PHYSIOLOGICAL CHARACTERISTICS, AMYLASE PROPERTIES AND METAGENOMIC ANALYSES OF BACTERIAL AND FUNGAL COMMUNITIES FROM IKOGOSI WARM SPRINGS

Abstract

Several warm and hot springs exist all over the world whose microbial diversity and physicochemical properties have been explored using both conventional and metagenomic methods to reveal biotechnologically relevant bacterial and fungal microbiomes. The Ikogosi Warm Springs (IWS) in Ekiti State, Nigeria is known for its unique warm and cold water outflow and tourism activities. Unlike other climes, there is paucity of information on the bacterial and fungal microbiome of IWS. This work was therefore designed to determine the taxonomic composition of the bacterial and fungal microbiome of IWS as well as prospect for alphaamylase of potential industrial importance. Sediment (20) and water (20) samples were collected from IWS during the wet and dry seasons for physicochemical analyses using standard procedures. The bacterial and fungal microbiome of IWS were evaluated using the V1–V3 regions of 16SrRNA and ITS1, respectively. Genomic libraries were constructed for paired-end sequencing and the microbial taxa present were analysed using DADA2 metagenomics pipeline, where alpha and beta diversity statistics and metrics were also computed. Bacteria were also isolated from samples employing culturedependent method and screened at 50°C for alpha-amylolytic properties using starchsupplemented media. Molecular identification of bacterial isolates, with the highest zone of hydrolysis, were done and alpha-amylase production and enzyme characteristics were determined using UV/visible spectrophotometry. Data were analysed using descriptive statistics, and ANOVA at α0.05. The pH and temperature of IWS ranged 6.36 to 7.93, and 28.3 to 38.0°C, in wet season, and 5.8 to 7.6, and 25.7 to 38.3°C, in dry season. In IWS water, Proteobacteria (84.4, 83.4%), Bacteroidetes (10.0, 16.0%), Firmicutes (2.4, 7.5%), Ascomycota (62.0, 89.0%) and Basidiomycota (37.7, 10.7%), were dominant in the wet and dry seasons; while the sediments (%) had 62.6 and 45.4; 21.4 and 53.5; 15.0 and 0.4; 65.1 and 65.1; 34.8 and 33.5, respectively. Alpha diversity measures indicated no significant phylogenetic diversity between the bacteriome and fungal microbiome of water and sediment, in both seasons. A significant difference occurred in the diversity between the bacteriome of water and sediment samples. Beta diversity showed significant differences in taxonomic similarity and relative abundance in water and sediment of both bacterial and fungal communities in both seasons. Vogesella, Aeromonas, Comamonas, Brevundimonas and Malassezia were present in both water and sediment samples. Culturedependent isolated amylolytic bacteria constituted 27.1% of 174 isolates. The best amylolytic isolates were Bacillus cereus-MPW3E and Bacillus siamensis-SW3F. Optimum alpha-amylase production of these bacilli was observed at 55°C; pH 7.0; Ca2+ supplementation; 1% seed inoculum and 48-hour incubation period. The alpha-amylase activities for Bacillus cereusMPW3E and Bacillus siamensis-SW3F were optimum at 1% substrate concentration, with pH 6.0 and 7.0, and at 50°C and 55°C, respectively. The Michaelis Menten constant was 0.197 and 0.052 mM, with maximum velocity of 6.305 and 25.974 µmol/min for Bacillus cereus-MPW3E and Bacillus siamensis-SW3F, respectively. Microbiome taxonomic diversity of Ikogosi warm springs predominantly contained phyla Proteobacteria, Bacteroidetes, Firmicutes, Ascomycota and Basidiomycota. Thermotolerant alpha-amylase-producing Bacillus cereus and Bacillus siamensis were also obtained.