COMMUNITY AND PREDICTIVE FUNCTIONAL PROFILING OF HEAVY METAL TOLERANT BACTERIA IN SOIL FROM A BITUMEN PRODUCTION SITE

Abstract

ABSTRACT Heavy Metal (HM) contamination in soil, particularly in areas surrounding bitumen production sites, poses significant environmental and health risks. Certain bacteria have developed mechanisms to tolerate and bioaccumulate HMs, offering potential bioremediation strategies but the specific mechanisms remain poorly understood. This study investigated community and predictive functional profiling of HM-tolerant bacteria in soils from a bitumen production site in Agbagu, Ondo State, Nigeria. Three soil samples were collected from the surface to a depth of 15 cm, and physicochemical properties were determined using standard analytical methods. HM-tolerant bacteria were isolated from the soil samples using a standard protocol, the isolates were further assessed for their tolerance to varying concentrations (20, 40, 60 μg/ml) of Pb, Cd, Cr, Cu, Zn while growth were monitored daily for 3 days using spectrophotometry at 630 nm. Minimum Inhibitory Concentration were determined for each metal. Biochemical identification of selected isolates was done using standard biochemical tests. Metagenomics and predictive functional profiling of heavy metal tolerant bacteria in the three soil samples were done. Bacteria DNA was extracted using a commercial bacterial extraction kit. Full-length hypervariable region of the 16S rRNA amplification gene from the isolates’ genomic DNA was amplified and sequenced using NGS. Sequences showing 97% similarity were grouped into identical Operational Taxonomic Units (OTUs). The Shannon and Simpson diversity α-diversity indices were obtained. Evolutionary history was inferred using unweighted pair-group method with arithmetic mean method. Functionalities of all OTUs were forecasted using Kyoto Encyclopaedia of Genes and Genomes databases. Five bacterial isolates (out of a total of eighteen isolates) exhibiting tolerance to varying concentrations of the HMs were identified as: Micrococcus sp., Pseudomonas sp., Pseudomonas fluorescence, Bacillus sp., and Alcaligens sp. The isolates showed varying tolerance to HMs in a dose-dependent manner over time. From the metagenomics, the OTUs of HM-tolerant soil bacteria were classified into 3 phyla, 5 classes, 6 orders, 9 families and 10 genera. Alpha diversity indices of 0.17, 0.61 and 0.18 (Shannon) and 0.06, 0.30 and 0.07 (Simpson) were obtained for the three samples respectively indicating less species diversity in samples (i) and (iii). The main HM-tolerant bacteria genera identified in the soil were species of Bacillus, Comamonas, Pseudomonas, Providencia, Brevibacillus and Lysinbacillus. The phylogenetic tree delineated the HM-tolerant bacteria into three related clusters. Predictive functional profile of the gene expression showed varying metabolic pathways to cope with HM stress. Aerobic respiration pathway (PWY-3781), beta fatty acid oxidation, toluene degradation (PWY-5180), and coenzyme Q (PWY-5856) synthesis were more expressed in the bacteria in sample (ii) while the super pathway of (R, R)-butanediol biosynthesis (P125-PWY), butyrate-producing pathway (PWY-5022), D-galactose degradation pathway (PWY-6317) were more expressed in sample (i); however, fatty acid salvage (PWY- 7094) and TCA cycle acetate producer were more expressed in sample (iii). The identified bacteria and their associated metabolic pathways provide valuable insights into the mechanisms of heavy metal tolerance and bioaccumulation, contributing to the development of effective bioremediation strategies.