Tolerance of Some Soil Fungi to the Content of Deep Cycle Battery and Their Bioremediation Potential

Abstract

Aims: The purpose of this study was to isolate and screen soil fungi that are able to tolerate the contents of spent deep cycle battery (inverter), and to test for their bioremediation potential. Place and Duration of Study: Sample: Department of Microbiology, Rivers State University, between June 2019 and February 2020. Methodology: Soil samples were collected from a mechanic village while spent inverter batteries were obtained from a waste vendor. The battery was forced open to extract its contents of the battery. Using standard microbiological techniques, fungi were enumerated and characterized. Stock solution of the battery content was prepared by dissolving the inverter battery content in sterile deionized water. This stock solution was used to carry out the screening test on the fungal isolates to ascertain the fungi that can tolerate the contents of the spent battery. Results: Total heterotrophic fungal counts for the polluted and unpolluted soil were 6.0 x 103 cfu/g and 7.5 x 104cfu/g respectively. The fungal isolates identified from the polluted soil samples were members of the genera Rhizopus, Mucor, Aspergillus, Penicillium, and Candida, while, the isolates identified from the unpolluted soil sample includes: Candida sp, Aspergillus niger, Penicillium sp, Aspergillus fumigatus, Aspergillus flavus, Mucor sp, Yeast, Fusarium sp and Aspergillus sp. After the screening, total heterotrophic fungal counts for the soil ranged from 1.0 x 102cfu/g to 9.5 x 102cfu/g. Two fungi of the genera: Rhizopus and Mucor had the highest counts during 72 hours of incubation for the screening test. The results obtained from this study indicated that species of Aspergillus, Penicillium, and Candida were the most inhibited by the contents of the spent battery while Rhizopus and Mucor spp were more tolerant to the contents of the inverter. Rhizopus and Mucor spp were therefore, adopted in the bioremediation of soil contaminated with contents from the battery. It was observed that Rhizopus and Mucor spp in a consortium had the highest percentage of heavy metal removal (or uptake) in the following order: Cadmium (66.66%) > Lead (38.15%) > Zinc (26.83%) > Nickel (20.83). Conclusion: These organisms can be used in the bioremediation of soil polluted with metals from spent deep cycle batteries.

Full text

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*Corresponding author: Email: sallyakdougy@yahoo.com;
South Asian Journal of Research in Microbiology
8(1): 34-46, 2020; Article no.SAJRM.62224
ISSN: 2582-1989
Tolerance of Some Soil Fungi to the Content of Deep
Cycle Battery and Their Bioremediation Potential
S. I. Douglas
1*
, C. U. Wellington
1
and T. G. Sokari
1
1
Department of Microbiology, Faculty of Science, Rivers State University, P.M.B. 5080, Nkpolu-
Oroworukwo, Port Harcourt, Rivers State, Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Author SID designed the study,
performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript.
Authors CUW and TGS managed the analyses of the study. Author TGS managed the literature
searches. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/SAJRM/2020/v8i130186
Editor(s):
(1) Dr. Ana Claudia Coelho, University of Tras-os-Montes and Alto Douro, Portugal.
Reviewers:
(1) Maria Maura Margarita Teutli León, Benemérita Universidad Autónoma de Puebla (BUAP), Mexico.
(2)
V. Madhavi, BVRIT Hyderabad College of Engineering for Women, India.
Complete Peer review History:
http://www.sdiarticle4.com/review-history/62224
Received 25 August 2020
Accepted 30 October 2020
Published 18 November 2020
ABSTRACT
Aims:
The purpose of this study was to isolate and screen soil fungi that are able to tolerate the
contents of spent deep cycle battery (inverter), and to test for their bioremediation potential.
Place and Duration of Study: Sample: Department of Microbiology, Rivers State University,
between June 2019 and February 2020.
Methodology: Soil samples were collected from a mechanic village while spent inverter batteries
were obtained from a waste vendor. The battery was forced open to extract its contents of the
battery. Using standard microbiological techniques, fungi were enumerated and characterized.
Stock solution of the battery content was prepared by dissolving the inverter battery content in
sterile deionized water. This stock solution was used to carry out the screening test on the fungal
isolates to ascertain the fungi that can tolerate the contents of the spent battery.
Results: Total heterotrophic fungal counts for the polluted and unpolluted soil were 6.0 x 10
3
cfu/g
and 7.5 x 10
4
cfu/g respectively. The fungal isolates identified from the polluted soil samples were
members of the genera Rhizopus, Mucor, Aspergillus, Penicillium, and Candida, while, the isolates
identified from the unpolluted soil sample includes: Candida sp, Aspergillus niger, Penicillium sp,
Aspergillus fumigatus, Aspergillus flavus, Mucor sp, Yeast, Fusarium sp and Aspergillus sp. After
Original Research Article

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35
the screening, total heterotrophic fungal counts for the soil ranged from 1.0 x 10
2
cfu/g to 9.5 x
10
2
cfu/g. Two fungi of the genera: Rhizopus and Mucor had the highest counts during 72 hours of
incubation for the screening test. The results obtained from this study indicated that species of
Aspergillus, Penicillium, and Candida were the most inhibited by the contents of the spent battery
while Rhizopus and Mucor spp were more tolerant to the contents of the inverter. Rhizopus and
Mucor spp were therefore, adopted in the bioremediation of soil contaminated with contents from
the battery. It was observed that Rhizopus and Mucor spp in a consortium had the highest
percentage of heavy metal removal (or uptake) in the following order: Cadmium (66.66%) > Lead
(38.15%) > Zinc (26.83%) > Nickel (20.83).
Conclusion: These organisms can be used in the bioremediation of soil polluted with metals from
spent deep cycle batteries.
Keywords: Fungal isolates; soil; inverter battery content; Rhizopus; Mucor.
1. INTRODUCTION
As a result of man’s quest to make his
environment more conducive for living, as well as
advances in information and communications
industries, various electronic devises are
manufactured, and used. This has led to large
scale generation of waste, called electronic or e-
waste at the end of their life span [1]. Most of its
components are harmful to the environment and
the living things in it [2]. When these wastes are
improperly disposed of they may pollute water
bodies; and also they may contaminate soil and
seep into groundwater. Owing to poor power
problem, there is an increased demand for
various charged batteries which are used in
diverse electronic gadgets, including inverters,
cell phones, laptops, television, refrigerators etc
[3].
Batteries and other energy storage devices store
energy so that it can be used when needed. In a
stand-alone power system, the energy stored in
batteries can be used when energy demand
exceeds the output from renewable energy
sources like solar and wind [4]. Battery types can
be divided into two basic categories namely, the
primary batteries (e.g. Mercury oxide, Lithium,
silver oxide, Zinc-carbon) which are disposable
and the secondary batteries (e.g. Lithium-ion,
Nickel-metal hydride, Nickel-Cadmium, Lead-
acid) which are rechargeable [5]. Lithium-ion
batteries are about half as toxic to humans as
Lead-acid batteries, and less toxic than nickel-
cadmium batteries. Nickel-metal hydride
batteries are the least toxic to humans [6].
Deep cycle batteries are rechargeable batteries
that could be drained of most of their power and
recharged repeatedly. Deep cycle batteries are
composed of thick solid lead plates. Because the
plates are thicker there is less surface area
producing less current. But this current can be
produced and maintained for longer periods [7].
Inverters are a class of deep cycle battery that
turns energy from one form to another. An
inverter is an electronic device or circuitry that
converts direct current (DC) to alternating current
(AC) [8].
Inverter batteries are classified based on the
chemistry of their cells. The four major categories
include; Nickel-Cadmium (NiCad), Lithium-ion,
Nickel-metal hydride and the Lead-Acid (L-A) (A1
Power Technologies, 2019) [9]. The L-A batteries
are the most common type and are more
hazardous and prone to leaks than the other
battery types [10]. Lead-Acid batteries contain
60-75% elemental lead, lead dioxide and a
sulphuric acid solution electrolyte. These heavy
metal elements make them toxic, and improper
disposal can be hazardous to the environment
(Green Earth Battery Recycling),
microorganisms, animalsand human health [1].
Heavy metals are elements that exhibit metallic
properties such as ductility, malleability,
conductivity, cation stability, and ligand specificity
with relatively high density and high relative
atomic weight and an atomic number greater
than 20 [11]. Some heavy metals that are toxic to
soil organisms, plants and humans include:
mercury, lithium, cadmium, chromium, lead,
nickel, selenium, and silver. These heavy metals
are toxic even at very low concentrations [11].
Bioremediation is a known biological technique
which relies on microorganisms and plants to
alter heavy metals bioavailability in the
environment and can be enhanced by addition of
organic amendments to soils [12]. It is effective,

Douglas et al.; SAJRM, 8(1): 34-46, 2020; Article no.SAJRM.62224
36
economical and environmentally friendly
compared to other remediation techniques [13].
Microbial activities are essential to how pollutants
in the ecosystem are transformed which is
reflected in biogeochemical cycles and food
webs. The mode in which microorganisms
respond to toxicants in an ecosystem will
partially, if not majorly, determine the fate of that
ecosystem when the assimilative capacity has
not been exceeded [2].
This study was conducted to evaluate the
tolerance of soil fungi to the content of deep
cycle battery as well as their bioremediation
potential in the removal of selected heavy metals
from a contaminated soil.
2. METHODOLOGY
2.1 Sampling
Polluted soil samples were collected from e
mechanic village located at Odo lane,
Rumuochita beside Kesioru playground in Obio-
Akpor Local Government, Rivers State, Nigeria.
The coordinates are 4º50ˈ46ʺ N and 6º59ˈ9ʺ E.
Also, uncontaminated soil samples were
collected from the school farm in Rivers State
University which is void of electronic waste
contamination. The soil samples were collected
into black polyethylene bags using soil auger.
Spent inverter battery was obtained from a
dumpsite worker at a major dumpsite at Location
road, off Ada George road, Port Harcourt.
2.2 Physicochemical and Heavy Metals
Analysis of the Soil Samples
The physicochemical parameters were
determined using Standard Methods according to
APHA [14]. The following parameters were
analyzed: Sulphate (SO
4
2-
), Phosphate (PO
4
3-
),
Electrical conductivity, pH, Temperature,
Moisture Content and Total Organic Carbon. Zinc
(Zn), Nickel (Ni), Lead (Pb) and Cadmium (Cd)
were the heavy metals analysed in this study
[15].
2.3 Analysis of Some Heavy Metals
Present in the Battery
The inverter battery content was analyzed for the
levels of some heavy metals and their quantity
(in mg/kg) using the API-RP45 method
(American Petroleum Institute Recommended
Practices). The heavy metals analyzed were
Zinc, Nickel, Lead and Cadmium, using an
Atomic Absorption Spectrophotometer (AAS)
calibrated daily with specific metallic standard
[15].
2.4 Enumeration of Total Heterotrophic
Fungi
The standard plate count method was used in
enumerating fungi in the soil samples. Serial
tenfold dilution was made from soil samples from
both the mechanic village and the school farm.
One gram (1g) of each the soil sample was
transferred into a test tube containing 9ml sterile
diluent (normal saline). Subsequent serial
dilutions were carried made up to 10
-4
. Using a
sterile pipette, 0.1ml amounts of dilutions 10
-1
,
10
-3
and 10
-4
were inoculated in duplicate on to
freshly prepared sterile Sabouraud dextrose agar
(SDA) plates, to which 0.2ml of 0.5% ampicillin
was added to prevent bacterial growth, and
incubated at 25°C for 2-5 days. The fungal
colonies were counted; average counts of the
duplicate plates were recorded. Discrete colonies
were subcultured unto freshly prepared SDA
plates to get pure fungal isolates, which were
preserved on SDA slants [1].
2.4.1 Identification of fungal isolates
The fungal isolates were identified based on
macroscopic examination of the colonies such
as: colour of colony, shape, and surface
appearance. The microscopic examination was
by the wet mount method as described by
Cheesebrough, [16]. From the pure culture
plates a small portion of the isolate was
picked using a sterile inoculating needle,
placed on a clean grease free slide, to
make a smear with lactophenol. Thereafter, the
slide was covered with cover slip and viewed
under the microscope at X10 and X40 to check
the hyphae (septate and non-septate) and
fruiting body according to Barnett and Hunter
[17].
2.5 Preparation of Stock Solution
The stock solution of the inverter battery was
prepared using the method of Odokuma and
Akponah[18] and Kpormon and Douglas [2] with
slight modifications. In this method, 4g of the
inverter battery content was dissolved in sterile
100 ml deionized water.

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Table 1. Experimental setups for bioremediation treatments
S/N
Set up label
Treatments
1 CONTROL 2.5 kg Soil + 125 ml Battery Content
2 SI/FA Rhizopus sp. + 2.5 kg soil + 125 ml battery content
3 SI/FB Mucor sp. + 2.5 kg soil + 125 ml battery content
4 SI/FA/FB Rhizopus sp. + Mucor sp + 2.5 kg soil + 125 ml inverter content
2.5.1 Preliminary screening test
The method of Odokuma and Akponah [18] was
adopted, where 9ml of contents from the inverter
battery stock solution was dispensed into sterile
labeled test tubes (test tubes were labeled
according to the isolates to be screened). One
milliliter (1ml) of 48 hours old fungal cultures in
Sabouraud dextrose broth were transferred into
respective labeled test tubes containing 9ml of
the battery content. Another set of test tubes
which contained only 9ml of normal saline and
1ml of the respective inoculum served as the
positive control while an uninoculated 10ml test
tube containing the battery content only served
as the negative control. The inoculated test tubes
were incubated at 25°C for 48 hours. After
incubation, aliquots of 0.1ml from the different
test tubes were drawn and inoculated on fresh
Sabouraud dextrose agar plates using the
spread plate method. Inoculated plates were
incubated at 25°C for 48 hours. Fungal isolates
that were able to proliferate after screening, as
seen in Table 1 were adopted for the
bioremediation set up [18].
2.5.2 Production of inoculum for
bioremediation experiments
The screened fungal isolates preserved in SDA
slants were subcultured to obtain 72hour old
cultures. One milliliter of each of the screened
fungal isolates was separately inoculated into
1000 ml of freshly prepared sterile Sabouraud
Dextrose Broth in a 1500 ml Erlenmeyer flask.
Incubation followed at 25°C for 72hours [18].
2.6 Bioremediation Experimental Setup
Bioaugmentation was performed using pure
cultures of Rhizopus sp and Mucor sp, where
unpolluted soil sample was prepared for the
bioremediation process and subdivided into the
various experimental setups, 2.5 kg each, in
which was added with 125 ml of stock solution
from inverter content and 25 ml of fungal culture
[19]. This was done for each set up using
Rhizopus and Mucor spp as bioaugmenting
organisms individually and in a consortium as
described in Table 1. After mixing properly,
microcosms were kept at ambient temperature in
green house. Sterile distilled water was used to
water every 5days to maintain water holding
capacity of 50% and properly tilled for proper
aeration and mixing using a sterile hand trowel.
Samples were taken out every seven days to
monitor the levels of the metals for 28 days.
Table 1, shows the various experimental set ups.
2.6.1 Percentage (%) heavy metal uptake
evaluation
The percentage (%) heavy metal removal is
calculated as follows:
% Toxin or Heavy Metal Removal = 100 x [(C
0
-
C
I
) /C
0
]
Where C
0
= initial concentration
C
I
= final concentration
2.7 Data Analysis
The data gathered in this study were subjected to
statistical analysis. The data were properly
arranged in the Microsoft excel (2016 version),
the means and standard deviations were
computed using the SPSS (Version 22).
3. RESULTS AND DISCUSSION
The total heterotrophic fungal counts obtained for
the polluted and unpolluted soil samples were
6.0 x 10
3
cfu/g and 7.5 x 10
4
cfu/g respectively.
The results of microbiological analyses
conducted on the soil samples showed that the
unpolluted soil had a higher fungal population,
when compared to the polluted soil. This may be
as a result of the presence of the pollutant in the
soil. When there is pollutant in an environment it
puts a selective pressure on the organisms
present, those that are able to with stand the
pollutant grow while others die off. Thereby,
affecting the fungal diversity and populations of
the soil polluted soil [1].
The fungal isolates identified from the polluted
soil samples were members of the genera

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38
Rhizopus, Mucor, Aspergillus, Penicillium, and
Candida. The results are presented in Table 2.
The fungal isolates identified from the unpolluted
soil sample includes: Candida sp, Aspergillus
niger, Penicillium sp, Aspergillus fumigatus,
Aspergillus flavus, Mucor sp, Yeast, Fusarium sp
and Aspergillus sp. The results are also
presented in Table 3. These results shown that
pollution affects fungal population and diversity.
Table 2. Cultural and morphological characteristics of fungal isolates from polluted soil
Isolates
Macroscopy
Microscopy
Probable Identity
A White cottony growth with
blackish spores, yellow reverse
Non septate hyphae with
non-septate
sporangiophores bearing
sporangia
Rhizopus spp
B White fluffy growth, white reverse Aseptate hyphae bearing
long sporangiosphore,
presence of bug
Mucor spp
C Blue-green powdery growth, pale
yellow reverse
Septate hyphae with
smooth-walled
conidiophores bearing
conidia in chains
Aspergillus spp
D White to grey-green flat cottony
growth, pale yellow reverse
Septate hyphae with long
conidiophores bearing
conidia in chains
Penicillium spp
E Smooth creamy colonies Branched pseudohyphae
with blastoconidia in small
clusters
Candida spp
Table 3. Cultural and morphological characteristics of fungi from the unpolluted soil
Isolates
Macroscopy
Microscopy
Prob. ID
A. Cream large round Oval budding
blastoconidia
Candida spp
B. Black spores surrounded by
cream background, brown
reverse
Septate hyphae with
aeseptateconidiosphore
bearing conidia
Aspergillus niger
C. Green powdery surface
surrounded by white lawn, brown
reverse
Septate hyphae with
septate conidiophores
bearing conidia
Penicillium spp
D. Black-brown suede surface, black
reverse
Septate hyphae with
aeseptateconidiosphore
and scattered conidia
Aspergillus
fumigates
E. Light green lawn surrounded by
white lawn-like growth
Septate hyphae with
aeseptateconidiosphore
bearing conidia
Aspergillus flavus
F. Fluffy white cottony, white reverse Aeseptate hyphae
bearing sporangiospores
Mucor spp
G. white small round Oval budding
blastoconidia
Yeast
H. White cottony lawn like growth,
with reverse yellow colour
Septate hyphae, with
presence of banana
shaped septate conidia
Fusarium spp
I. Black spores surrounded by
cream background, brown
reverse
Septate hyphae with
aeseptateconidiosphore
bearing conidia
Aspergillus spp

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39
Table 4, shows the selected heavy metals
analysis of the inverter content which showed
that Lead concentration was 1.577 mg/kg, Nickel
was 0.292 mg/kg, Cadmium was 0.059 mg/kg,
Zinc was <0.005 mg/kg. This confirms that the
Luminous inverter battery used in this study is a
Lead-Acid battery (L-A battery), not a Nickel-
Cadmium battery (NiCad battery) or Mercury
battery. The Intervention Values for
Micropollutants for a Standard Soil in Nigeria is
Lead: 530 mg/kg, Nickel: 210 mg/kg, Zinc: 720
mg/kg and Cadmium: 380 mg/kg [20]. This
implies that currently Lead-Acid inverters are not
likely to pose any threat of heavy metal pollution
to our environment.
Table 4. Heavy metal content of the inverter
battery
Heavy Metals Battery Content(mg/kg)
Zinc <0.005
Nickel 0.292
Lead 1.577
Cadmium 0.059
Results of the preliminary screening for the
tolerance of the fungal isolates to the contents of
the battery (Table 5), showed that two of the
isolates (Rhizopus and Mucor spp) were able to
tolerate the toxic contents of the inverter while
the other three isolates (Aspergillus, Penicillium,
and Candida) were inhibited by the contents of
the inverter, hence showed no growth. Rhizopus
and Mucor spphave been found to be among the
list of fungi to have the highest metal adsorption
capacities as reported by Zaidi et al., [21]. This
may also be due to the fact that apart from the
presence of heavy metals, some other
components contained in the battery content may
be toxic, which completely inhibited the growth of
Aspergillus, Penicillium, and Candida spp. These
fungi may lack the mechanisms required to
tolerate the toxic contents of the inverter battery.
Microorganisms found in polluted environment
possess astonishing metabolic pathways which
tolerate and possibly utilizes various toxic
compounds as a source of energy for growth and
development, through respiration, fermentation,
and cometabolism [22]. According to Ayangbenro
and Babalola (2017) [23], the majority of heavy
metals disrupt microbial cell membranes, but
microorganisms can develop defense
mechanisms that assist them in overcoming the
toxic effect and also transform pollutants in the
environment.
The mechanism of heavy metal uptake in fungi
(living fungal cells) is basically metabolism-
independent uptake, which involves adsorption
processes such as ionic, chemical and physical
uptake. A variety of ligands located on the fungal
cell walls are known to be involved in metal
chelation [24].
The results of the physicochemical parameters
for the polluted and unpolluted soil samples are
presented in Table 6. The results showed that the
pH of the polluted and unpolluted soils used in
this study were 8.5 and 7.5 respectively. The
polluted soil has a higher pH than the unpolluted
soil which is agreement with the results obtained
by Klimek and Niklinska[25] who observed that
the pH of polluted soils is significantly higher than
that of unpolluted soils. As seen from the results
of the total heterotrophic fungal counts, the
unpolluted soil is still capable of supporting a
wider range of soil fungi.
The results of electrical conductivity of the
polluted and unpolluted soils are 0.51µS/cm and
0.05µS/cm respectively. Soil electrical
conductivity (EC) is a measure of the amount of
salts in soil (salinity of soil) [26]. It is an important
indicator of soil health and can serve as an
indirect indicator of the moisture content and
water-soluble nutrients available for plant
removal such as nitrate, sulphate and phosphate
[26]. Thus, this confirms the results of the
moisture content of polluted and unpolluted soils
(6.21% and 11.48% respectively), meaning a
lower moisture content for the polluted soil with a
high electric conductivity. Soil microbial activity
declines as EC increases [26]. This impacts
important soil processes such as respiration,
residue decomposition, nitrification and
dentrification [26]. According to the United States
Department of Agriculture (USDA, 2013), When
EC readings are less than 1 dS/m, soil are
considered non-saline and do not impact soil
microbial processes while EC readings greater
than 1 dS/m means the soil are considered
saline and impact important microbial processes,
such as nitrogen cycling, production of nitrous
and other N-oxide gases, respiration, and
decomposition; increased nitrogen losses;
populations of plant-parasitic nematodes can
increase [27]. The both soil samples showed EC
readings less than 1 dS/m meaning the soil
microbial processes are not impacted despite the
presence of the metals.
The phosphate (PO
4
3-
) levels of the polluted and
unpolluted soils are 0.22 mg/kg and 0.86 mg/kg

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40
respectively. According to Ademola et al., [28],
phosphate presents itself as a nutrient to soil
microflora and readily sequesters metals and
reduces their bioavailability via the formation of
insoluble metal phosphate species. This
explains why the phosphate level in the
polluted soil is lower than that of the unpolluted
soil. The results for nitrate (NO
3
2-
) of the polluted
and unpolluted soils (26.00 mg/kg and 33.00
mg/kg respectively) are in line with the
findings made by Tanee et al., [29]; with nitrate
values of 23.04 mg/kg and 61.76 mg/kg for
polluted and unpolluted soils respectively and
Ataikiru et al., [30], with nitrate values of <0.001
mg/kg and 15.42 mg/kg for polluted and
unpolluted soils respectively, showing that
most heavy metal polluted soils usually have
lower nitrate concentrations compared to non-
polluted soils. In polluted sites, the limiting
nutrients, (nitrate and phosphate) which are
essential for biodegradation to occur are
usually released to the microorganisms involved,
thereby causing a reduction in the concentration
of these nutrients (Ataikiru et al. [30]. The
sulphate (SO
4
2-
) of the polluted and
unpolluted soils (14.37 mg/kg and 36.26 mg/kg
respectively) is an indicator that the fungi present
in the soil might be involved in inorganic nutrition
[31].
3.1 Bioremediation Potential of Screened
Fungal Isolates
The treatments for the bioremediation
(bioaugmentation) process are described in
Table 1. The results of the individual heavy metal
uptake by the isolates are presented in Tables 7 -
10. The results revealed that the concentration
of Zinc in the contaminated soil was reduced
greatly by the combination of Rhizopus and
Mucor spp used in a consortium when compared
to their individual performances. This was also
observed in the reduction of the concentrations
of the other metals - Nickel, Lead and Cadmium.
Rhizopus and Mucor spp in a consortium showed
heavy metal percentage removal in the following
order: Cadmium (66.66%) > Lead (38.15%) >
Zinc (26.83%) > Nickel (20.83). This is observed
in Figs. 1 to 4. This implies that a combination of
potent microorganisms in a consortium has
higher efficiency in the removal of heavy metal
compared to single species, and the final
removal efficiency for the consortium could be
reached in a considerably shorter time [32].
Table 5. Fungal counts after screening
Isolates With toxicant (cfu/ml) Without toxicant (cfu/ml)
Rhizopus sp 2.1x10
2
1.0 x10
2
Mucor sp 2.3x10
2
1.0 x10
2
Aspergillus sp No growth 1.5 x10
3
Penicillium sp No growth 9.0x10
2
Candida sp No growth 9.5x10
3
Table 6. Physicochemical properties of the soil samples
Parameters Unit Polluted soil Unpolluted soil
Nitrate (NO
3
2-
) mg/kg 26.00 33.00
Sulphate (SO
4
2-
) mg/kg 14.37 36.26
Phosphate (PO
4
3-
) mg/kg 0.22 0.86
Electrical conductivity µS/cm 0.51 0.05
pH
8.6
7.5
Temperature °C 29.2 29.3
Moisture Content % 6.21 11.48
Total Organic Carbon
mg/kg
0.38
0.35
Zinc (Zn) mg/kg 6.80 0.83
Nickel (Ni) mg/kg 3.00 0.71
Lead (Pb) mg/kg 3.82 0.54
Cadmium (Cd) mg/kg 1.00 0.03

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41
Table 7. Rate of metal uptake of zinc in the setup
Setup Identity
Unit
Initial concentration
Day 0
Day 7
Day 14
Day 21
Day 28
CONTROL mg/kg 0.83±0.02
a
0.83±0.01
a
0.78±0.01
d
0.74±0.01
f
0.70±0.01
de
0.69±0.01
e
SI/FA mg/kg 0.83±0.02
a
0.80±0.01
a
0.74±0.01
abc
0.71±0.01
ef
0.70±0.01
de
0.65±0.01
e
SI/FB mg/kg 0.83±0.02
a
0.82±0.01
a
0.75±0.01
bcd
0.73±0.01
f
0.72±0.01
e
0.66±0.01
de
SI/FA/FB mg/kg 0.83±0.02
a
0.82±0.01
a
0.72±0.01
ab
0.67±0.01
cd
0.62±0.01
b
0.60±0.01
c
Means with similar superscripts within columns show no significant difference at P ≥ 0.05
Table 8. Rate of metal uptake of nickel in the setup
Setup Identity
Unit
Initial concentration
Day 0
Day 7
Day 14
Day 21
Day 28
CONTROL mg/kg 0.72±0.01
a
0.72±0.01
a
0.70±0.01
d
0.70±0.01
e
0.68±0.01
f
0.66±0.01
e
SI/FA mg/kg 0.72±0.01
a
0.70±0.01
a
0.67±0.01
cd
0.64±0.01
d
0.61±0.01
e
0.57±0.01
d
SI/FB mg/kg 0.72±0.01
a
0.70±0.01
a
0.67±0.01
cd
0.64±0.01
d
0.60±0.01
de
0.58±0.01
d
SI/FA/FB mg/kg 0.72±0.01
a
0.72±0.01
a
0.65±0.01
abc
0.62±0.01
cd
0.56±0.01
bc
0.57±0.01
d
Means with similar superscripts within columns show no significant difference at P ≥ 0.05
Table 9. Rate of metal uptake of lead in the setup
Setup Identity
Unit
Initial concentration
Day 0
Day 7
Day 14
Day 21
Day 28
CONTROL mg/kg 0.54±0.01
a
0.54±0.01
a
0.47±0.01
d
0.47±0.01
f
0.48±0.01
e
0.47±0.01
c
SI/FA mg/kg 0.54±0.01
a
0.55±0.01
a
0.55±0.01
e
0.38±0.01
d
0.34±0.01
c
0.35±0.01
b
SI/FB mg/kg 0.54±0.01
a
0.54±0.01
a
0.48±0.01
d
0.43±0.01
e
0.40±0.01
d
0.35±0.01
b
SI/FA/FB mg/kg 0.54±0.01
a
0.54±0.01
a
0.51±0.01
d
0.38±0.01
d
0.35±0.01
c
0.33±0.01
b
Means with similar superscripts within columns show no significant difference at P ≥ 0.05
Table 10. Rate of metal uptake of cadmium in the setup
Setup Identity
Unit
Initial concentration
Day 0
Day 7
Day 14
Day 21
Day 28
CONTROL mg/kg 0.03±0.01
a
0.03±0.01
a
0.03±0.01
a
0.03±0.01
a
0.02±0.01
a
0.02±0.01
a
SI/FA mg/kg 0.03±0.01
a
0.03±0.01
a
0.03±0.01
a
0.02±0.01
a
0.02±0.01
a
0.01±0.00
a
SI/FB mg/kg 0.03±0.01
a
0.03±0.01
a
0.03±0.01
a
0.03±0.01
a
0.02±0.01
a
0.01±0.00
a
SI/FA/FB mg/kg 0.03±0.01
a
0.02±0.01
a
0.03±0.01
a
0.03±0.01
a
0.02±0.01
a
<0.01±0.00
a
Means with similar superscripts within columns show no significant difference at P ≥ 0.05

Fig
Key: SI/FA = Rhizopus sp. + 2.5
kg soil + 125
inverter content;
SI/FA/FB =
Fig
SI/FA = Rhizopus sp. + 2.5
kg soil + 125
content; SI/FA/FB =
Rhizopus sp. + Mucor sp
The increase in the microbial load of the control
was not significant (at P
≥ 0.05) while
significant increase was observed in the fungal
load of the set up
containing a combination of
Rhizopus + Mucor
spp in a consortium
(2.67±0.38
a
<3.78±0.03
a
< 3.89±0.27
3.93±0.21
a
< 4.17±0.06
a
) load during the 28 days
compared to the individual performances of the
31%
PERCENTAGE (%) ZINC UPTAKE
32%
PERCENTAGE (%) NICKEL UPTAKE
Douglas et al.; SAJRM, 8(1): 34-46, 2020
; Article no.
42
Fig
. 1. Percentage zinc removal
kg soil + 125
ml inverter content; SI/FB = Mucor sp. + 2.5
kg soil + 125
SI/FA/FB =
Rhizopus sp. + Mucor sp + 2.5
kg soil + 125ml inverter content
Fig
. 2. Percentage nickel uptake
kg soil + 125
ml inverter content; SI/FB = Mucor sp. + 2.5
kg soil + 125
Rhizopus sp. + Mucor sp
+ 2.5
kg soil + 125ml inverter content
The increase in the microbial load of the control
≥ 0.05) while
a
significant increase was observed in the fungal
containing a combination of
spp in a consortium
< 3.89±0.27
ab
<
) load during the 28 days
compared to the individual performances of the
fungi.
This implies that the spent inverter content
was inhibitory to some of the fungal species in
the control while the fungi adopted for the study
as well as some fungal species present in the
soil were able to tolerate the spent battery
content, thereby incre
asing their fungal counts.
similar observation was made by Ataikiru
[30], in their research on Bioremediation of
20%
24%
25%
31%
PERCENTAGE (%) ZINC UPTAKE
CONTROL
SI/FB
SI/FA
SI/FA/FB
13%
26%
29%
32%
PERCENTAGE (%) NICKEL UPTAKE
CONTROL
SI/FB
SI/FA
SI/FA/FB
; Article no.
SAJRM.62224
kg soil + 125
ml
kg soil + 125ml inverter content
kg soil + 125
ml inverter
kg soil + 125ml inverter content
This implies that the spent inverter content
was inhibitory to some of the fungal species in
the control while the fungi adopted for the study
as well as some fungal species present in the
soil were able to tolerate the spent battery
asing their fungal counts.
A
similar observation was made by Ataikiru
et al.,
[30], in their research on Bioremediation of
CONTROL
SI/FA/FB
CONTROL
SI/FA/FB

Bonny light crude oil polluted soil by
bioaugumentation using yeast isolates. They
observed a significant increase in the fungal load
of microcosm A of which
Rhizopus
Fig
Key: SI/FA = Rhizopus sp. + 2.5 kg
soil + 125
inverter content; SI/FA/FB =
Rhizopus sp. + Mucor sp
Fig.
4
Key: SI/FA = Rhizopus sp. + 2.5
kg soil + 125
inverter content;
SI/FA/FB =
30%
PERCENTAGE (%) LEAD UPTAKE
30%
PERCENTAGE (%) CADMIUM UPTAKE
Douglas et al.; SAJRM, 8(1): 34-46, 2020
; Article no.
43
Bonny light crude oil polluted soil by
bioaugumentation using yeast isolates. They
observed a significant increase in the fungal load
Rhizopus
and Mucor
spp were inclusive (6.21±0.15
a
< 7.70±0.12
7.28±0.25
ac
) over a period of 28
days, based on
monitoring fortnightly.
Fig
. 3. Percentage lead uptake
soil + 125
ml inverter content; SI/FB = Mucor sp. + 2.5
kg soil + 125
Rhizopus sp. + Mucor sp
+ 2.5 kg soil + 125
ml inverter content
4
. Percentage uptake of cadmium
kg soil + 125
ml inverter content; SI/FB = Mucor sp. + 2.5
kg soil + 125
SI/FA/FB =
Rhizopus sp. + Mucor sp + 2.5
kg soil + 125ml inverter content
10%
32%
28%
30%
PERCENTAGE (%) LEAD UPTAKE
CONTROL
SI/FB
SI/FA
SI/FA/FB
14%
28%
28%
30%
PERCENTAGE (%) CADMIUM UPTAKE
CONTROL
SI/FB
SI/FA
SI/FA/FB
; Article no.
SAJRM.62224
< 7.70±0.12
a
<
days, based on
kg soil + 125
ml
ml inverter content
kg soil + 125
ml
kg soil + 125ml inverter content
CONTROL
SI/FA/FB
CONTROL
SI/FA/FB

Douglas et al.; SAJRM, 8(1): 34-46, 2020; Article no.SAJRM.62224
44
Fig. 5. Mean fungal counts (cfu/g) of the bioremediation setup during the 28 days
Key: SI/FA = Rhizopus sp. + 2.5 kg soil + 125 ml inverter content; SI/FB = Mucor sp. + 2.5 kg soil + 125ml
inverter content; SI/FA/FB = Rhizopus sp. + Mucor sp + 2.5 kg soil + 125 ml inverter content
4. CONCLUSION
Soil fungi play very important role in the soil
structure, production of humus, decomposition,
nutrient recycling and organic matterproduction
of the soil. Mucor and Rhizopus spp were among
the indigenous fungal species identified in the
polluted soil that have the ability to tolerate the
contents of deep cycle battery. The fungal
isolates - Mucor and Rhizopus spp in a
consortium were found to be more efficient
in the removal of the selected heavy metals
during the 28days monitoring period, yielding a
higher percentage of heavy metal uptake
compared to their individual performances.
Therefore, since Mucor and Rhizopus spp
were able to tolerate contents of deep
cycle batteries, they could be used as
bioaugumenting organisms to bioremediate soil
polluted with contents from spent deep cycle
batteries.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
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