Ecology, Diversity and Metabolites Analysis of Extremophiles

Our earth is naturally bestowed with extreme ecological niches which rarely support any life form to exist there. Microbes harboring such habitats are unusual and exhibit specific characters to cope with stress conditions. Present research is conducted to study the diversity, ecology and metabolites of such microbes i.e. extremophiles. Water, soil and sediment samples were collected from selected extreme sites i.e. salt mine, acid seep, and hot water spring. Total DNA was extracted from soil and sediment samples for metagenome sequencing. Microbial diversity was investigated by barcoded amplicon sequencing of metagenome. Sequencing output files were submitted in the National Centre for Biotechnology Information (NCBI) database under the accession numbers SAMN08026743, SAMN08026744, SAMN08398365, and SAMN08398366 for saline, hot spring, and acid seep samples respectively. Metagenomic analysis revealed that microbial communities were dominated by Proteobacteria in all samples. Other than Proteobacteria, Firmicutes constituted the most abundant population in hot spring, Euryarchaeota were abundant in saline soil and Cyanobacteria dominated the acid seep sample. Owing to the complexities of extreme sites, some unidentified sequences were also found in samples. For isolation and characterization of extremophiles, cultivation based method was used. Eleven halophilic bacteria i.e. MB588, MB589, MB590, MB591, MB592, MB593, MB594, MB595, MB596, MB597, and MB598 were isolated from saline soil samples on LB medium with 4M NaCl concentration. Isolates were characterized morphologically, biochemically and physiologically. On the basis of 16S rDNA gene analysis, MB590, MB591, MB593, MB594, MB595, and MB596 were identified as Halomonas elongata. Whereas, MB588, MB589, MB592, MB597 and MB598 were identified as Halobacillus karajiensis, Alkalibacillus almallahensis, Aquisalibacillus elongatus, Salinicoccus sesuvii and Halomonas aquamarina respectively. Most of the strains exhibited multiple heavy metal and antibiotic resistance. Multiple antibiotics and heavy metal resistance indices i.e. MAR and MHR, revealed Halomonas elongata MB593 as a most resistant strain. Strong association among heavy metal resistance and antibiotic tolerance was observed in halophilic bacteria. No significant extremozyme production was observed in halophilic bacteria. However, biosurfactant production was indicated in nine halophilic strains.Most of the strains significantly reduced surface tension up to 35 dynes/cm and explicated emulsification activities of different hydrocarbons i.e. hexane, canola, xylene and petrol. Moreover, all the strains exhibited xviii oil displacement. Structural characterization of biosurfactants revealed mixture of glycolipids. Three bacterial strains i.e. MB592, MB597 and MB598 exhibited pigment production. Characterization of pigments using different analytical techniques i.e. UV spectroscopy, IR spectroscopy, and HPLC analysis revealed bacterioruberin carotenoids. Methanolic extracts of pigments showed strong antibacterial and antioxidant activities. From hot spring sample, seven hyper-thermophilic bacteria were isolated at 80˚C. Isolates were characterized morphologically, biochemically and physiologically. On the basis of 16S rDNA sequence analysis, isolates were identified as Geobacillus jurassicus MB599, Geobacillus jurassicus MB605, Geobacillus sp. MB601, Geobacillus sp. MB604, Geobacillus stearothermophilus MB600, Geobacillus thermocatenulatus MB602 and Geobacillus thermoparaffinivorans MB603. All the strains exhibited significant protease and amylase production. From acid seep sample, a gram negative, iron(II) oxidizing, acidophilic bacterium IO2C was isolated at pH 2 and 25˚C. On the basis of 16S rDNA analysis, it was identified as Acidithiobacillus ferrooxidans. Strain produced biominerals by oxidation of iron substrates like iron sulfate and iron ammonium sulfate. SEM, XRD and FTIR analysis of bio-minerals indicated schwertmannite and jarosite. Whole genome sequence analysis of Acidithiobacillus ferrooxidans IO-2C revealed 58.73% of G+C content, 2,716,894 base pairs in 23 contigs and 2,927 protein coding sequences. In addition to this, it contained functional genes for iron and sulfur metabolism, multidrug resistance, and heavy metal resistance. Present study provided a comprehensive examination of microbial diversity of extremophiles by consolidating culture dependent and independent techniques to get more realistic portrayal of microbial communities. This study also identified bacterial metabolites and enzymes which will provide industry with more cost-effective biomolecules for specific purposes. Future in depth studies on genomics and metabolomics of extremophilic bacterial strains identified in this study will provide insight to their adaptive mechanisms and their potential roles in bioremediation of toxic pollutants, food processing, and biomedicine.