Chapter 2.4 - Exploration of Bacterial Alkaline Protease Diversity in Chilika Lake Wetland Ecosystem

Abstract

Alkaline proteases (APs) are one of the most widely used hydrolase enzymes in industrial processes, which remain active between neutral to alkaline ranges. The diversity of bacterial APs was explored for the first time in the brackish water wetland ecosystem of Chilika Lake on the east coast of India using culture-dependent and culture-independent methods. Using a culturable approach, 62 selected bacterial strains were identified (based on protease activity at pH (5–12) and NaCl (2%–10%) and 16S rRNA sequence) and found to be affiliated to Actinobacteria, Proteobacteria, and Firmicutes phyla, which contained various genera of Bacillus, Holomonas, Acinetobacter, Halobacillus, Exiguobacterium, Escherichia, Fictibacillus, Jeotgalicoccus, Pseudomonas, Rheinheimera, Serratia, Staphylococcus, Virgibacillus, Isoptericola, Aeromonas. Halobacillus marinus KGW1T, Rheinheimera pleomorphica PKS7T, and Bacillus sp. KGS4 was taxonomically characterized (polyphasic approach and whole genome sequencing) and classified as novel species. The culturable approach focused on potential isolates with bacterial AP genes, 16S rRNA sequence-based identification and phylogenetic diversity, whole genome comparison, protease type, and a minimum set of pathway reconstruction to confirm the novelty and classification of strains. In the nonculturable approach, the spatial and temporal community diversity of bacterial APs in the brackish water sediments of Chilika Lake was analyzed using 16S rRNA-based (V4-V6 regions) amplicon sequencing (454 pyrosequencing) from 24 monitoring stations spread over all four ecological zones (central, north, south, and outer channel) and three distinct seasons of summer, rainy, and winter from Chilika Lake to obtain taxonomic diversity and phylogenetic information based on comparison with MEROPS database. Bacterial AP diversity of the entire lake differed from that of Nalabana Inner and outer regions during winter and rainy seasons, respectively, which houses the bird sanctuary receiving migratory birds resulting in high organic sedimentary nitrogen and proteolytic activity. Whole metagenome sequencing (Illumina) of 12 selected regions of the lake across four zones in the winter season showed Proteobacteria (30%) was the most abundant phylum with the major classes as Gamma-proteobacteria and genus Caldilinea sp. (12%) was the most abundant genus with the highest variability and 40% sequences were unassigned. Based on KEGG classification the industrially relevant APs of the S8 family were abundant in stations Nalabana (central), Bhasramundia (central), CDA 19 (north), Bhabakundaleshwar (outer channel), and Godhimukh (central) where diversity of the alkaline proteolytic bacteria and nitrogen metabolism genes were found to be higher in comparison to other regions in Chilika Lake. Nonculturable approaches enabled the identification, abundance, and distribution of protease types of several OTUs containing bacterial APs of the S8 family from Chilika Lake. This study opens doors to understanding the delicate balance of the environment as maintained by microbes and for future exploration of microbial communities for biotechnological and industrial applications for the benefit of mankind.