Biotechnology Journal International
25(1): 14-22, 2021; Article no.BJI.60179
ISSN: 2456-7051
(Past name: British Biotechnology Journal, Past ISSN: 2231–2927, NLM ID: 101616695)
Microbial Diversity of Nigerian Sludge and Its
Potential for Use as Biofertilizer
Opeyemi Fatunla1,2*, Joseph Essien1,2, Utibe Ofon1, Solomon Shaibu2,3
and Emmanuel Dan2,3
1
2
Department of Microbiology, University of Uyo, Uyo, Nigeria.
International Centre for Energy and Environmental Sustainability Research (ICEESR),
University of Uyo, Uyo, Nigeria.
3
Department of Chemistry, University of Uyo, Uyo, Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Author OF designed the study,
performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript.
Authors JE and UO managed the analyses of the study. Authors SS and ED managed the literature
searches. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/BJI/2021/v25i130130
Editor(s):
(1) Dr. Anil Kumar, Devi Ahilya University, India.
(2) Prof. Chung-Jen Chiang, China Medical University, Taiwan.
Reviewers:
(1) Oscar Monroy Hermosillo, Universidad Autónoma Metropolitana, Mexico.
(2) Lorena De Medina Salas, Universidad Veracruzana (UV), Mexico.
Complete Peer review History: http://www.sdiarticle4.com/review-history/60179
Original Research Article
Received 10 June 2020
Accepted 13 August 2020
Published 26 February 2021
ABSTRACT
Sludge samples were collected from a wastewater treatment plant in Nigeria for characterization
and evaluation for agricultural applications. Conventional and Molecular techniques were adopted
for the isolation and identification of indigenous microorganisms and resulting isolates were
characterized and identified by consulting Bergey’s manual of determinative bacteriology and
subjected to further screenings to assess their biofertilizer potential using standard microbiological
techniques. The viable cells obtained were enumerated and were found to be in the range of 1.03 ±
0.09 x103 cfu/g to 7.45 ± 0.78 x103 cfu/g for heterotrophic Bacteria and 1.63 ± 0.74 x103 cfu/g for
fungal community. The Molecular analysis carried out revealed a rich assemblage of diverse
species of microorganisms with Bacteria (99.40%) being the most dominant group, followed by
Fungi (0.39%) and others (0.21%). Thirty (30) isolates belonging to four (4) Phyla was recovered
culturally and identified with Firmicutes 9(30%) being the most dominant group, followed by
_____________________________________________________________________________________________________
*Corresponding author: E-mail: opeyemifatunla@uniuyo.edu.ng;
Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
Proteobacteria 8(26.7%) and Zygomycota 1(3.33%) was the least dominant. The phosphate
solubilization index range from 0.86 to 6.3 for bacterial and 2.5 to 3.8 for fungal isolates
respectively. The molecular analysis also revealed microbes adept at improving soil fertility to
include those in the order Rhizobiales and Actinomycetales. Although pathogens are of a concern
in the land application of sludge, our findings have revealed rich microbial consortia of
heterotrophic microorganisms whose beneficial attributes can be harnessed to produce nutrient rich
biofertilizer and soil amendment.
Keywords: Sludge; characterization; molecular sequencing; biofertilizer.
1. INTRODUCTION
most especially molecular techniques have been
developed to assess the unculturable microbial
diversity in order to forestall the existing barriers
that prevent the estimation of biodiversity. In the
past decades, new biochemical and molecular
techniques have been developed to identify and
classify microorganisms especially bacteria.
Measuring microbial diversity is difficult because
of the limited knowledge about bacteria species
and classification through families and orders.
Molecular techniques broaden our knowledge
about microbial diversity and help the taxonomy
of species. Measuring and monitoring soil
microbial communities can lead us to better
understanding of their composition and function
in many ecosystem processes [6]. Sewage
sludge is the solid, semi-solid, or liquid residue
generated from the treatment of domestic
sewage. It is very rich in nutrients (nitrogen and
phosphorus), organic matter and some trace
elements needed for plant growth. The treatment
of the large amounts of wastewater produced in
the society of today generates large quantities of
sludge. The wastewater is treated in such a way
that undesirable substances are separated from
the water. The first treatment is often mechanical
and it removes the bigger particles from the
wastewater. Substances can also be removed
biologically which is often the case in for example
nitrogen and carbon, which is measured in
biological oxygen demand (BOD). Chemical
treatment is sometimes used and it encourages
small particles and dissolved substances to form
larger particles which facilitate separation. This is
called chemical precipitation. Sludge is formed
when these larger particles clump together
during suitable separation methods [7]. All the
sludge that is separated during these treatment
methods (mechanical, biological and chemical)
are referred to as raw sludge, which has to
undergo varies kinds of further treatment.
The fact that microbes play a direct or an indirect
role albeit incognito, in many biological activities
that is beneficial to mankind has been
established and cannot be overemphasized [1-3].
Microbes has served as a powerful tool in
modern agriculture by being useful especially in
combating crop diseases, production of plant
growth promoting substances and in bioaugmentation.
Diverse
species
of
soil
microorganisms has been found to be closely
associated with the rhizosphere of plants where
they stimulate plant growth through several
mechanisms. These bacteria are collectively
known as PGPR (plant growth promoting
rhizobacteria). The search for PGPR and
investigation of their modes of action are
increasing at a rapid pace as efforts are made to
exploit them commercially as biofertilizers [4,5].
Biofertilizer has been identified as a cheap
alternative to chemical fertilizer in a bid to
increasing soil fertility while also boosting
production of crop in sustainable farming.
Biofertilizers contain live microbes, which are
adept at converting nutritionally important
elements from their unavailable form to forms
easily available and accessible to plants during
biological processes [4]. The utilization of
biofertilizers has several advantages over
conventional chemicals for agricultural purposes.
World agriculture has been found to be heavily
dependent on synthetic fertilizers as the source
of plant nutrients to meet the increasing demand
for food [3]. However, persistent and uncontrolled
use of chemical fertilizers often results in
unexpected
negative
impacts
on
the
environment. Poor soil fertility and imbalanced
soil microbial activity could be an implication of
unabated use of chemical fertilizers. Thus, the
use of organics including biofertilizer has gained
attention of recent in the sustainability of soil
fertility and plant productivity [3]. As many as
99% of the microorganisms present in certain
environment cannot be detected by regularly
used cultural techniques, therefore, protocols,
Thus, in this current study, critical cultural,
biochemical characterization and molecular
identification of the diverse species of microbes
in sludge were carried out and based on these
15
Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
findings;
work
has
access the biofertilizer
microorganisms.
been
done
to
potential of these
the sludge was carried out and previously
reported by Fatunla K et al. [9].
2.3 Isolation
of
organisms
2. MATERIALS AND METHODS
2.1 Study Site
Culturable
Micro-
A sub-sample (50 g) of raw sludge was added to
450 ml of sterile phosphate-buffered saline (PBS)
solution aseptically and homogenized. As
-1
-3
described by Cappuccino JG et al. [10], 10 –10
dilutions were made using sterile PBS solutions.
Total culturable heterotrophic bacterial and
fungal counts were determined by pour and
streak plate techniques respectively following the
methods of APHA [8]. Viable cells obtained after
isolation and enumeration were purified using
nutrient agar medium (NA). Following repeated
sub-culturing by streak method Cheesbrough M
[11], pure colonies of the isolates obtained were
kept in McCarthy bottles containing freshly
prepared agar in slants and incubated at 30 +
2°C for 18 to 24 hours before storage at 4°C for
future use. Identification of isolates will be carried
out on the basis of its growth characteristics on
differential media and biochemical properties
using standard protocol as described by
Cheesbrough M [11] and Holt JG [12].
Sludge samples were obtained from Lower
Usuma Dam Wastewater Treatment Plant
(LUDWTP) located in Abuja Nigeria between
latitude 9°01’ 12” N and longitude 7°25’ 16” E
(Fig. 1). It has a capacity to process 120 million
litres of waste water and provide Abuja and its
neighboring areas with the same amount of clean
drinking water per day.
2.2 Collection of Samples
Grab sludge samples (25 Litres) were collected
from
the
study
site
using
standard
Microbiological technique described by APHA [8].
The samples were collected with the aid of a
sterile hand trowel and stored in sterile amber
bottles. All samples were transported in icecooled chest to the laboratory for analysis.
Extensive physicochemical characterization of
Fig. 1. Delineated map of Abuja, showing the location of lower usuma dam water treatment
plant, Abuja, Nigeria
16
Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
2.4
x103 cfu/g to 2.19 ± 0.88 x103 cfu/g for Total
3
Heterotrophic Bacterial Count, 1.50 ± 0.42 x10
3
cfu/g to 2.27 ± 0.03 x10 cfu/g for Salmonella
3
3
Count, 3.45 ± 0.49 x10 cfu/g to 5.45 ± 0.35 x10
3
cfu/g for Total Coliform Count, 2.60 ± 0.14 x10
3
cfu/g to 7.45 ± 0.78 x10 cfu/g for Pseudomonad
3
Count and 1.63 ± 0.74 x10 cfu/g for Total Yeast
and Mould Count. Further examinations were
carried out on the cultures and comparison of
results with Bergey’s Manual showed that of the
eight gram-negative bacilli isolated, two were
identified as genus of Pseudomonas while the
remaining six belong to the following genera
Salmonella, Shigella, Aeromonas, Klebsiella,
Citrobacter, and Providentia. All gram-positive
bacilli isolated were identified as genus of
Bacillus, while the Gram Positive cocci isolated
were identified as genus of Micrococcus,
Staphylococcus, Streptococcus, Enterococcus
respectively (Table 2).
Molecular
16S
rRNA
Whole
Community Genomic Sequencing of
Sludge Sample
DNA Extraction from Sludge Sample was
performed using ZYMO soil DNA extraction Kit
(Model D 6001, Zymo Resaerch, USA) following
to the manufactures instructions. The filtered
DNA obtained was then used for PCR and DNA
sequencing. DNA sequencing was performed by
Next Generation Sequencing Technique to
determine the nucleotide sequence of all
microorganism present in the soil sample using
sequencing
primer
-16S:
27F:
5’GAGTTTGATCCTGGCTCAG-3’ and 518R: 5’ATTACCGCGGCTGCTGG-3’. The sequencing
was performed by Next Generation Sequencing
Technique to determine the nucleotide sequence
of all microorganisms present in the sludge
sample using automated PCR cycle- Genome
Sequencer™ MiSeq (Illuminar). Analysis and
alignment was performed using Vecton NTI suite
9 (InforMax, Inc.). Overall bioinformatic analysis
was done using NCBI-BLAST-2.2.24 and CLC
bio Genomics workbench v7.5.1 [13].
2.5 Screening for the
Potential of Isolates
The sequencing results of 16S rRNA gene based
analysis showed a total of fifteen (15)
Operational Taxonomic Unit (OTUs) in the
sludge sample. As shown in Fig. 2, phylum
Firmicutes was the most abundant with a relative
abundance of 65.39% represented mainly by the
families
Listeriaceae,
Lactobacillaceae,
Bacillaceae and Clostridiaceae (Fig. 3) followed
by phylum Proteobacteria with a relative
abundance of 10.31% represented mainly by the
families Enterobacteriaceae, Rhizobiaceae and
Bradyrhizobiaceae (Fig. 3). The least abundant
phylum in the sludge sample was Euryarchaeota
with a relative abundance of <0.01% represented
mainly by the families Methylobacteriaceae and
Thermococcaceae (Fig. 3).
Bio-fertilizer
All isolates were screened initially based on their
capability to solubilize phosphate when cultured
on modified pikovskaya’s medium (0.50gL-1 yeast
extract, 10.0 gL-1 dextrose, 5.0 gL-1 Ca3PO4, 5.0
-1
-1
-1
gL (NH4)2SO4, 0.1 gL MgSO4 .7H2O, 0.2 gL ,
-1
KCl, 15.0 gL agar agar and 1000mL of distilled
water at pH 7.0) as described by Karpagam T et
al. [14]. Isolates showing phosphate solubilizing
ability were spot inoculated at the centre of
°
Pikovskaya’s agar plate and incubated at 37 C.
Diameter of clearance zone were measured
successively after 24 hours, up to 7 days. The
Phosphate Solubilization Index (PSI) which is the
ratio of total diameter .i.e. clearance zone
including bacterial growth and the colony
diameter were measured [14].
A typical result of a confirmation assay for
phosphate solubilizing potentials of the 10
isolates (selected due to their ability to solubilize
phosphate in Pikovskaya’s broth medium was
carried out on Pikovskaya’s agar plates.
Phosphate solubilization index ranged from 0.86
to 6.3 for bacterial isolates (Fig. 5) and from 2.5
to 3.8 for fungal isolates (Fig. 6) after incubation
for a period of 24hrs to 120hrs. Streptococcus
pyogenes had the highest solubilization index of
6.3 at incubation period of 24hrs followed by
Shigella sp with solubilization index of 3.9 at the
same incubation period. The least (0.86)
solubilization
index
was
observed
in
Enterococcus faecium after 24 hours of
incubation (Fig. 5). Among the fungal isolates,
Candida sp had the highest solubilization index
of 3.8 at incubation period of 96 hours followed
by Candida famata with solubilization index of
Phosphate Solubilization Index (SI)
=
Colony diameter + halo zone diameter
Colony diameter
3. RESULTS
The quantitative estimates of the microbial
population (Table 1) have shown that the
culturable bacterial community present in 1 g of
sludge samples were in the range of 1.03 ± 0.09
17
Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
3.0 at incubation period of 72hours. On the other
hand, Rhizopus sp had the least solubilization
index of 1.3 at incubation period of 48 hours
(Fig. 6).
plays a critical role in determining the health of
living organisms including microbes and plants.
For nodulating legumes, the Nitrogen demand is
fulfilled through symbiotic Nitrogen fixation (SNF)
wherein atmospheric N2 is converted to usable
forms by nitrogenase of Rhizobia [16]. The
biological nitrogen fixation accounts for about
65% of the total nitrogen currently utilized in
agricultural practices which of course is believed
to be continuously required in future sustainable
crop production systems [17]. Other plant
growth-enhancing traits for which Rhizobia have
been exploited includes synthesis of siderophore
and solubilization of inorganic phosphorous [18]
and as biocontrol agents. Candida sp,
Aspergillus sp and Penicillum sp represent the
fungal communities encountered in this study.
Although these strains were not found to be
pathogenic, the presence of other pathogenic
species has been reported in sewage sludge
[19].
4. DISCUSSION
As revealed by the metagenomics analysis (Figs.
2-4) carried out in this study, Rhizobia species of
the
genera
Rhizobium,
Mesorhizobium,
Bradyrhizobium were found in abundance in
LUDWTPS. Rhizobia species were probably
gotten from the soil via the influent wastewater
from which sludge was produced. They are
normal flora of soil where they work in close
association with leguminous plants to fix nitrogen
[15]. The role of the Rhizobia group in biological
nitrogen fixation cannot be overstated. Nitrogen
is one of the prime elements required essentially
for the synthesis of enzymes, proteins,
chlorophyll, DNA and RNA. And hence, Nitrogen
Table 1. Microbial counts of the sludge samples obtained from lower Usuma dam water
treatment plant Abuja Nigeria
Sample
1
2
3
3
THBC X 10
mean
count + S.D
(CFU/g)
2.19 + 0.88
1.34 + 0.04
1.03 + 0.09
3
TYMC X 10
mean
count +
S.D (CFU/g)
1.63 + 0.74
-
3
TCC X 10
mean count
+ S.D
(CFU/g)
3.45 + 0.49
5.45 + 0.35
-
3
TSC X 10
mean count
+ S.D
(CFU/g)
4.40+ 1.60
4.90 + 1.13
2.55 + 0.35
3
TPC X 10
mean count
+ S.D
(CFU/g)
3.96 + 3.5
2.6 + 0.14
7.45+ 0.78
3
TSSC X 10
mean count
+ S.D
(CFU/g)
1.50 + 0.42
2.27 + 0.03
-
Values are means of duplicate plate counts with standard deviation
Key: THBC: Total Heterotrophic Bacterial Count; TYMC: Total Yeast and Mould Count; TCC: Total Coliform
Count; TSC: Total Staphylococcal Count; TPC: Total Pseudomonad Count; TSSC: Total Salmonella Shigella
Count; S. D = Standard deviation
Fig. 2. Distribution and percentage occurrence by phylum of the whole microbial community in
sludge sample obtained from lower Usuma dam Water treatment plant located in Abuja Nigeria
18
Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
Table 2. Culturable microbial communities recovered from sludge obtained from Lower Usuma
dam Water treatment plant located in Abuja Nigeria
Phylum
Firmicutes
Family
Micrococcaceae
Bacillaceae
Genera
Micrococci
Bacilli
Streptococcaceae
Streptococci
Enterococcaceae
Enterococci
Staphylococcaceae
Staphylococci
Enterobacteriaceae
Pseudomonadaceae
Salmonellae,
Shigellae, Klebsiellae,
Citrobacter,
Providentiae
Pseudomonae
Ascomycota
Aeromonadaceae
Saccharomycetaceae
Aeromonae
Candida
Zygomycota
Trichocomaceae
Mucoraceae
Penicillium
Rhizopus
Proteobacteria
Species
Micrococcussp
Bacillus cereus, Bacillus
megaterium
Streptococcus pyogenes,
Streptococcus sp
Enterococcus faecium,
Enterococcus sp,
Staphylococcus aureus,
Staphylococcus epidermidis,
Salmonella sp,
Shigella sp
Klebsiella oxytoca, Citrobacter
diversus, Providentia rettgeri
Pseudomonas aeruginosa,
Pseudomonas flourescens
Aeromonas sp
Candida famata, Candida sp,
Candida succiphila
Penicillium sp
Rhizopus sp
Fig. 3. Distribution and percentage occurrence by family of the whole microbial communities
in sludge sample obtained from lower Usuma dam Water treatment plant located in Abuja
Nigeria
insoluble forms of phosphates and lost in the soil
due to adsorption, precipitation, or conversion to
organic phosphates. Soil microorganisms play an
important role in making the phosphorus
available to plants by mineralizing the organic
phosphorus in the soil. Several researchers have
reported the crucial role of microbes in making
phosphorous available to plants in utilizable form
Phosphorus is a plant macronutrient that plays a
significant role in plant metabolism, ultimately
reflected on crop yields. It is important for the
functioning of key enzymes that regulate the
metabolic pathways [20]. The uptake of
phosphorus by the plant is only a small fraction
of what is actually added as phosphate fertilizer.
The remaining phosphorus is later converted to
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Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
Vatsyayan N et al. [2], Yoon MH et al. [3],
Karpagam T et al. [14], Poonam AS et al. [21],
Mardad I et al. [22]. The phosphate solubilizing
activity is determined by the microbial
biochemical ability to produce and release
organic acids, which through their carboxylic
groups chelate the cations (mainly Ca), bound to
phosphate converting them into the soluble forms
Kpomblekou K et al. [23]. In this study, the
solubilization index recorded ranged from 0.92 to
6.3.
PSB1
(Bacillus
sp)
3.0,
PSB3
(Bacillus megaterium) 3.0 and PSB 8
(Enterococcus sp) 6.3 exhibited the best
phosphate solubilisation on pikovskaya medium.
Although phosphate solubilisation index was
generally higher than those reported by Yoon
MH et al. [3] and Karpagam T et al. [14]. PSF1
(Candida sp) 3.7 and PSF3 (Candida succiphila)
2.7 exhibited the best phosphate solubilisation on
Pikovskaya medium. The research findings
have shown that members of genus
Bacillus
displayed
higher
solubilisation
capabilities over the course of the experiment
compared to Enterococcus and the fungal
solubilizers. This finding agrees with the work of
Vatsyayan N et al. [2], Yoon MH et al. [3],
Karpagam T et al. [14], Mardad I et al. [22] and
Chen YP et al. [24]. Hence, presence of these
organisms in rhizosphere might be beneficial to
plant phosphorous nutrition and growth. The
findings have also shown that few sludge
microbes have poor phosphate solubulization
efficiency (PSE), and it is suggestive of their
weak or little potential to enhance or stimulate
heterotrophic mineralization of organic sources of
nutrients.
Fig. 4. Distribution and percentage occurrence by order of the whole microbial communities in
sludge sample obtained from Lower Usuma dam Water treatment plant located in Abuja
Nigeria
Fig. 5. Solubilization index of phosphate solubilizing bacterial isolates on pikovskaya plates.
Values given are the means (n = 3) ± standard deviation
20
Fatunla et al.; BJI, 25(1): 14-22, 2021; Article no.BJI.60179
Fig. 6. Solubilization index of phosphate solubilizing fungal isolates on pikovskaya plates.
Values given are the means (n = 3) ± standard deviation
5. CONCLUSION
the Institute of Science and Technology for
Sustainability (UNU and GIST Joint Programme).
The research findings have shown the nitrogen
fixing bacteria from the paraphyletic group
Rhizobia represented by Bradyrhizobiaceae,
Rhizobiales and Mesorhizobium and bacteria
from the Bacillus genera to represents the bulk of
heterotrophic bacteria in sludge. The role of
these
versatile
microorganisms
in
biogeochemical cycling has been researched
extensively and established. Although sewage
sludge is rich in plant nutrients, the heterotrophic
potential of these beneficial microorganisms
could be harnessed to enhance the use of sludge
as a biofertilizer and soil conditioner for land
reclamation and bioremediation, especially of the
crude oil contaminated wetlands in the Niger
Delta region of Nigeria.
COMPETING INTERESTS
Authors have
interests exist.
declared
that
no
competing
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DISCLAIMER
The products used for this research are
commonly and predominantly use products in our
area of research and country. There is absolutely
no conflict of interest between the authors and
producers of the products because we do not
intend to use these products as an avenue for
any litigation but for the advancement of
knowledge. Also, the research was not funded by
the producing company rather it was funded by
personal efforts of the authors.
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This study was supported by the Ministry of
Science and Technology in South Korea through
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© 2021 Fatunla et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Peer-review history:
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http://www.sdiarticle4.com/review-history/60179
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