Gurdeep Rastogi

Characterization of thermostable cellulases produced by Bacillus and Geobacillus strains.

The composition of thermophilic (60 degrees C) mixed cellulose-degrading enrichment culture initiated from compost samples was examined by constructing a 16S rRNA gene clone library and the presence of sequences related to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria were identified. Eight isolates capable of degrading cellulose, carboxymethyl cellulose (CMC), or ponderosa pine sawdust were identified as belonging to the genera Geobacillus, Thermobacillus, Cohnella, and Thermus. A compost isolate WSUCF1 (Geobacillus sp.) was selected based on its higher growth rate and cellulase activity compared to others in liquid minimal medium containing cellulose as a source of carbon and energy. Strain WSUCF1 and a previously isolated thermophilic cellulose-degrading deep gold mine strain DUSELR13 (Bacillus sp.) were examined for their enzyme properties and kinetics. The optimal pH for carboxymethyl cellulase (CMCase) activity was 5.0 for both isolates. The optimum temperatures for CMCase of WSUCFI and DUSELR13 were 70 and 75 degrees C, respectively. For CMC, the DUSELR13 and WSUCF1 CMCases had K(m) values of 3.11 and 1.08mg/ml, respectively. Most remarkably, WSUCF1 and DUSELR13 retained 89% and 78% of the initial CMCase activities, respectively, after incubation at 70 degrees C for 1day. These thermostable enzymes would facilitate development of more efficient and cost-effective forms of the simultaneous saccharification and fermentation process to convert lignocellulosic biomass into biofuels.

Molecular Techniques to Assess Microbial Community Structure, Function, and Dynamics in the Environment

Culture-based methods are important in investigating the microbial ecology of natural and anthropogenically impacted environments, but they are extremely biased in their evaluation of microbial genetic diversity by selecting a particular population of microorganisms. With recent advances in genomics and sequencing technologies, microbial community analyses using culture-independent molecular techniques have initiated a new era of microbial ecology. Molecular analyses of environmental communities have revealed that the cultivable fraction represents <1% of the total number of prokaryotic species present in any given sample. A variety of molecular methods based on direct isolation and analysis of nucleic acids, proteins, and lipids from environmental samples have been discovered and revealed structural and functional information about microbial communities. Molecular approaches such as genetic fingerprinting, metagenomics, metaproteomics, metatranscriptomics, and proteogenomics are vital for discovering and characterizing the vast microbial diversity and understanding their interactions with biotic and abiotic environmental factors. This chapter summarizes recent progress in the area of molecular microbial ecology with an emphasis on novel techniques and approaches that offer new insights into the phylogenetic and functional diversity of microbial assemblages. The advantages and pitfalls of commonly used molecular methods to investigate microbial communities are discussed. The potential applications of each molecular technique and how they can be combined for a greater comprehensive assessment of microbial diversity has been illustrated with example studies.

Microbial Diversity in Uranium Mining-Impacted Soils as Revealed by High-Density 16S Microarray and Clone Library

Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.

Molecular Studies on the Microbial Diversity Associated with Mining-Impacted Coeur d’Alene River Sediments

The prokaryotic diversity associated with highly metal-contaminated sediment samples collected from the Coeur d’Alene River (CdAR) was investigated using a cultivation-independent approach. Bacterial community structure was studied by constructing an RNA polymerase beta subunit (rpoB) gene library. Phylogenetic analysis revealed that 75.8% of the rpoB clones were associated with β-Proteobacteria while the remaining 24.2% were with γ-Proteobacteria. All phylotypes showed close similarity to previously reported cultivable lineages from metal or organic contaminant-rich environments. In an archaeal 16S rRNA gene library, 70% of the clones were affiliated to Crenarchaeota, while 30% belonged to Euryarchaeota. Most of the Euryarchaeota sequences were related to acetoclastic lineages belonging to Methanosarcinales. A single phylotype within the Euryarchaeota showed no association with cultivable euryarchaeotal lineages and might represent novel taxon. Diversity indices demonstrated greater diversity of Bacteria compared to Archaea in CdAR sediments. Sediment characterization by the X-ray fluorescence spectroscopy revealed high amount of toxic metals. To our knowledge, this is the first culture-independent survey on the prokaryotic diversity present in mining-impacted sediments of CdAR.

Spatiotemporal distribution and composition of phytoplankton assemblages in a coastal tropical lagoon: Chilika, India

The Asia’s largest lagoon, Chilika, is a shallow water estuary and a designated “Ramsar” site located in the east coast of India. The spatiotemporal diversity of phytoplankton based on the monthly sampling between July 2011 and June 2012 was investigated in relation to physicochemical variables of the surface water column from 13 stations. The salinity was minimum (average 9) during the monsoon which was primarily due to riverine discharge. As the season progressed towards post-monsoon, average salinity of the whole lagoon reached to 10 which further increased to 20 during pre-monsoon season. A total of 259 species of phytoplankton, mostly dominated by the Bacillariophyta (138 species) followed by Dinophyta (38 species), Chlorophyta (32 species), Cyanophyta (29 species), Euglenophyta (18 species), and Chrysophyta (4 species), were recorded in this study. Different ecological sectors of the lagoon (except the northern sector) were dominated by diatoms, while the northern sector due to its freshwater regime supported large population of euglenoids. Based on the multivariate ordination analysis, salinity regime and light availability played important role in determining the distribution, diversity, and composition of phytoplankton communities. Overall, this study documented a very high diversity of phytoplankton and highlighted the importance of taking extensive sampling in getting a clearer understanding of phytoplankton community structure in less-studied environments such as Chilika lagoon.

Interannual and cyclone-driven variability in phytoplankton communities of a tropical coastal lagoon

One of the main challenges in phytoplankton ecology is to understand their variability at different spatiotemporal scales. We investigated the interannual and cyclone-derived variability in phytoplankton communities of Chilika, the largest tropical coastal lagoon in Asia and the underlying mechanisms in relation to environmental forcing. Between July 2012 and June 2013, Cyanophyta were most prolific in freshwater northern region of the lagoon. A category-5 very severe cyclonic storm (VSCS) Phailin struck the lagoon on 12th October 2013 and introduced additional variability into the hydrology and phytoplankton communities. Freshwater Cyanophyta further expanded their territory and occupied the northern as well as central region of the lagoon. Satellite remote sensing imagery revealed that the phytoplankton biomass did not change much due to high turbidity prevailing in the lagoon after Phailin. Modeling analysis of species-salinity relationship identified specific responses of phytoplankton taxa to the different salinity regime of lagoon.

An inventory of free-living marine nematodes from Asia’s largest coastal lagoon, Chilika, India

The present study revealed an inventory of free-living marine nematode species from the Asia’s largest coastal lagoon, Chilika (lat. 19°28′ and 19°54′N; long. 085°05′ and 085°38′E), located in the East Coast of India and facing the Bay of Bengal. This study provided a checklist of 64 free-living marine nematode species belonging to 32 genera and 13 families. Among these, marine nematode species Oncholaimus oxyuris has been reported as new distributional record from Indian waters. The distribution of encountered nematode species was investigated in relation with environmental variables such as salinity and sediment texture throughout the lagoon. Most of the nematode species were found in stations which had high salinity (>6) and nature of sediments varied from sandy to silt/clay. However, few nematode families (e.g. Oncholaimidae and Microlaimidae) were also observed in low salinity stations of the lagoon indicating tolerance to variation in salinity.

Salinity and macrophyte drive the biogeography of the sedimentary bacterial communities in a brackish water tropical coastal lagoon

Brackish water coastal lagoons are least understood with respect to the seasonal and temporal variability in their sedimentary bacterial communities. These coastal lagoons are characterized by the steep environmental gradient and provide an excellent model system to decipher the biotic and abiotic factors that determine the bacterial community structure over time and space. Using Illumina sequencing of the 16S rRNA genes from a total of 100 bulk surface sediments, we investigated the sedimentary bacterial communities, their spatiotemporal distribution, and compared them with the rhizosphere sediment communities of a common reed; Phragmites karka and a native seagrass species; Halodule uninervis in Chilika Lagoon. Spatiotemporal patterns in bacterial communities were linked to specific biotic factors (e.g., presence and type of macrophyte) and abiotic factors (e.g., salinity) that drove the community composition. Comparative assessment of communities highlighted bacterial lineages that were responsible for segregating the sediment communities over distinct salinity regimes, seasons, locations, and presence and type of macrophytes. Several bacterial taxa were specific to one of these ecological factors suggesting that species-sorting processes drive specific biogeographical patterns in the bacterial populations. Modeling of proteobacterial lineages against salinity gradient revealed that α- and γ-Proteobacteria increased with salinity, whereas β-Proteobacteria displayed the opposite trend. The wide variety of biogeochemical functions performed by the rhizosphere microbiota of P. karka must be taken into consideration while formulating the management and conservation plan for this reed. Overall, this study provides a comprehensive understanding of the spatiotemporal dynamics and functionality of sedimentary bacterial communities and highlighted the role of biotic and abiotic factors in generating the biogeographical patterns in the bacterial communities of a tropical brackish water coastal lagoon.

Multiple Spatial Scale Analysis Provide an Understanding of Benthic Macro-Invertebrate Community Structure Across a Lagoonal Ecosystem

Chilika, the largest coastal lagoon of Asia, represents a complex transitional zone between terrestrial and marine ecosystems. In this study benthic macro-invertebrate community structure was elucidated based on multiple spatial scales approach (station-wise, sector-wise and region-wise) across Chilika from June 2013 to February 2015 (temporal scale), encompassing 23 pre-selected stations in link with measurement of abiotic parameters. Sixty two macro-invertebrate species representing seven macrofaunal groups were encountered, of which gastropods were the most abundant in terms of density (~96%) and species composition (~43%), and crustaceans were the least. The structure of the macrofaunal community (in terms of abundance) showed significant differences across spatial (station-wise, sector-wise and region-wise), and temporal scales (month-wise). While macro-invertebrate species composition revealed heterogeneity both station-wise and month-wise but it was found to be homogenous within the sector-wise and region-wise scales. BIO-ENV analysis indicated that salinity together with sediment nature were major abiotic variables associated with observed macro-invertebrate patterns. The results together demonstrated that macro-invertebrate communities are adapted to prevailing environmental conditions of Chilika lagoon; homogeneous and heterogeneous distribution and diversity patterns across multiple spatial scales revealed assemblages distinct to this ecosystem. The multiple scales approach could prove to be useful for ecological monitoring of lagoons.

Presence of glucose, xylose, and glycerol fermenting bacteria in the deep biosphere of the former Homestake gold mine, South Dakota.

Eight fermentative bacterial strains were isolated from mixed enrichment cultures of a composite soil sample collected at 1.34 km depth from the former Homestake gold mine in Lead, SD, USA. Phylogenetic analysis of their 16S rRNA gene sequences revealed that these isolates were affiliated with the phylum Firmicutes belonging to genera Bacillus and Clostridium. Batch fermentation studies demonstrated that isolates had the ability to ferment glucose, xylose, or glycerol to industrially valuable products such as ethanol and 1,3-propanediol (PDO). Ethanol was detected as the major fermentation end product in glucose-fermenting cultures at pH 10 with yields of 0.205–0.304 g of ethanol/g of glucose. While a xylose-fermenting strain yielded 0.189 g of ethanol/g of xylose and 0.585 g of acetic acid/g of xylose at the end of fermentation. At pH 7, glycerol-fermenting isolates produced PDO (0.323–0.458 g of PDO/g of glycerol) and ethanol (0.284–0.350 g of ethanol/g of glycerol) as major end products while acetic acid and succinic acid were identified as minor by-products in fermentation broths. These results suggest that the deep biosphere of the former Homestake gold mine harbors bacterial strains which could be used in bio-based production of ethanol and PDO.