Culture independent molecular analysis of bacterial communities in the mangrove sediment of Sundarban, India

Background: Sundarban is the world's largest coastal sediment comprising of mangrove forest which covers about one million hectares in the south-eastern parts of India and southern parts of Bangladesh. The microbial diversity in this sediment is largely unknown till date. In the present study an attempt has been made to understand the microbial diversity in this sediment using a cultivation-independent molecular approach. Results: Two 16 S rRNA gene libraries were constructed and partial sequencing of the selected clones was carried out to identify bacterial strains present in the sediment. Phylogenetic analysis of partially sequenced 16 S rRNA gene sequences revealed the diversity of bacterial strains in the Sundarban sediment. At least 8 different bacterial phyla were detected. The major divisions of detected bacterial phyla were Proteobacteria (alpha, beta, gamma, and delta), Flexibacteria (CFB group), Actinobacteria, Acidobacteria, Chloroflexi, Firmicutes, Planctomycetes and Gammatimonadates. Conclusion: The gammaproteobacteria were found to be the most abundant bacterial group in Sundarban sediment. Many clones showed similarity with previously reported bacterial lineages recovered from various marine sediments. The present study indicates a probable hydrocarbon and oil contamination in this sediment. In the present study, a number of clones were identified that have shown similarity with bacterial clones or isolates responsible for the maintenance of the S-cycle in the saline environment

Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions

Degradation of raw feather by a novel high molecular weight extracellular protease from newly isolated Bacillus cereus DCUW

Biotreatment of feather wastes and utilization of the degraded products in feed and foodstuffs has been a challenge. In the present study, we have demonstrated the degradation of feather waste by Bacillus cereus DCUW strain isolated during a functional screening based microbial diversity study on East Calcutta Wetland Area. A high molecular weight keratinolytic protease from feather degrading DCUW strain was purified and characterized. Moreover, utilization of degraded products during feather hydrolysis was developed and demonstrated. The purified keratinolytic protease was found to show pH and temperature optima of 8.5 and 50°C, respectively. PMSF was found to inhibit the enzyme completely. The purified enzyme showed molecular weight of 80 kDa (from SDS-PAGE). The protease was found to have broad range substrate specificities that include keratin, casein, collagen, fibrin, BAPNA and gelatin. The protease was identified as minor extracellular protease (Vpr) by RT-PCR and northern blotting techniques. This is the first report describing the characterization of minor extracellular protease (Vpr) and its involvement in feather degradation in B. cereus group of organisms

Bacterial diversity of east Calcutta wet land area: possible identification of potential bacterial population for different biotechnological uses

The extent of microbial diversity in nature is still largely unknown, suggesting that there might be many more useful products yet to be identified from soil microorganisms. This insight provides the scientific foundation for a renewed interest in examining soil microorganisms for novel commercially important products. This has led us to access the metabolic potential of soil microorganisms via cultivation strategy. Keeping this in mind, we have performed a culture-dependent survey of important soil bacterial community diversity in East Calcutta Wetland area (Dhapa Landfill Area). We describe isolation of 38 strains, their phenotypic and biochemical characterization, and finally molecular identification by direct sequencing of polymerase chain reaction (PCR)-amplified 16S rRNA gene products. We have isolated and identified strains able to fix nitrogen, produce extracellular enzymes like protease, cellulase, xylanase, and amylase, and solubilize inorganic phosphates. Some isolates can synthesize extracellular insecticidal toxins. We find a good correlation between biochemical and phenotypic behavior and the molecular study using 16S rRNA gene of the isolates. Furthermore, our findings clearly indicate the composition of cultivable soil bacteria in East Calcutta Wetland Area

Evaluation of polymerase chain reaction-based ribosomal DNA sequencing technique for the diagnosis of mycotic keratitis

Purpose: To evaluate the polymerase chain reaction (PCR) and sequencing-based ribosomal deoxyribonucleic acid (rDNA) typing technique for rapid and sensitive diagnosis of fungal keratitis. Design: Laboratory investigation. Methods: The present study included culture-based methods along with PCR and ITS2/5.8S rDNA sequencing to evaluate fungal infections in 32 corneal scrapings from patients with suspected mycotic keratitis. Results: Good correlation was found between the culture and PCR rDNA sequencing-based methods. We also identified fungal strains that were noncultivable in routine culture medium and fungal strains, which have been reported scarcely in previous reports from eastern Indian studies. Conclusions: The PCR and rDNA sequencing-based approaches may be useful alternatives to culture-based techniques for the rapid and sensitive diagnosis of fungal keratitis. We found that clinically significant pathogenic fungal strains can be detected and differentiated by this method in a short period. This method therefore is useful for the early treatment of fungal infection

Thermodynamic characterization of a highly thermoactive extracellular pectate lyase from a new isolate Bacillus pumilus DKS1

An extracellular pectate lyase (EC 4.2.2.2) was purified from the culture filtrate of a newly isolated Bacillus pumilus DKS1 grown in pectin containing medium. Using ion-exchange and gel filtration chromatography, this enzyme was purified and found to have a molecular weight of around 35 kDa. The purified enzyme exhibited maximal activity at a temperature of 75°C and pH 8.5. The presence of 1 mM calcium and manganese enhanced pectate lyase activity and was strongly inhibited by zinc, nickel and EDTA. The thermal inactivation studies revealed an entropy-enthalpy compensation pattern below a critical temperature. The alkaliphilicity and high thermostability of this pectate lyase may have potential implications in fibre degumming

Bacterial diversity assessment of pristine mangrove microbial community from Dhulibhashani, Sundarbans using 16S rRNA gene tag sequencing

The global knowledge of microbial diversity and function in Sundarbans ecosystem is still scarce, despite global advancement in understanding the microbial diversity. In the present study, we have analyzed the diversity and distribution of bacteria in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. Metagenome is comprised of 1,53,926 sequences with 108.8 Mbp data and with 55 ± 2% G + C content. Metagenome sequence data are available at NCBI under the Bioproject database with accession no. PRJNA245459. Bacterial community metagenome sequences were analyzed by MG-RAST software representing the presence of 56,547 species belonging to 44 different phyla. The taxonomic analysis revealed the dominance of phyla Proteobacteria within our dataset. Further taxonomic analysis revealed abundance of Bacteroidetes, Acidobactreia, Firmicutes, Actinobacteria, Nitrospirae, Cyanobacteria, Planctomycetes and Fusobacteria group as the predominant bacterial assemblages in this largely pristine mangrove habitat. The distribution of different community datasets obtained from four sediment samples originated from one sampling station at two different depths providing better understanding of the sediment bacterial diversity and its relationship to the ecosystem dynamics of this pristine mangrove sediment of Dhulibhashani in, Sundarbans. Keywords: Mangrove sediment, Dhulibhashani, Metagenome, Pyrosequencing, Bacterial diversit