MOLECULAR IDENTIFICATION AND THE ANTIMICROBIAL PROPERTIES OF MICROORGANISMS PRESENT IN LOCUST BEANS EFFLUENTS (PARKIA BIGLOBOSA) JACQUE BENTH

Abstract

Molecular identification of microorganisms associated with locust been effluents and their antimicrobial properties was carried out to isolate and identify the microorganisms associated with its effluent. In addition to the molecular method employed in the identification of microorganisms isolated from locust beans effluent; the conventional methods were also carried out on the isolates. Antimicrobial properties and the phytochemical constituents of the effluents were also determined in this study. Locust beans was cooked until the coat was soft and the effluent was decanted, the chaffs were removed and cooked again and the effluent was also decanted. Both effluents were used in the course of this research. The conventional method of identification was carried out using the pour plate technique of culturing and the isolates were later subjected to cultural and biochemical characterization using the standard method. The conventional identification was carried out by extracting the DNA of the isolated microorganisms, subjecting the DNA to Polymerase chain reaction (PCR) and sequencing, the sequenced genes were blasted using the NCBI database. The effluents were tested against some pathogenic microorganisms such as; Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Escherichia coli and Streptococcus pyogene for their antimicrobial properties using the conventional antibiotics as the control. The pH of the effluents was tested using a pH meter and the effluents were further subjected to phytochemical screening. Bacillus subtilis, Enterobacterfaecalis, Enterobacter aerogenes, Alcaligenes feacalis, Lactobacillus sp., Aspergillus flavus, Geotrichum candidum, Saccharomyces sp. and Penicillum sp. were isolated using conventional method. The microorganisms identified using molecular method include: Bacillus subtilis, Lactobacillus paracasei, Enterobacter ludwigii, Aspergillus flavus, Geotrichum candidum, and Trichomonacaea sp. When the pathogenic microorganisms were subjected to antimicrobial tests using both effluents at 100mg/ml; both effluents were able to inhibit Staphylococcus aureus (ATCC 43300), Salmonella typhi (ATCC 35240), Escherichia coli (ATCC 35218) while Pseudomonas aeruginosa (ATCC 27853) and Klebsiella pneumoniae (ATCC 48891) were resistant to the effluent at 100mg/ml. The clinical isolates were more resistant to both effluents. However, the effluents were able to inhibit Staphylococcus aureus, Salmonella typhi, Streptococcus pyogenes and Escherichia coli, while Pseudomonas aeruginosa and Klebsiella pneumoniae were resistant to the effluent at 100mg/ml. Pseudomonas aeruginosa and Klebsiella pneumoniae were also resistant to the effluent at 200mg/ml. The extract without chaffs was found to possess more bactericidal effect on the test microorganisms when compared with the effluent containing chaffs. The highest antibacterial activity of the locust beans effluent without chaffs was recorded for Salmonella typhi with 18.00 ± 0. 00 inhibition zones and 14.00 ± 0.00 with chaffs respectively. The lowest activity was observed on Pseudomonas aeruginosawith the zones of inhibition ranging from 9.67 ± 0.58 and 7.67 ± 0.58 respectively. However, there was no antibacterial activity observed on Staphylococcus aureus and Klebsiella pneumoniae. The phytochemical components of both effluents were saponin, tannin, flavoid, terpernoid, and alkaloid. Saponin had the highest concentration when the effluents were subjected to quantitative phytochemical analysis with 39.82mg/g and 39.27mg/g for effluent with chaffs and effluent without chaffs respectively while tannin had the lowest concentration with 4.17mg/g for both effluents. The pH of the Parkia biglobosa effluent without chaffs ranged from acidity to alkalinity (6.3 - 8.2), while that of the effluent with chaffs ranged from 6.4 - 6.9. This study has provided useful information on microorganisms associated with the effluent; the antibacterial activity of the effluents against Salmonella typhi, Pseudomonas aeruginosa and other microorganisms used in this study. Further work can be carried out on the effluent such as determining the toxic dose and extraction of the bioactive component for use in production of drugs.