Bioremediation Potential and Molecular Characterization of Heavy Metal-resistance Profiles of Bacterial Isolates from Industrial Effluents in Mojo and Bishoftu area, Ethiopia

Abstract

Various heavy metals (HM) can be removed from the environment by various bacterial species, which are developing resistance and reduction through various mechanisms. Bacteria bioaccumulate Heavy metals (HMs) inside their cell structures. This study aimed to isolate and characterize heavy metal–resistant bacterial isolates (HMRBI) from environmental samples collected in Ethiopia’s Bishoftu Eastern Industrial Zone and Mojo Tannery Industry, with the objective of mitigating environmental pollution. Soil and wastewater samples were collected and transported to the Molecular biology and Biotechnology laboratory, Haramaya University. The HMRBIs were subsequently screened and identified based on their resistance to selected heavy metals. These isolates were characterized and identified using biochemical assays, protein profiling through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and molecular analysis based on 16S rRNA gene sequencing. Furthermore, several computational approaches were employed to investigate the expression patterns and regulatory mechanisms of heavy metal resistance genes (HMRG). These included comparative genomes, predicting promoter regions, & common sequence motifs, identifying transcriptional sites, re-annotating genes, and identifying transcriptional factors. The Multiple Em Motif FOR Elicitation (MEME Suite), Rapid Annotation using Subsystem Technology (RAST), Orthologous Average Nucleotide Identity (OANI), and EziBIO databases were key tools utilized in this analysis. Cell deformation, bio-absorption, and infrared spectra corresponding to heavy metal uptake by cells were examined using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) and Fourier transform infrared spectroscopy (FTIR). Atomic Absorption Spectrophotometry (AAS) was also utilized to assess the biodegradability of HMRBI. Among seventeen characterized bacterial isolates, Pseudomonas putida-HMRBI-2 showed the highest ability to bioaccumulate cobalt, with removal rates of 67.38%. Exiguobacterium aurantiacum strain HMRBI-7 was the most effective at removing copper and chromium, with bioaccumulation rates of 65.02 ± 0.002% and 70.04 ± 0.001%, respectively. Aeromonas hydrophila strain HMRBI-12 and Delftia acidovorans-HMRBI strain 7 demonstrated good cadmium and zinc removal abilities, with bioaccumulation rates of 65.02% and 70.32%, respectively. Although each bacterial isolate exhibited variable levels of HM resistance, they all showed unique capabilities for the bioaccumulation of HMs. It is believed that the isolate most likely possesses a combination of intracellular and extracellular biosorption mechanisms to degrade HMs. The study, which utilized xvieight 16 rRNA gene sequences, successfully identified three microbial isolates: P. benzoelyticum strain HMRBI-1, Citrobacter strain HMRBI-5, and Klebsiella strain HMRBI-14 as having the highest HM removal capacity. Furthermore, the minimum inhibitory concentration (MIC) results highlighted that Klebsiella spp. exhibited an MIC of 700 ppm, whereas Citrobacter spp. presented an MIC of 800 ppm. Moreover, the maximum bioaccumulation efficiencies were noted at 89.74, 85.21, and 79.27% for Klebsiella strain HMRBI-14, Citrobacter strain HMRBI-5, and P. benzoelyticum strain HMRBI-1, respectively. Additionally, 14 transcription factors were identified and predicted from the reliable and lowest candidate motif with an e-value of 3.0e-009. Further investigation was conducted on the 14 identified transcription factors concerning activation, repression, and dual functions and regulation in microorganisms to thrive in harsh environmental conditions. As the data suggest, the identified transcription factors were used for the activation of gene regulation rather than repression. Therefore, the study highlights the importance of bacteria in HM bioremediation at contaminated sites due to the genes they possess. Analyzing regulatory elements provides a crucial foundation for understanding the genes the key to HM bioremediation, and the combined approach of laboratory experiments and bioinformatics effectively harnesses native microorganisms to clean heavy metal-contaminated environments.