
estimation of residual.
3.1 Materials and Method
Maize husk was collected, Rice straw and Cabbage
byproducts were purchased from Epe farm opposite
Yaba College of Technology Epe Campus. The 3
(Three) sample wastes were cut to 1–2 cm in length,
which weighed approximately 3 to 3.5Kgs and were
left to be decomposed in a specific environment for 4
weeks. The raw material concentrations are presented
in Table 2, and the microbiota samples that grew on
solid waste were carefully collected after that.
Aseptically obtained soil samples were stored at 4 oC
in a sterile generic plastic zip-lock pouch bag before
being stamped by their source. In the lab, soil
microorganisms and moisture content were isolated,
as well as the final PH reading.
3.2 Evaluation of Humid Concentration and
pH in Water Samples
The collected 3 samples were placed on a filter paper
and combined weight was recorded. The measured
samples were stored in a hot air oven held at a
temperature of 110oC
The AWPA [9] preceding analysis was used to
confirm the humidity of the sample.
(%) Percentage = MC =
For example, MC denotes the amount of moisture, W
indicates the initial weight, and w represents the
weight constant prior to oven drying. The pH was
measured via the electroanalytical method using a pH
meter and the mix glass terminal method.
3.3 Bacterial Constituent Separation from
Waste Samples
For the containment of microorganisms, the serial
dilution technique was adopted. In this process, a test
suspension was formed by adding soil combined with
the different wastes (the samples) and addition of 1g
to 10 ml distilled water and fast mixing for no less
than 1 minute to generate a test solution. For a brief
amount of time, the weakening method was effective.
Cabbage leaf Corn flour [28]
59.4 ± 0.7 877.3 ± 1.9 [28]

Beginning with the sample and progressing through
10-1 to 10-4, sterile weakening gaps were stamped
sequentially. Using a clean sterile pipette, 1ml of the
sample was swapped with 1ml of the 10-1 dilution
clearance. For each successive step, 1ml from the 10 -1
dilution was transferred to the 10-2 tube, then from the
10-2 to the 10-3, and finally from the 10-3 to the 10-4. In
each dilution tube, 0.1ml of dilution liquid was put
into Nutrient Agar society media and incubated for 24
hours at 37 °C. NA society medium comprised 0.5
percent peptone, 0.3 percent yeast separate, 0.5
percent NaCl, 0.25 percent glucose, 1.5 percent agar,
distilled water, and a pH of 7 at room temperature.
The naturally existing microscopic organisms were
sub-refined in NA inclines after significant breeds of
microorganisms were sub-refined; incubated at 37°C
to achieve enthusiastic growth and after that protected
in 20 percent glycerol vials at - 80 °C.
3.4 Isolated Bacteria's Microbiological and
Biochemical Characteristics
3.4.1 The Cellulase-producing bacteria
Our bacteria of interest were bacteria capable of
degrading agriculture waste. These bacteria contain
different enzymes in degrading the organic material.
Bacteria containing cellulose enzyme was the
organism of interest. CMC as selective media were
used in the isolation of bacteria found in the soil
sample having cellulose enzyme. The samples were
kept at 4°C then Carboxymethyl cellulose (CMC)
agar plates were prepared using 10.0; yeast extract,
1.0; (NH4)2SO4, 2.5; K2HPO43H2O, 0.25; NaCl,
0.1; MgSO47H2O, 0.125; FeSO4-7H2O, 0.025;
MnSO44H2O, 0.025; agar, 10(g/L each) and media
adjusted to pH of 7. The samples were prepared by
serial dilution using 1gram and dilutions were made
using sterile water. The CMC agar plates were
inoculated with the sample using pour plate method
technique and were incubated at 37°C for two days.
Then the positive colonies were isolated in nutrient
agar for biochemical test.
3.4.2 The bacterial isolate identification
The plates were observed and the colonies showing
clear zones were isolated. The clear zones were due to
the organism cellulase enzyme hydrolyzing the
cellulose in the CMC media. Identification was done
first by Gram staining and culture characteristics.
According to Bergey's Manual of Determinative
Bacteriology, the isolates were described and
identified up to the genus level (8th edition). Gram-
positive bacteria were identified by using an API 20E
kit as well. API strips were injected with 24-hour-
grown cultures and then incubated at 37°C. In
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Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
accordance with the manufacturer's instructions, the
findings were evaluated against known Bacillus
subtilis strains. Motility, sporulation, catalase, and the
Gram response of the clones were all tested according
to protocol.
4. FINDINGS AND DISCUSSION
This study involved the extraction and analysis of
microbial isolates from Yaba College of Technology
Epe farm wastes. Bacterial growth is dependent on
several physicochemical conditions, such as medium,
pH, temperature, incubation time, presence of carbon
supply, and so on. As a result, alternative
circumstances under which microscopic organisms
are cultured in shared living space should be explored
before proceeding to large-scale augmentation for use
as a decomposer. In this manner, the component
parameters were considered.
4.1 Characteristics of Agricultural Solid
Waste of Both Physical and Chemical
Properties
Microbes thrive in different levels of wetness,
whereby research done at Yaba College of
Technology Epe farm found that water content in the
wastes was 70.51% and 72.32% respectively where
bacteria growth was different between the waste
samples collected depending on their soil content.
About 75% of bacteria thrive in moist, high-oxygen
environments that degrade waste. Despite the fact that
some species, such as Pseudomonas, E. coli, and
Bacillus, are obligate anaerobes, these organisms can
survive in either an oxygen-rich or oxygen-depleted
environment. Low oxygen clostridium species, for
example, require a lack of oxygen in order to thrive.
Two media (CMC and NA) were chosen for bacterial
refinement because they had higher pH values for
Bacillus subtilis. PH values ranging from 5-9 were
found in both samples of the collected material. The
pH of a simulated environment is critical to the
growth of microorganisms. Two media, Supplement
Agar (NA) and CMC agar were used to advance the
pH. It was found that the pH ranges of 7.2 and 7.6 for
NA and CMC media were ideal for the most bacterial
growth. The results showed that the sample had a pH
of roughly 7.79, making it likely that the organism
will thrive in an in vitro environment with a pH range
of 7-8 in CMC and NA.
4.2 Screening of Cellulolytic Bacteria
It was found that three samples of decomposed soil on
the campus of Yaba college of technology Epe
campus contained cellulolytic bacteria. CMC agar
plates were used for each sample dilution.
 Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022

Transferring positive clones that showed strong and CMCase activity test, their cellulolytic abilities
colonial growth and a clearly defined clearing zone were established on CMC agar media and in liquid
was done. Using the trypan blue-staining technique cultures.

Fig. 1. Bacterial cell growth and CMCase activity on trypan blue-containing CMC agar plates. S52-2
isolates from maize husk; SL9-9 isolates from rice straw waste; C5-16 isolates from cabbage; and SL9-9
isolates from rice straw waste. Clear halos caused by cellulolytic activity could be seen surrounding the
colonies. Biochemical tests, microscopy, and culture characterization were done on positive colonies
(showing cellulose hydrolysis) by culturing n Nutrient agar. The API strip tests were recorded below
Source: Kim, et al, 2012

Table 3. Biochemical test results (API) and culture characteristics

Organism B. subtilis B. cereus B. thuringiensis E. coli Pseudomonas

spp,
Gram Gram-positive Gram-Positive (+) Gram-Positive (+) Gram-Positive Gram-
characteristic (+) (+) Positive (+)
ONPG test Positive (+) Positive (+) Negative (-) Positive (+) Negative (-)
ADH Positive (+) Positive (+) Positive (+) Negative (-) Negative (-)
LDC Negative (-) Negative (-) Negative (-) Positive (+) Negative (-)
ODC Negative (-) Negative (-) Negative (-) Positive (+) Negative (-)
CIT Positive (+) Positive (+) Positive (+) Negative (-) Negative (-)
H2S Negative (-) Negative (-) Negative (-) Negative (-) Negative (-)
URE Negative (-) Positive (+) Positive (+) Positive (+) Negative (-)
TDA Negative (-) Negative (-) Negative (-) Negative (-) Positive (+)
IND Negative (-) Negative (-) Positive (+) Positive (+) Positive (+)
VP Positive (+) Positive (+) Positive (+) Positive (+) Positive (+)
GEL Positive (+) Positive (+) Positive (+) Negative (-) Positive (+)
GLU Positive (+) Negative (-) Positive (+) Positive (+) Positive (+)
MAN Positive (+) Negative (-) Positive (+) Positive (+) Positive (+)
INO Positive (+) Negative (-) Negative (-) Negative (-) Positive (+)
SOR Positive (+) Negative (-) Negative (-) Negative (-) Positive (+)
RHA Negative (-) Negative (-) Negative (-) Positive (+) Positive (+)
SAC Positive (+) Positive (+) Positive (+) Positive (+) Positive (+)
MEL Positive (+) Negative (-) Negative (-) Positive (+) Negative (-)
AMY Positive (+) --- Negative (-) Negative (-) Positive (+)
ARA Positive (+) Negative (-) Negative (-) Positive (+) Positive (+)
API 20E Kit was used to determine positive (+) and negative (-); variables

24

Days of Incubation
45
% Degradation over control
40
35
30
25
20
15
10
5
0
cabbage
Days of incubation
Fig. 3. B. subtilis Strains of Waste Degradation Capabilities.
Compiled by Author
4.3 The Potency of Microorganisms
Degrade Waste Sample
In the analysis, it can be seen that cabbage has the
greatest degradation potential, followed by maize
husks and rice straw waste samples. As
microorganisms (microbes) break down the waste, the
waste’s weight decreases. After being broken down
and converted to a simple form by bacteria, the treated
wastes were found to be lighter than untreated wastes.
Examples of waste weight-loss rates were expanded
when the degradation operation was moved forward,
as seen in Fig. 3.
5. CONCLUSION AND RECOMMENDA-
TION
B. subtilis is a terrestrial microbe that forms an
endospore when the heat elevates above the threshold
and is known to produce commercially beneficial
substances. However, it is nonpathogenic in nature, B.
subtilis produces several extracellular enzymes like α-
amylase, a levansucrase, several β-glucanases, and at
least two different lipolytic enzymes, which
specifically cleave the bonds for the degradation of
organic wastes. Moreover, it is a plant-promoting
bacteria, which has the biofertilizer functions as it
suppresses a disease by producing a secondary
metabolite, hormones, cell-wall-degrading enzymes,
and also antioxidant that helps the plant in its defense
mechanism again pathogens.
Hence, B. subtilis can be used industrially like paper
industry to digest cellulose, in ethanol production. So,
Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022
% Degradation over control
maize husk rice straw
to therefore, there are strains of B. subtilis isolated that
possesses a potential cellulase, microcrystalline
cellulose-hydrolytic activity, cell-bound β-
glucosidases, and hemicellulases in addition to
endoglucanases. Thus, B. subtilis has great potential
in various fields including biofertilizers, degradation
of cellulose or organic pollutants, and ethanol
production also to some extent. There are special
conditions in waste sites or pits to allow microbiota to
grow or can grow naturally; Microbiota can only
thrive and multiply in a limited range of
environmental conditions. Temperature, pH, dissolved
gases, osmotic pressure, and water availability may all
impact whether or not microbiota can grow.
5.1 Recommendation
Microbes like B. subtilis transform the sample through
their cellulose enzymatic or metabolic process, and
this includes two processes such as Growth and
Cometabolism. During the growth, microbes use the
organic wastes as a source of carbon and energy, thus
resulting in the complete degradation or
mineralization of pollutants. Hence the use of this
process called bioremediation is very eco-friendly and
effective. However, bioremediation may not be very
effective in treating inorganic wastes as it takes a
longer time to detoxify the pollutants. Also, the
Negative impacts of microbiota activities in the soil
can promote the growth of bacteria that create
enzymes in waste materials which as a result can
pollute the environment. This results in air pollution,
which alters the composition of gut microbiome and
this can affect human health. However, when
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 Omoikhoje et al.; AJMAB, 7(1): 17-27, 2022

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