Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

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Case Report

Bioremediation of lead contaminated environment by Bacillus cereus strain

BUK_BCH_BTE2: Isolation and characterization of the bacterium
Fatima Abdullahi Harun a, b, Muhammad Rabiú Yusuf a, Shehu Usman c, Dayyabu Shehu a,
Kamaluddeen Babagana a, Aminu Jibril Sufyanu a, Muhammad Mustapha Jibril a,
Aliyu Maje Bello a, Kabiru Abubakar Musa a, Ahmad Hussaini Jagaba d, **, Mohd Yunus Shukor e,
Hafeez Muhammad Yakasai a, f, *
a
Department of Biochemistry, Faculty of Basic Medical Sciences, College of Health Sciences, Bayero University Kano, P.M.B. 3011, Kano, Nigeria
b
Department of Biochemistry, Faculty of Science and Computing, Capital City University Kano, P.M.B. 3409, Kano, Nigeria
c
Department of Plant Science and Biotechnology, Federal University Dutsin-Ma, Katsina, Nigeria
d
Interdisciplinary Research Centre for Membranes and Water Security, King Fahd University of Petroleum and Minerals, Dhahran, 31261, Saudi Arabia
e
Department of Biochemistry, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400, Serdang Selangor, Darul Ehsan, Malaysia
f
Skyline University Nigeria, No. 2 Zaria Road, Kano State, Nigeria

A R T I C L E I N F O A B S T R A C T

Keywords: Lead is one of the most toxic heavy metals ever known by man today, which has zero biological role. It is known
Lead to cause anemia and affect the nervous and reproductive systems with irreversible effects. Its continual usage
Heavy metals leads to its high accumulation polluting the soil and water bodies with devastating health effects. This research,
Isolation
aimed at isolation and characterization of indigenous lead tolerant bacteria from contaminated soil of Anka.
Bacteria
Results of Atomic Absorption Spectrophotometry (AAS) showed high concentration of lead (738 mg/kg) of the
Lead-tolerant
affected sites above the EPA standard (400 mg/kg). Similarly, a mineral salt media was used to isolate the
bacteria following serial dilution. An indigenous bacterial isolate (Ac) with potential to tolerate up to 3000 mg/L
Pb(NO3)2 was isolated and molecularly identified based on 16s rRNA sequencing as Bacillus cereus strain
BUK_BCH_BTE2 with the accession number MT160412. The isolate was further characterized for its optimum
growth and tolerance conditions using one factor at a time (OFAT). Sucrose with an optimum concentration of 5
g/L was the best carbon source for the isolate. The isolate was found to utilize ammonium sulphate as the best
nitrogen source at a concentration of 2.5 g/L, with an optimum pH and tempertaure of 7.0 and 37 ◦ C respec­
tively. A concentration of 1000 mg/L Pb(NO3)2 was found to be the optimum concentration for the isolate. The
optimum incubation time and inoculum size were found to be 48 hrs and 100 μL respectively. The fact that the
isolate could tolerate high Pb(NO3)2 concentration makes it suitable for future bioremediation work involving
lead.

1. Introduction concentration. Heavy metals such as Hg, Pb, Cr, Cd are non-essential and
thus toxic [2]. Lead is toxic even at low concentration as it has no known
The rapid increase in population as well as in industrialization and biological role in the system. Lead-poisoning remains an important and
urbanization are the major cause of environmental pollution we are common condition that contributes to 0.6 % of the global burden of
facing today [1]. Anthropogenic activities such as mining, smelting, as illness and causes diseases with an environmental origin [3]. Lead
well as refining to mention but few are some of the events that increased poisoning typically affects children under the age of six and can have a
the level of heavy metals to toxic level above the traditional negative impact on their physical and mental growth. Inflammation of

* Corresponding author. Department of Biochemistry, Faculty of Basic Medical Sciences, College of Health Sciences, Bayero University Kano, P.M.B. 3011, Kano,
Nigeria.
** Corresponding author. Interdisciplinary Research Centre for Membranes and Water Security, King Fahd University of Petroleum and Minerals, Dhahran, 31261,
Saudi Arabia.
E-mail addresses: ahjagaba@gmail.com (A.H. Jagaba), hmyakasai.bch@buk.edu.ng (H.M. Yakasai).

https://doi.org/10.1016/j.cscee.2023.100540
Received 23 September 2023; Received in revised form 24 October 2023; Accepted 1 November 2023
Available online 2 November 2023
2666-0164/© 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

the kidneys, seizures, brain damage, anaemia, muscle weakness, and

gastro-intestinal symptoms are just a few of the major health disorders
that can be caused by elevated blood lead levels (BLL) [4,5]. Minimal
lead levels are found to be connected to poorer IQ scores and behav­
ioural problems [3].
Several strategies have been employed for clean-up of the lead
contaminated environment such as physical, chemical, and biological
remediation of soil. A variety of techniques are available, including
excavation, in-situ vitrification, soil dressing, and washing. But these
methods are not effective as they incur high cost and they might lead to
the production of secondary pollutants which have negative impact on
the environment [6,7]. Hence, the need for a safer, more reliable and
cost effective remediation technique. Research conducted by authors [8]
reported high lead levels in water samples analyzed using atomic ab­
sorption spectrophotometer of the gold-mine sites after one year of
clean-up activities in that site.
Researchers have focused on the bioremediation approach since both
physical and chemical procedures have fallen short of satisfying the
economic need for soil remediation [9]. Bioremediation is a
cost-effective remediation strategy. To minimize the toxicity of heavy
metals, specific microbial species are used to ingest, precipitate, oxidize,
or reduce the metals. For the bio-mineralization of heavy metals, a wide
Fig. 1. Flowchart showing the design of the research work.
range of fungi, algae, and bacteria are employed as biosorbents; some of
these organisms induce metal transitions and lessen toxicity. Toxic
heavy metals can be removed by a variety of microbes [10,11]. from the soil. The approach of Ref. [15] was modified to create serial
To date, literatures have dwelled on the isolation of microbes with dilution. An aliquot (100μ L) of 2 out of the six diluted samples were
lead-tolerant and/bioremediation potential. Thus, this research focused spread and plated in triplicates each (direct plating, 10− 3, and 10− 4) on
on not only isolation but also on characterization using one factor at a Luria-Bertani (LB) agar medium and incubated for 24 hrs at 37 ◦ C. The
time (OFAT) of the indigenous lead tolerant isolate from gold mining media contained lead nitrate at a concentration of 500 mg/L. The col­
areas of Anka, Zamfara Nigeria. onies were counted using a colony counter and purified by sub-culturing
onto freshly prepared LB agar plates by making use of the streak-plate
2. Materials and methods technique [9].

Materials that are of quality were used for this research work. Class II 2.1.3. Identification of bacterial isolates
biosafety cabinet was used for microbiological works. All glasswares Six bacterial isolates were obtained. Hence gram staining test and
were cleaned with 2 N HNO3 and rinsed with double distilled water. microscopic examination were performed for identification of the iso­
lates. From 24 hrs LB culture, sample was used to make a smear on a
2.1. Soil collection glass slide, the gram-stained preparation was examined with the oil
immersion objective of the bright-field microscope and identified based
Soil from four (4) sampling sites (Bagega, Dareta, Abare, and War­ on morphological characteristics [16,17].
amu) in Anka LGA,Zamfara State were collected, alongside samples
from Gusau Zamfara State and Sharada industrial area of Kano State as 2.1.4. Screening of lead-tolerant bacterial isolates
control. Using a soil auger, samples were obtained 0–10 cm down the To screen the bacterial isolates for lead tolerance, an aliquot (100 μL)
surface. The sampled soils were labeled, placed in a tidy, sterile poly­ of the bacterial isolates were inoculated in Luria Bertani (LB) agar
thene bag, and kept at room temperature. Fig. 1 shows the study containing varying concentrations of Pb(NO3)2 (500, 700, 900, 1100,
methodology. 1300, 1500, and 3000 mg/L) and incubated at 37 ◦ C for 24 hrs.
Following the required incubation, the plates were observed for grown
2.1.1. Assay of soil lead levels colonies; those isolates that could not tolerate a higher lead concentra­
Assay of the sampled soils was determined using an atomic absorp­ tion failed to grow, while the isolate that could tolerate up to 3000 mg/L
tion spectrophotometer (AAS). The sampled soils were digested by acid was selected for further analysis.
extraction process using the method described by Ref. [12] with modi­
fications in order to quantify the lead content of the soil. 2 g of soil 2.2. Molecular identification of the bacterial isolates
sample was treated with 24 ml mixture of hydrochloric and nitric acids
in the ratio of 3:1. The acid-treated soil was heated using a hot plate for 2 2.2.1. Genomic DNA extraction
hrs at 100 ◦ C in a conical flask. The contents of the conical flask after Using DNA extraction kits from QIAGEN, the genomic DNA of the
digestion were filtered into a 50 mL sterile container and topped up to bacterial isolate was extracted from a pure bacterial culture that was
the mark with deionized water. The filtrates were then analyzed for lead grown on Luria-Bertani Agar medium at 37 ◦ C for 24 hrs. The extraction
content using atomic absorption spectrophotometer [9]. A standard procedure was in accordance with the manual’s recommendations
calibration plot method was used to determine the corresponding lead (QIAGEN kit) and stored frozen at − 4 ◦ C until a PCR reaction was run.
concentration after measuring the absorbance by AAS, the metal con­
centration was recorded in milligrams per kilogram (mg/kg) after 2.2.2. Polymerase chain reaction (PCR)
extrapolation from the standard curve [13]. The 16S rRNA of the bacterial isolate was amplified using the uni­
versal primers 1447-F (AGAGTTGATCCTGGCTCAG) and 1492-R
2.1.2. Isolation of lead-resistant bacteria (GGTTACCTTGTTACGACTT), by PCR reaction using KapaTaq DNA
10 g of the sampled soil was added to 100 mL of deionized water, and polymerase. Bioer Gene Touch thermal cycler was used for the poly­
the mixture was shaken for three hrs [14] to separate microorganisms merase chain reaction. 15 L was used for the reaction as a whole. The

2
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

reaction mixture consists of 1.25 mM (0.75 L) MgCl2, 0.25 mM (0.15 L) determined using MSM media containing 500 mg/L Pb(NO3)2 alongside
dNTP mixes, 0.12 L of TaqDNA polymerase in dH2O, 0.4 M (0.5 L) of the best carbon and nitrogen sources, where the pH was varied from the
forward and reverse primers, 1.5 L of 10 TaqA buffer, and 1 L of genomic range of 3–8. A bacterial aliquot (100 μL) was used for the incubation at
DNA [18,19]. The following conditions were used for the amplification: 37 ◦ C for 24 hrs. After the required incubation, O.D. was measured
a five-minute initial denaturation at 95 ◦ C, 35 cycles of 30 sec each spectrophotometrically at 600 nm to determine growth. The pH was
denaturation, primer annealing, and extension, each lasting one min. A adjusted using 1 N NaOH and acetic acid [21].
final extension of 10 min at 72 ◦ C came next. To estimate the size of the
DNA, the PCR result was run on a 1.5 % agarose gel stained with 2.3.4. Effect of temperature
ethidium bromide and a side marker. A 14 well-comb was placed in the The optimum temperature was determined by varying the temper­
gel where it was allowed to settle for about 30 min. the comb was ature range (25, 30, 35, 37, 40, and 50 ◦ C) for the incubation of the
removed after solidification of the gel, hence the gel tray was placed in bacterial isolate using MSM broth containing 500 mg/L Pb(NO3)2
the buffer tank. The gel was submerged to a depth of about 2–5 mm by alongside the best concentrations of both carbon and nitrogen sources
pouring 0.5 × TBE into the tank. Following purification, the 16S rRNA and optimum pH for the isolate. Bacterial concentration (100 μL) was
amplicon (16S rRNA) was forwarded for sequencing to MYTACG incubated in the media over 24 hrs at 37 ◦ C. After the required incu­
Bioscience Enterprise in Selangor Darul Ehsan, Malaysia. bation, optical density (OD) was measured spectrophotometrically at
600 nm to determine the growth [15].
2.2.3. Phylogenetic analysis
The sequence was identified using the BLAST search software on the 2.3.5. Effect of substrate concentrations (Pb(NO3)2) on the lead-tolerant
NCBI website. The sequence was aligned using BIOEDIT version 7.2.5, isolate
after which all the closely related identities were downloaded for Bacterial isolate was tested for lead tolerance by growing it on MSM
additional determination from the NCBI website [20]. Sequence was with varying concentrations of Pb(NO3)2 (500, 1000, 2000, and 4000
aligned, and phylogenetic tree was built using neighbor-joining tech­ mg/L), incubated at pH 7.0 and temperature of 37 ◦ C for 24 hrs,
nique on Mega version 7 software. The accession number was then absorbance was measured spectrophotometrically at 600nm [21,22].
requested from NCBI by sending the sequences there [21].
2.3.6. Effect of incubation time
2.3. Characterization of lead-tolerant isolate The bacterial isolate (100 μL) was separately incubated in MSM
broth containing the optimum Pb(NO3)2 concentration (1000 mg/L),
Mineral salt media (MSM) was made in accordance with the di­ along with the best carbon and nitrogen sources, at an optimum pH of
rections provided by Ref. [22] in order to characterize the growth and 7.0 and a temperature of 37 ◦ C. An aliquot (2 ml) from the culture
tolerance of the isolate on lead nitrate (Pb(NO3)2) using one factor at a medium was periodically taken (6, 24, 30, 48, 54, 72, 78, and 96 hrs)
time (OFAT). The media contains: Na2HPO4, 4.0g; NaH2PO4, 1.5g; and optical density (OD) was measured spectrophotometrically at 600
FeCl3⋅6H2O 0.06g; MgSO4•7H2O, 0.2g; diammonium citrate, 0.2g; in 1 L nm to determine growth.
of deionized water. This content was in addition with 2 mL of modified
Hoagland trace elements solution comprising of (BH3, 11.0g; 2.3.7. Effect of inoculum size
MnCl4•4H2O, 7.0g; AlCl3, 1.0g; CoCl2, 1.0g; CuCl2, 1.0g; KI, 1.0g; NiCl2, The inoculum volume of the isolate was varied (25, 50, 100, 200, and
1.0g; ZnCl2, 1.0g; BaCl2, 0.5g; KBr, 0.5g; LiCl, 0.5g; Na2MoO4, 0.5g; 400 μL) and incubated in MSM broth containing the optimum Pb(NO3)2
SeCl4, 0.5g; SnCl2•2H2O, 0.5g; NaVO3•H2O, 0.1 g; in 3.6 L of deionized concentration (1000 mg/L), along with the best carbon and nitrogen
water) and adjusted to pH 7.0. sources, at an optimum pH of 7.0 and a temperature of 37 ◦ C for a period
between 24 and 96 hrs. After the required incubation, the culture me­
2.3.1. Effect of various carbon sources on the growth of lead-tolerant isolate dium was read using a spectrophotometer at 600 nm to determine
In MSM containing 500 mg/L Pb(NO3)2, various carbon sources, growth.
including glucose, fructose, sucrose, lactose, starch, and glycerol, were
independently used. In order to determine the optimal carbon source 2.4. Effect of other heavy metals
necessary for the growth of lead-tolerant bacteria, a bacterial aliquot
(100 μL) from the isolate was injected into the media and incubated at The bacterial isolate was tested for growth in the presence of other
37 ◦ C for 24 hrs. After the necessary incubation, the growth was assessed metal ions at a concentration of 1 ppm. The isolate was incubated in
using a spectrophotometer at an optical density (OD) of 600 nm. The MSM broth containing Pb(NO3)2 and various metal salts at 37 ◦ C over a
optimal concentration of the carbon source in the isolate was then period of 96 hrs, the optical density was measured spectrophotometri­
determined by optimizing the best carbon source at varying concen­ cally at 600 nm to determine growth [15].
trations (2.5, 5, 10, 20, 40, and 80 g/L) [15].
2.5. Statistical analysis
2.3.2. Effect of nitrogen sources on the growth of lead-tolerant isolate
The lead-tolerant isolate was incubated (100 μL) in MSM media GraphPad InStat and Microsoft Excel were used to analyze all the
containing 500 mg/L Pb(NO3)2, and supplemented with different ni­ data. One-way analysis of variance with Tukey’s post hoc analysis was
trogen sources; ammonium sulphate, ammonium chloride, urea, glycine, used to compare between the groups. Data was significant at P < 0.05.
and valine, and incubated at 37 ◦ C for 24 hrs. The samples were
measured spectrophotometrically at 600 nm to determine the best ni­ 3. Results and discussions
trogen source for the growth of the bacterial isolate. The best nitrogen
source was then optimized at various concentrations (1.25, 2.5, 5, 10, 3.1. Determination of soil lead levels
20, and 40 g/L) to find the optimum concentration for the bacterial
growth and incubated at 37 ◦ C for 24 hrs. The growth of the isolate was The lead (Pb) concentration in the sampled soils from Anka and
then determined by measuring absorbance spectrophotometrically at Gusau Local governments of Zamfara State ranged between 522.64 and
600 nm [15,23]. 738.43 mg/kg (Fig. 1). The result showed that samples from lead
affected areas (test groups; Bagega, Dareta, Abare, Waramu) had
2.3.3. Effect of initial pH significantly (P < 0.05) higher soil lead concentration compared to soil
The initial pH for the optimal growth of the bacterial isolate was samples from Kano state (control soil) and that of Environmental

3
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

Protection Agency (EPA) standard (Fig. 2). The significantly higher lead Table 1
concentration above EPA standard found in soil samples from Bagega, Screening of lead-tolerant bacterium.
Dareta, Abare, Waramu of Zamfara state (test samples) relative to that of Sampling Site Colony count (CFU)
Kano state (control sample) (Fig. 2), could be possible due to higher lead
Bagega 658 × 103
contamination resulting from artisanal gold mining and processing. A Dareta 480 × 103
study conducted by Ref. [2] reported a high lead concentration of 2.936 Abare 477 × 103
± 1.851 in ore processing site than in control site (1.509 ± 1.936) of Waramu 429 × 103
Anka, Zamfara state. It was however deduced that, the high lead con­ Gusau 338 × 103
Kano 501 × 103
centration resulted from the fact that lead is a waste product of gold ore
processing and hence leached into the environment. The fact that sam­
ple from Gusau, Zamfara state having highest lead level at P < 0.05 strain BUK_BCH_BTE 2 (Fig. 3). The sequence was deposited in the
could be resulting from the fact that the sample was taken from the road GeneBank under the accession numbers MT160412. The 16S rRNA
side, where the soil got contaminated due to accumulation of lead from sequence accession numbers are listed alongside each species name. The
motor exhaust. This finding is in line with the report by authors [6], numbers at branching points are nodes based on 1000 resamples that
where lead contamination was high at sites closer to road junctions. The represent bootstrap values. The resulting dataset contained 938 loca­
present finding shows that there is high concentration of the heavy metal tions altogether and consisted of 23 nucleotide sequences that were
lead in gold mine area of Zamfara as well as areas far from the gold mine analyzed. The outgroup was Serratia marcescens. The most reported
(Gusau) which could possibly be because lead is mobile. bacteria isolated from soil are the Bacillus sp. and Pseudomonas sp. They
have been reported to have high tolerance to heavy metals and other
3.2. Isolation and morphological identification of lead-tolerant bacteria related substances that are present in the environment [26]. To date, a
number of Bacillus sp. with lead tolerance and degrading capacity were
Table 1 shows the result of colony forming units (CFU) by direct isolated from the soil of Nigeria and around the world. Interestingly, this
plating, after 24 hrs of incubation at 37 ◦ C. The total CFU following paper is the first to isolate Bacillus sp. from a Nigerian soil that could
inoculation of the serially diluted soil samples on LB media from Bagega, tolerate up to 3000 mg/L lead nitrate and quite a number of other toxic
Dareta, Abare, Waramu, Gusau and Kano were found to be 658, 480, heavy metals like Co, Cu, Li, Ni, and Sn.
477, 429, 338 and 501( × 103) CFU respectively. Following Gram-
staining, it was observed that isolate Ac was Gram positive, thick, 3.4. Characterization of lead-tolerant isolate using one-factor-at-a-time
short and rod-shaped. The observed higher CFU in samples from Bagega, (OFAT)
Dareta and Kano (Table 1), could be from the fact that, the concentration
of lead in soil of that locations were relatively low. Hence more observed 3.4.1. Effect of carbon sources
bacterial counts in those sites. Soil parameters such as heavy metal The effect of six (6) different carbon sources on the growth of isolate
content are major influencers of microbial diversity [24]. Research re­ (Ac) was investigated (Fig. 4a). Sucrose was found to be the best carbon
ported by authors [25] indicated low heavy metal contents in the soil of source that supports the growth of the isolate with a significant differ­
Riyadh region, Saudi Arabia and hence high counts of bacterial and ence (p < 0.05), followed by lactose, fructose and glucose respectively.
fungal isolates from that soil was observed. However, soil lead levels However, starch and glycerol did not support the growth of the isolate
were relatively higher in Abare, Waramu and Gusau with corresponding after 24 hrs incubation at 37 ◦ C. Fig. 4b shows the various sucrose
lower CFU. This was proven by the research conducted by authors [9], concentrations that supported the optimal growth of the bacterial
which shows soil with higher lead level have tendency of having low isolate. Isolate Ac tends to grow optimally at sucrose concentration of 5
microbial biomass. g/L. The utilization of sucrose as the most preferred carbon source for
growth by the isolate (Fig. 4a) could be because sucrose is a simple sugar
utilized by most microbes. This finding deviates from the work of [15],
3.3. Molecular identification and phylogenetic analysis which reported glucose as the best carbon source for lead-tolerant iso­
lates (Bacillus sp. and Proteus sp.). Study conducted by Refs. [27–31] on
The 16S rRNA sequence of the isolate following amplification and molybdenum reduction reported the utilization of sucrose as the sole
subsequent phylogenetic analysis of the 16S rRNA gene sequences using carbon source by the thermophilic bacteria Bacillus tequilensis strain
neighbor-joining method revealed low bootstrap values of less than 50 Pharon2 (MK078034) obtained from Egypt as well as Serratia sp. Strain
% similarity. Thus, isolate Ac was tentatively assigned as Bacillus cereus HMY1 and Serratia sp. Strain HMY3 both obtained from Nigeria. The
fact that starch did not support growth of the isolate after 24 hrs of in­
cubation could be as a result of starch being a complex carbohydrate
which requires the involvement of other carbohydrate digesting en­
zymes to convert it to simple sugar for use by most bacteria. Thus, most
microbes prefer simple assimilable carbon sources such as glucose, su­
crose and fructose [32]. The requirement of carbon source concentration
by isolate (Ac) (Fig. 4b) signifies that organisms have different optimal
requirement for nutrients. The linear increase in growth of isolate Ac at
sucrose concentration between 0 and 5 g/L shows that lower concen­
tration of this sugar best supports the growth of the bacteria. Interest­
ingly [32] reported the utilization of sucrose by most bacteria at a
concentration less than 5 % due to osmotic effect. This could be observed
in the drastic decline in bacterial growth after 5 g/L sucrose concen­
tration in the present research work. This result shows that the bacteria
utilize simple sugar for its growth and metabolism, making it to be a
Fig. 2. Soil lead concentrations from the sampled sites. Samples from Gusau potential and effective remediation tool, because bioremediation does
and Kano are controls (1 and 2) and EPA is a reference standard. Bars represent not require the utilization of complex substances/compounds.
mean ± standard deviation (n = 3). Values with different alphabets over the
bars are significantly different (ANOVA, Tukey’s test, P<0.05).

4
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

Fig. 3. A phylogram indicating genetic relationship between Bacillus Cereus strain BUK_BCH_BTE 2 and 16s rRNA sequences of related groups.

significant difference (p < 0.05) with ammonium chloride and glycine.

There was no observable significant difference between ammonium
sulphate, valine and urea at (p < 0.05) (Fig. 5a). The observed best ni­
trogen source obtained in Fig. 5a for the isolate was varied into different
concentrations (Fig. 5b), isolate Ac grows optimally at ammonium sul­
phate concentration of 2.5 g/L. Although most previous works on lead
bioremediation [2,9,11,15,21,22,33,34] did not report the requir­
ement/utilization for nitrogen source. However, the utilization of
ammonium sulphate as the best nitrogen source by isolate Ac (Fig. 5a)
indicated that cheaper nitrogen sources are the most preferred. This is
advantageous as other amino acid based nitrogen sources such as valine
and glycine are expensive and not recommended for use in bioremedi­
ation. Nitrogen is the building block of amino acids and nucleic acids
(DNA and RNA) needed for cellular development and function. A study
Fig. 4a. Effect of various carbon sources on the growth of isolate Ac on MSM by Ref. [35] showed the utilization of urea as the best nitrogen source for
media at 37 ◦ C after 24 hrs of incubation. Bars represent mean ± standard
a lead tolerant bacterium which is also a cheap and even waste product.
deviation (n = 3). Values with different alphabets over the bars are significantly
In Fig. 5b there was an observed linear increase in growth due to in­
different (ANOVA, Tukey’s test, P<0.05).
crease in substrate concentration from 0 to 2.5 g/L where it reached its
optimum. An observable decline and subsequent unchanged or sta­
tionary phase occurred at a concentration of 4–40 g/L. This could be
because at a relatively low concentration of substrate, initial velocity

Fig. 4b. Effect of different sucrose concentrations on the growth of isolate Ac

on MSM media at 37 ◦ C for 24 hrs. Bars represent mean ± standard deviation
(n = 3).

3.4.2. Effect of nitrogen sources on the growth of lead-tolerant isolates Fig. 5a. Effect of various nitrogen sources on the growth of isolate Ac on MSM
The result of the present study shows that ammonium sulphate was media at 37 ◦ C after 24 hrs of incubation. Bars represent mean ± standard
deviation (n = 3). Values with different alphabets over the bars are significantly
the best nitrogen source supporting growth of isolate Ac having a
different (ANOVA, Tukey’s test, P<0.05).

5
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

India. Perhaps, pH is one of the influencers of the rate of some chemical

and biological processes [36]. In addition, authors [2] also reported a
pH of 7.0 for lead biosorption potential of a resident bacteria (Pontoea
agglomerans) in Abare village of Anka Zamfara state Nigeria. Change in
pH affect cellular activities. Drastic change in pH may damage cell and
membrane proteins, which in turn may debilitate the permeability
barrier of cell [37].

3.4.4. Effect of temperature

The effect of various temperature ranges on the growth of isolate
(Ac) was evaluated in Fig. 7. Growth was slow at temperature between
25 and 35 ◦ C for the isolate, which attained optimum growth at tem­
perature of 37 ◦ C and decline at a temperature above 37 ◦ C (p < 0.05).
Temperature is one of the critical parameters that influence rate of
chemical reaction and lead tolerance of the bacteria. Temperature below
Fig. 5b. Effect of different nitrogen sources concentrations on the growth of and above optimum temperature result in decline in enzymatic activity
isolate Ac on MSM media at 37 ◦ C after 24 hrs of incubation. Bars represent of the bacteria. Chemical reactions depend on enzyme efficiency; hence
mean ± standard deviation (n = 3). an optimum temperature increases the activation energy of enzyme [38,
39]. The optimal temperature of 37 ◦ C found in the isolate (Ac) (Fig. 6)
increases linearly with an increase in substrate concentration, a further could be due to the fact that the bacteria are mesophiles isolated from a
increase in substrate concentration increases the rate of reaction at a temperate region, hence, grow optimally at near human body temper­
slower rate and finally reaches a steady state where substrate concen­ ature. This is in conjunction with the works of [15,22] where a tem­
tration has virtually no effect on the reaction rate. Hence the observed perature of 37 ◦ C was the optimum for Bacillus sp. and Proteus sp. as
effect of ammonium sulphate concentration on the lead-tolerant isolate. well as Aspergillus niger and Aspergillus fumigatus. Authors [2] re­
ported an optimum temperature of 35 ◦ C for the lead biosorption Pon­
3.4.3. Effect of initial pH toea agglomerans isolated from Abare village of Zamfara state Nigeria.
The effect of initial pH was evaluated to determine the pH that best In conjunction to this study, both [40,41] reported a similar temperature
supports the growth of the isolate (Fig. 6). Isolate (Ac) grew optimally at of 37 ◦ C for Burkholderia sp. Strain Neni-11 and Enterobacter sp. Strain
a neutral pH of 7.0 at 37 ◦ C over 24 hrs. Growth was not supported at a Aft-3 respectively on mo-reduction. High temperature can alter the
pH of 3.0 for the isolate, instead the isolate picked up a linear growth growth of some bacterial strain and obviously impede tolerance of the
pattern from pH of 4.0. A decline in growth was observed after pH of 7.0 bacteria to lead. Because high temperature leads to inactivation or even
(p < 0.05). pH is a critical parameter that influence bacterial growth. It denaturation of enzymes that are responsible for cellular activities.
may affect the enzymatic activity and tolerance capacity of the bacteria. Extreme temperatures in turn harm DNA as well as membrane structure
Enzymes play a major role in bacterial interaction with heavy metal. [42]. Thus, due to the persistence of heavy metals in the environment,
Chemical reaction depends on ionization states of the amino acid resi­ heavy metals have been assigned as important inorganic pollutants that
dues of the active site of an enzyme as well as the ionic state of a sub­ need drastic and immediate effects [43].
strate. A slight change in pH can alter the optimum activity of both the
enzyme as well as the substrate. pH was evaluated on the bacterial 3.4.5. Effect of substrate concentration (Pb(NO3)2 on the isolate
growth and tolerance capacity at different pH ranges (3–8). The optimal Fig. 8 shows optimum growth of isolate Ac at 1000 mg/L of (Pb
pH of 7.0 found in isolate (Ac) in Fig. 5 could be because, most Bacillus (NO3)2 after 24 hrs of incubation at 37 ◦ C. Growth of the isolate linearly
sp. are neutrophiles and very sensitive to slight change in hydrogen ion increases at lead nitrate concentration of 500 mg/L, attaining optimum
concentration in their environment. Similar finding was reported by at 1000 mg/L (Pb(NO3)2 and significantly decreased at concentration
Ref. [15] in lead-tolerant Bacillus sp. and Proteous sp. with optimal above 1500 mg/L. Perhaps, no growth was observed at 4000 mg/L Pb
growth at neutral pH of 7.0, however deviate from the findings of (NO3)2. Researches on lead-tolerant microbes has been done over the
Ref. [9] in which acidic pH was reported. Ref [21] also reported an years and the maximum tolerable concentration so far has been reported
optimal pH of 5.0 for Klebsiella sp. VITKAS-2, a member of Enterobac­ by Ref. [15]. The author reported 1600 mg/L tolerance to lead by a
teriaceae isolated from tannery effluent of Ranipet, Vellore district of

Fig. 7. Effect of temperature on the growth of isolatisolatedMSM media at

Fig. 6. Effect of initial pH on the growth of isolate Ac on MSM media at 37 ◦ C 37 ◦ C after 24 hrs of incubation. Bars represent mean ± standard deviation (n
after 24 hrs of incubation. Bars represent mean ± standard deviation (n = 3). = 3).

6
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

report by Ref. [45] showed an optimum incubation time of 48 hrs

required for bacterial chromium-reducing ability cultivated on TSB.
Another study by Ref. [46] also observed an optimum growth after 48
hrs by atrazine degrading B. safensis in MSM broth. A 2014 study by
Ref. [47] stated that to achieve an optimal reproducibility of bacterial
cells at physiological condition time factor must be adhered to.

3.4.7. Effect of inoculum size

The result obtained in Fig. 10 shows that growth in isolate (Ac) was
higher after 24–48 hrs when large inoculums size (400 μL) was used.
However, after a prolong incubation of above 48 hrs the culture media of
the isolate (Ac) reveals optimum growth when 100 μL inoculum was
used (72 and 96 hrs). The significant decrease in bacterial growth in
culture with initial inoculum above 100 μL could be due to rapid in­
Fig. 8. Effect of various substrate concentrations (lead nitrate) on the growth of crease in the cell population surviving on the limited amount of nutrient
isolated Ac on MSM media at 37 ◦ C after 24 hrs of incubation. Bars represent which creates competition and eventual death of less competent cells
mean ± standard deviation (n = 3). (Fig. 9).

Proteus sp. isolated from sewage water in India. However this study
reported a concentration of up to 3000 mg/L tolerated by Bacillus cereus 3.5. Effect of other heavy metals
strain BUK_BCH_BTE2 (Fig. 7) which could be due to the soil where the
isolate was obtained is highly contaminated by lead, hence making them The result obtained in Fig. 11 revealed that growth in isolate Ac in
to have inherent tolerance for this metal. The linear increase in growth the presence of 1 ppm other interacting metals increases linearly with
(Fig. 7) at lower concentration of 500–1000 mg/L indicates that low increase in incubation time. Potassium significantly increases the
concentration of lead nitrate is not toxic to the bacterial isolate. How­ growth of Ac at p < 0.05. There was no significant increase (p < 0.05) in
ever, an observed rapid decrease at a concentration above 1000 mg/L growth of the isolate by cobalt. Mercury (Hg) however shows a different
reveals that concentrations higher than the optimum is toxic to bacterial pattern in growth of the isolate by lowering the growth significantly (P
growth, hence slowing down the growth rate of the isolate. This study < 0.05) (6–96 hrs). The result obtained from Fig. 10 clearly shows that,
however, deviated from 320 mg/kg reported by Ref. [9] and also 250 in the presence of potassium and cobalt, the bacterium growth
ppm reported by Ref. [11]. Similarly, authors [33] reported lead toler­ improved, whereas Cu, Li, Mn, Se, Ni, Sn and Zn hinders growth of the
ance level of 1.0 mM by Pseudomonas sp. W6 isolated from hot water isolate. Mercury was observed to have great impact in retarding the
spring in India. Microbial growth is influenced by substrate concentra­ growth of the isolate, this could be because, mercury is very toxic, hence
tion. Very high concentration of substrate hinders bacterial growth and decrease the growth of the lead-tolerant isolate by slowing down their
eventually metabolism [44]. This result shows that this isolate tolerate metabolism and increasing the lag period. This is in conjunction with the
high concentration of lead ever noted, and hence could be a potential work of Ref. [48], where Molybdenum reduction in strain KIK-12 was
bioremediation tool. Enzymes play a critical role in biosorption of heavy found to be inhibited by mercury, copper and silver.
metals in bacterial cells, hence assay of the enzyme responsible for the Mining, which is an anthropogenic activity is the major cause of
heavy metal tolerance could be done in the laboratory. heavy metal pollution in the environment leading to serious and dele­
terious health effects. Heavy metals like Hg, Pb, Co. Cd Cr and Zn are
3.4.6. Effect of incubation time being released into the environment. Inorganic pollutants-heavy metals
The result obtained in Fig. 9 shows that bacterial growth was slow are stable in their structure hence resulting in their non-biodegradable,
and linear between 6 and 36 hrs attaining optimum after 48 hrs by the long-lasting and harmful nature even in minute concentrations [49,50].
isolate (Ac), and significantly decreases afterwards (p < 0.05). Time is a These heavy metals bioaccumulate in the living systems, whereby the
factor in bacterial growth and metabolism. A linear increase in the concentration increases with time, causing deleterious effects to health
growth of the isolate from 0 to 48 hrs indicates that time might be a and life in general [51–58]. Hence need to be detoxified from the
significant criterion for lead tolerance in bacteria. The optimum growth environment. Conventional methods of remediation have been difficult
observed after 48 hrs of incubation by isolate (Ac) (Fig. 8) could be due and costly, hindering its wide usage [59]. Hence the need for a better
to the fact that most bacteria grow well after 24 hrs of incubation. A and cost effective means of remediating heavy metal polluted environ­
ment. The need to assess the effect of other interacting heavy metals on

Fig. 9. Effect of various incubation times on the growth of isolate Ac on MSM Fig. 10. Effect of inoculum size on the growth of isolate Ac on MSM media at
media at 37 ◦ C. Bars represent mean ± standard deviation (n = 3). 37 ◦ C over time period. Bars represent mean ± standard deviation (n = 3).

7
F.A. Harun et al. Case Studies in Chemical and Environmental Engineering 8 (2023) 100540

Fig. 11. Effect of other interacting metals concentrations (1 ppm) on the growth of isolate Ac on MSM media at 37 ◦ C over time. Bars represent mean ± standard
deviation (n = 3).

the growth of this lead-tolerant isolate result from the fact that these less cost and more effective than the physical and chemical methods
heavy metals does not occur freely in nature, however they occur as of remediation.
co-contaminants in the polluted sites. Non-essential inorganic metals
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