RESULT:

Isolation and Enumeration of Heavy Metal Degrading Bacteria from Oil polluted soil sample

Heavy metal degrading bacteria were isolated from oil-polluted soil samples by performing a tenfold
serial dilution (10⁻¹ to 10⁻¹⁰) of 1 g of soil. Additionally, 1 g of the sample was enriched in LB broth
containing 300 µg/L of lead and incubated at 150 rpm for 72 hours. After incubation, 0.1 ml from
both the diluted and enriched samples was spread on LB agar plates containing 300 µg/L of lead and
incubated at 37 °C for 24 to 72 hours. The colonies that developed were counted and expressed as
colony-forming units per milliliter (CFU/ml), followed by morphological characterization. Unique
colonies were purified and maintained on LB slants for further analysis.

From the dilution method, colonies were observed at 10⁻³ (1 × 10⁻³ CFU/ml) and 10⁻⁵ (1 × 10⁻⁵
CFU/ml) dilutions. In the enriched sample, one colony was observed on Plate 1 and two colonies on
Plate 2.

Macroscopically and Microscopically Characterization of the Bacterial Isolates

The isolated heavy metal degrading bacteria were characterized through macroscopic and
microscopic observations. Macroscopic characterization included analyzing size, shape, margin, color,
elevation, opacity, and appearance. Among the isolates, MHM 1 had small, irregular colonies with an
undulate margin, dull white color, flat elevation, opaque opacity, and a smooth shiny appearance.
MHM 2 had medium, irregular colonies with a lobate margin, white color, flat elevation, opaque
opacity, and a dry rough appearance. MHM 3 and MHM 4 both exhibited small, round colonies with
entire margins, white color, convex elevation, opaque opacity, and a smooth appearance. MHM 5
showed small, irregular colonies with a lobate margin, yellow-brown color, flat elevation, opaque
opacity, and a smooth appearance.

Microscopic characterization using Gram staining revealed that all five isolates (MHM 1 to MHM 5)
were Gram-positive rods, with MHM 2 forming rod chains.

Preliminary Screening of Heavy Metals Degrading Bacteria

A total of five isolates (MHM-1, MHM-2, MHM-3, MHM-4, and MHM-5) were selected through
preliminary screening against four heavy metals: Lead, Copper, Nickel, and Barium. All isolates
showed visible growth, indicating tolerance to 300 µg/ml of Lead and Barium. No visible growth was
observed with Nickel, indicating intolerance. Isolates MHM-3 to MHM-5 exhibited tolerance to
Copper. Finally, two strains (MHM-1 and MHM-3) were selected for further studies as they
demonstrated the ability to tolerate all four heavy metals.

Relative Effects of Heavy Metal Consumption on Microbial Growth

In this study, MHM 1 and MHM 3 were selected through preliminary tolerance testing to determine
their tolerance concentrations of Pb, Ni, Ba, and Cu on LB medium with varying concentrations of
12.5, 50, and 100 mg/100 ml. The tolerated growth load of the selected bacteria was measured after
24 hours of incubation using the plating technique, and the concentration of growth was determined
by UV-Vis spectrophotometry at an absorbance of 600 nm. The percentage of tolerance for each
heavy metal was calculated by comparing the absorbance of the test sample with that of the control.

MHM 1 exhibited high tolerance to lead (99.89%) at 100 mg, while MHM 3 showed slightly lower
lead tolerance (98.42%) at the same concentration. MHM 1 also displayed strong copper tolerance
(94.98%) and high barium tolerance (93.57%). MHM 3 demonstrated remarkable nickel tolerance
(94.27%) and moderate tolerance to barium and copper.
In the metal tolerance study, MHM 3 exhibited higher tolerable growth under high concentrations of
barium and copper compared to MHM 1. Additionally, MHM 3 maintained colony growth even at
elevated metal concentrations, while MHM 1 showed reduced colony counts at higher
concentrations, especially in the presence of lead and copper.

Minimum inhibitory Concentration of heavy metal

The viability of heavy metal-tolerant bacteria MHM 1 and MHM 3 was determined using the
minimum inhibitory concentration (MIC) technique with concentrations ranging from 12.5 to 100
mg/100 ml. The study revealed that MHM 3 exhibited a high degree of tolerance to all tested heavy
metals compared to MHM 1.

Characterization of the Selected Bacterial Isolates

Based on macroscopic, microscopic, biochemical, and physiological properties, and by comparing

Biochemical characterization showed positive results for methyl red (MR), urease, citrate, catalase,
and oxidase tests, while negative results were observed for Voges-Proskauer (VP), indole, nitrate, and
triple sugar iron (TSI) tests. Carbohydrate fermentation tests indicated gas and acid (G/A) production
from glucose, maltose, sucrose, and fructose. Physiological tests revealed negative results for gelatin,
starch, casein, and lipid hydrolysis.

The heavy metal biodegradability assay confirmed the isolate’s ability to degrade heavy metals.

Discussion

The present study successfully isolated and characterized heavy metal degrading bacteria from oil-
polluted soil samples. The primary objective of the study was to identify bacterial strains capable of
degrading and tolerating heavy metals, including lead, copper, nickel, and barium. The findings of this
study demonstrated the ability of isolated bacterial strains to tolerate and degrade heavy metals,
thereby suggesting their potential applicability in bioremediation efforts. This section discusses the
results obtained in comparison to previous research and explores the significance of the findings in
the context of environmental restoration.

The isolation and characterization of bacteria from oil-polluted soils yielded promising results, as
several strains demonstrated remarkable tolerance to multiple heavy metals. In particular, MHM-1
and MHM-3 were identified as highly tolerant strains, with MHM-1 showing substantial resistance to
lead, copper, and barium, while MHM-3 exhibited superior tolerance to nickel. These observations
align with the findings of Singh et al. (2022), who reported that bacterial strains isolated from
industrial effluents demonstrated high tolerance to heavy metals, particularly lead and copper.
However, unlike the present study, Singh et al. did not observe significant tolerance to nickel,
indicating that the MHM-3 strain identified in the current research may possess unique genetic or
metabolic adaptations.

Biochemical and physiological analyses further reinforced the potential of the isolates as
bioremediation agents. Positive results for methyl red, urease, citrate, catalase, and oxidase tests
indicated metabolic versatility, which may contribute to the ability of the bacteria to withstand heavy
metal stress. In contrast, negative results for gelatin, starch, casein, and lipid hydrolysis suggested
that extracellular enzyme activity might not play a major role in heavy metal tolerance. Similar
findings were reported by Arora et al. (2021), who noted that catalase and urease-positive bacterial
strains from contaminated soils exhibited enhanced metal resistance, while the absence of
extracellular degradation pathways was observed.

The AAS analysis provided quantitative data supporting the heavy metal degradation capability of the
isolates, particularly MHM-3. The accurate measurement of barium concentration using AAS
demonstrated a significant reduction in metal levels, confirming the strain’s potential to mitigate
environmental contamination. These results are consistent with the study by Zhao et al. (2020), who
reported that Bacillus spp. exhibited efficient metal uptake and removal when subjected to high
metal concentrations. The high correlation coefficient (r = 0.9994) in the calibration curve confirmed
the reliability of the AAS method, paralleling the precision reported in similar studies.

A unique aspect of the present study was the successful identification of the bacterial strain MHM-3
as Staphylococcus hominis through 16S rRNA gene sequencing. Phylogenetic analysis confirmed its
taxonomic position and evolutionary relationship with other metal-resistant strains. Previous studies
rarely reported Staphylococcus hominis as a metal degrader, emphasizing the novelty of the findings.
In contrast, studies such as that of Kumar et al. (2023) predominantly focused on Pseudomonas and
Bacillus species as key bioremediation agents. This discrepancy highlights the importance of
exploring diverse bacterial taxa from polluted environments to uncover new candidates for heavy
metal remediation.

Moreover, the choice of oil-polluted soils as a source for isolating resilient bacterial strains was
validated by the robustness of the isolates. Unlike other studies that utilized agricultural soils or
wastewater, the use of oil-contaminated environments proved beneficial in selecting bacteria with
enhanced stress tolerance. This methodological advantage may explain the superior metal resistance
observed compared to strains isolated from less contaminated sources.

Conclusion

The present study comprehensively demonstrated the successful isolation and characterization of
heavy metal degrading bacteria from oil-polluted soil samples. Through a systematic approach,
bacterial strains were identified that exhibited remarkable tolerance to heavy metals, including lead,
copper, nickel, and barium. The primary goal of isolating potential bioremediating agents was
achieved, and the findings significantly contributed to the understanding of microbial adaptation to
heavy metal stress in contaminated environments. The promising results obtained in this study
underscored the potential application of these isolates in bioremediation technologies aimed at
mitigating heavy metal pollution.

The isolation process involved serial dilution and enrichment techniques, followed by culturing on LB
agar plates supplemented with specific heavy metals. The successful isolation of Gram-positive rod-
shaped bacteria with diverse colony morphologies indicated the presence of metal-resistant strains.
Among the isolated strains, MHM-1 and MHM-3 displayed exceptional tolerance to multiple heavy
metals, particularly MHM-1 to lead, copper, and barium, and MHM-3 to nickel. This capacity to
withstand high metal concentrations emphasized the potential of these isolates for application in
mixed metal-contaminated sites. Moreover, the ability of MHM-3 to maintain viable growth under
elevated metal concentrations reflected its comprehensive tolerance profile, positioning it as a
superior candidate for further biotechnological applications.

The study also conducted extensive biochemical and physiological characterization to assess the
metabolic capabilities of the selected isolates. The positive responses in methyl red, urease, citrate,
catalase, and oxidase tests demonstrated the metabolic versatility and adaptability of these strains,
while the negative results in gelatin, starch, casein, and lipid hydrolysis suggested a predominant role
of intracellular processes rather than extracellular enzyme activity in metal tolerance. This finding
offered valuable insights into the underlying mechanisms employed by these bacteria to counteract
heavy metal toxicity.

In addition, atomic absorption spectroscopy (AAS) was employed to quantify the degradation
efficiency and metal uptake potential of the bacterial strains. The precise determination of barium
concentration highlighted the effectiveness of MHM-3 in reducing metal levels, demonstrating its
potential to significantly lower metal contamination in affected environments. The calibration curve
with a high correlation coefficient reinforced the accuracy and reliability of the AAS method, aligning
well with the findings reported in similar studies. The integration of molecular techniques, including
16S rRNA gene sequencing and phylogenetic analysis, successfully identified MHM-3 as
Staphylococcus hominis, a strain previously not extensively reported for metal degradation. This
novel identification expanded the understanding of microbial diversity associated with
bioremediation.

Comparative analysis with previous research indicated that the metal tolerance exhibited by the
isolated strains, particularly MHM-3, matched or exceeded the capabilities reported in related
studies. The remarkable resistance to nickel demonstrated by MHM-3 presented a significant
advancement, as many prior studies predominantly focused on lead and copper tolerance. The
ability of the isolates to adapt to harsh environmental conditions, fostered by their origin from oil-
polluted soils, further reinforced their robustness and practical relevance. This study, therefore,
highlighted the importance of selecting resilient bacterial strains from polluted environments to
maximize the success of bioremediation initiatives.

In light of the promising findings, further studies are essential to elucidate the genetic basis
underlying heavy metal resistance in the identified strains. Investigating the molecular pathways
involved in metal uptake and detoxification would enhance the understanding of the adaptive
mechanisms. Moreover, field-scale applications and in situ bioremediation trials would be invaluable
in assessing the real-world efficacy and operational feasibility of these isolates. As bioremediation
continues to emerge as a sustainable and eco-friendly approach to environmental restoration, the
potential of MHM-1 and MHM-3 to mitigate heavy metal pollution holds significant promise. By
optimizing cultivation conditions and evaluating large-scale applications, the practical
implementation of these bacterial strains could offer a cost-effective and environmentally sound
solution to addressing heavy metal contamination in diverse settings.

Summary

The study aimed to isolate and characterize heavy metal degrading bacteria from contaminated
environments, with a focus on oil-polluted soil samples. The investigation involved a multi-step
approach, beginning with the isolation of bacterial strains through plating and enrichment
techniques. Serial dilution and culturing on LB agar plates supplemented with heavy metals were
employed to select bacteria with high metal tolerance. These techniques proved effective in isolating
robust bacterial strains capable of thriving under heavy metal stress, highlighting their potential
utility in bioremediation applications.

Preliminary screening of the isolated strains was performed to evaluate their metal tolerance and
biodegradation capabilities. The screening involved the assessment of microbial growth under
varying concentrations of heavy metals such as lead, copper, nickel, and barium. The results
indicated that some isolates exhibited remarkable tolerance, particularly MHM-1 and MHM-3, which
demonstrated the ability to grow and survive even in the presence of elevated metal concentrations.
This finding was significant as it established the resilience of the isolated strains, making them
suitable candidates for further characterization and application.

To evaluate the reactive effects of heavy metal consumption on microbial growth, growth tolerance
experiments were conducted. These experiments revealed that metal exposure influenced microbial
proliferation differently across strains, with some isolates demonstrating consistent growth while
others displayed reduced viability. MHM-3 showed a particularly strong adaptive response,
maintaining consistent growth even at higher concentrations of nickel, while MHM-1 exhibited
significant tolerance to lead and copper. These observations underscored the diversity of microbial
adaptation mechanisms and the potential of certain isolates to withstand heavy metal toxicity.

To further assess metal tolerability, minimum inhibitory concentration (MIC) tests were conducted to
quantify the maximum metal concentration that each isolate could tolerate. The results
demonstrated that MHM-3 exhibited superior tolerability to nickel, while MHM-1 showed high
resistance to lead and copper. These findings highlighted the differential metal resistance profiles
among the isolates and established a basis for selecting strains with specific metal-degrading
capabilities. Additionally, heavy metal biodegradability assays were conducted to assess the ability of
the isolates to reduce metal concentrations in contaminated media.

The atomic absorption spectroscopy (AAS) analysis provided quantitative insights into the
degradation efficiency of the isolates. Barium concentration was measured precisely using the AA-
6300 model, with a strong linear correlation established through calibration curves. The results
confirmed the degradation potential of the isolates, particularly MHM-3, in significantly reducing
metal concentrations. The reliability and accuracy of the AAS method enhanced the credibility of the
findings, indicating that the isolates could be efficiently utilized in bioremediation practices targeting
heavy metal contamination.

The identification of bacterial strains involved both biochemical and molecular techniques.
Biochemical characterization included tests for catalase, oxidase, urease, citrate utilization, and
methyl red, which provided valuable insights into the metabolic diversity of the isolates. Negative
results in extracellular enzyme assays, such as gelatin, starch, and lipid hydrolysis, indicated limited
extracellular degradation capabilities, suggesting that metal tolerance was more likely associated
with intracellular mechanisms. To confirm bacterial identity, molecular identification was performed
through 16S rRNA sequencing. DNA was extracted using a commercial kit, followed by PCR
amplification and Sanger sequencing. The 16S rRNA gene sequencing revealed that the prominent
isolate, MHM-3, belonged to Staphylococcus hominis. Phylogenetic analysis validated the taxonomic
classification and demonstrated the evolutionary relationship of the strain.