Understanding the Diversity, Carbon Metabolism, Bioprospecting Potential and Impact of Environmental Alterations on Heterotrophic Bacteria from Bhitarkanika Mangrove Ecosystem

Abstract

The thesis illustrates the bacterial diversity of the Bhitarkanika mangrove ecosystem, Odisha, India with potential cellulose degradation and antibiotic production by mangrove bacteria. Additionally, the thesis also focusses on the impacts of anthropogenic and environmental stressors on cellulose degradation and bioactive secondary metabolite production of the bacteria. Sampling was carried out from five study sites of Bhitarkanika mangrove ecosystem, i.e. Dangmal, Gupti, Habalikhati, Ekakula and Kalibhanjadiha. Bacterial diversity was elucidated utilizing both culture-dependent and culture- independent approaches. Overall, culture-dependent total heterotrophic bacteria varied from 2.16 ± 0.5 to 168.25 ± 1.73 × 103 cfu/g in sediment and 1.53 ± 0.915 to 38.33 ± 0.108 × 103 cfu/ml in water. Key regulatory factors influencing aquatic bacterial abundance included nitrate phosphate, conductivity, and dissolved oxygen in different seasons. Culture-independent study depicted that operational taxonomic unit (OTU) varied from 5077 to 21,207 in sediment and 14,213 to 17,501 in water. The dominance of Bacilli, Gammaproteobacteria, and Betaproteobacteria was observed from culture-based approach while the dominance of Gammaproteobacteria, Alphaproteobacteria and Bacilli was recorded from culture-independent study. α and ß diversity analysis revealed that in sediment, Dangmal site hold the most diverse bacterial community whereas in water all the sampling sites showed distinctly different community composition compared to the other. The study also aims to explore the cellulose degradation process of Bacillus haynesii DS7010 isolated from Bhitarkanika mangrove ecosystem. Optimization revealed highest cellulose degradation by the bacterium at 48 h of incubation and 41°C incubation temperature, with 1% substrate concentration. PCR amplification and homology modelling confirmed the presence of both exoglucanase (glycoside hydrolase 48 or GH48) and endoglucanase (glycoside hydrolase 5 or GH5) within the bacterial genome. Native PAGE, along with a subsequent zymogram assay, unveiled the presence of eight isoforms of cellulase ranged from 78kDa to 245kDa within the bacterium. Anthropogenic and environmental stressors such as pH, salinity, and lead (Pb) affected the bacterial metabolism. Most detrimental effect was observed under Pb followed by pH stress which was determined by reduced bacterial growth, increased intracellular ROS production along with reduced enzyme activity and downregulation of cellulase producing genes (celA and celB). Salinity augmented bacterial growth and cellulose metabolism up to 3% concentration of NaCl. Microcosm study revealed 4.05% reduction in total carbon (C%) content in natural condition while 0.97% decrease in C% under combined stress condition. The study further emphasizes on bioactive compound producing bacteria B. velezensis ES8024 and characterization of bioactive compound effective against potential fish pathogen Aeromonas hydrophila ATCC 35654. Optimization revealed maximum production of bioactive compound at 48 h of incubation at 37°C. Characterization using TLC and ATR-FTIR indicated the lipopeptidic nature of the compound, belonging to the surfactin family lipopeptide. The 1H NMR spectrum highlighted characteristic features of a lipopeptide compound, with a long aliphatic chain of the lipid moiety and aliphatic and amide bonds of the peptide moiety. HRMS data confirmed four surfactin isomers within the compound, with specific peak signals denoting C13 (1030.83 m/z) and C14 (1044.85 m/z) isomers and two C15 (1058.87 m/z and 1059.87 m/z) isomers. Two surfactin synthase genes (srfAA and srfAB) were identified in the bacterial genome. External modifiers were found to impact both bacterial growth and secondary metabolite production, with Pb at 1600 ppm concentration having the most adverse effect. Structural analysis using ATR- FTIR and 1H NMR revealed modifications in both protein and lipid moieties under stress conditions. Functional analysis through well diffusion assay demonstrated variable inhibition zones under different stress conditions. The expression of srfAA and srfAB showed 11.44 fold and 17.41 fold downregulations respectively in response to 1600 ppm Pb concentration. Thus, the overall outcome suggests that Bhitarkanika mangrove ecosystem harbors an overwhelming bacterial diversity. Bacillus is a potential genus within this mangrove ecosystem possessing cellulose degradation and antibacterial compound production capabilities. Both B. haynesii DS7010 and B. velezensis ES8024 are able to sustain under stressors such as pH, salinity and Pb. However, stressed condition can drastically affect both the primary and secondary metabolisms in bacteria.