Study of microbiota in biomass and tobacco smoke associated lung disease

Chronic Obstructive Pulmonary Disease (COPD) is a disease of public health concern globally. It is the third leading cause of death worldwide, and the second leading cause in India. Tobacco and biomass smoke are the main causes leading to different lung diseases. Chronic exposures to tobacco smoke and biomass smoke have been shown to be associated with the risk of developing COPD. In India, there is high prevalence of COPD due to biomass smoke and tobacco smoke exposure. Microbial diversity in tobacco-smoke associated COPD has been studied earlier, microbiota in biomass smoke associated COPD was not yet explored.

A collaborative study was conducted by researchers at KEM hospital Pune, NCMR Pune and Chest Research foundation Pune to understand the biomass and tobacco smoke associated microbiota in COPD. It was not well studied whether there is a difference in the microbiota between healthy subjects and biomass smoke-associated COPD (BMSCOPD) subjects and between BMSCOPD and tobacco smoke-associated COPD (TSCOPD) subjects. The aim of the study was to compare nasal and oral microbiota between healthy, TSCOPD and BMSCOPD from rural population in India. This study suggests that disruption or imbalance in microbial community happens which may further contribute to the progression of COPD.

Study subjects were recruited from the population of KEM Hospital research centre, Vadu, HDSS . Subjects who were diagnosed with COPD were randomly selected from the cohort of COPD subjects. Nasal swabs and oral washings were collected from all the 31 subjects. Amplicon sequencing of bacterial 16S rRNA gene, bioinformatics and statistical analysis was done.

Results indicated that microbial communities differed significantly between nasal and oral samples. Greater diversity and higher interindividual variations were observed in nasal samples as compared to oral samples. The findings also indicated that, not all but few specific taxa contribute to the alteration in microbial community structure from healthy to disease state.

To compare the microbial diversity between healthy and disease state, all the COPD samples (TSCOPD and BMSCOPD) were pooled together and compared with healthy subjects. In the nasal samples, researchers found significant increase in the abundance of Actinomyces, Actinobacillus, Megasphaera, and Selenomonas in COPD group, whereas Propionibacterium was significantly higher in the healthy subjects. In the oral samples, they noted a significant increase in the abundance of Corynebacterium, Selenomonas, and Actinomyces among COPD subjects compared to healthy subjects. In the nasal samples, they found a significantly higher abundance of the phyla Planctomycetes and Armatimonadetes in TSCOPD as compared to BMSCOPD, while Acidobacteria were significantly abundant in BMSCOPD as compared to TSCOPD in the oral samples. Between healthy and BMSCOPD, in nasal samples, they found that 15 genera were significantly different in their abundance between the healthy and BMSCOPD. In oral samples, they found 14 genera which were significantly diferent in their abundance between the healthy and BMSCOPD. A significant difference was found for Haemophilus in oral samples.

It is the first study that has examined differences in the nasal and oral microbiota between healthy, TSCOPD, and BMSCOPD subjects in an Indian rural population. Although there were no significant differences in the overall microbial community structure, they reported significant differences in the microbiota in some key taxa between healthy and COPD subjects and also between TSCOPD and BMSCOPD subjects. Between healthy and COPD subjects, they reported a significant increase in the abundance of Actinomyces, Actinobacillus, Megasphaera, and Selenomonas in the nasal samples of COPD subjects, while Corynebacterium, Selenomonas, and Actinomyces were found to be increased in the oral samples of COPD subjects. Between TSCOPD and BMSCOPD subjects, they reported that Gallibacterium and Methanosaeta were significantly higher in the nasal samples of BMSCOPD subjects, while Acinetobacter was significantly higher in the oral samples of TSCOPD subjects.

The difference in the bacterial communities in BMSCOPD and TSCOPD suggests difference in pathophysiology of the disease and also suggests clinical phenotypic differences between these two groups. Thus, the pathophysiology of TSCOPD and BMSCOPD may differ due to the differences in microbial communities and, this should be considered carefully before designing the treatment method for COPD.


On the Occasion of upcoming National Science Day 2021!

National Science Day is celebrated in India every year on February 28. The day is celebrated to commemorate the discovery of Raman Effect by Sir C.V. Raman on the same day in 1928. Sir C.V. Raman was awarded the Nobel Prize in Physics in 1930 for this important discovery. The Government of India honored him with the highest civilian award, the Bharat Ratna in 1954. The very first National Science Day was celebrated on February 28, 1987.

The objective for celebration of this day is to inspire students about science, spreading the knowledge related to science, scientific activities and scientific achievements. Many events are organized on this day to popularize Science and Technology. The theme for the National Science Day 2021 is ‘ Future of STI: Impact on Education, skills and work.’

Every year NCMR-NCCS Pune arranges Microbial exhibition for school students to celebrate National Science Day, which is not possible this year due to covid-19 situation. On the Occasion of upcoming National Science Day, we are taking an opportunity to describe National Centre for Microbial Resource (NCMR), the largest culture collection of the world.

National Centre for Microbial Resource (NCMR) is a national microbial facility funded by Department of Biotechnology (DBT), Government of India and is affiliated with National Centre for Cell Science (NCCS) Pune. NCMR started as Microbial Culture Collection (MCC) in 2009 has an authorization to preserve and catalogue diversity of microorganisms collected from different ecological niches from all over India and to make them available for exploitation by researchers. NCMR supplies authentic cultures of archaea, bacteria, fungi and plasmids to both academic institutions and industry.

The aim of the Centre is to serve as a leading world-class Microbial Resource Repository and to provide authentic high-quality services for microbial preservation, characterization and authentication and supply to industry and academic institutions. The Centre is built on “Service for Science, Science for Service” model. It is serving the nation in biodiversity conservation, biotechnological research and education by providing services of the highest international standards and conducting research in the related areas of microbial ecology and systematics, and human resource development. The Centre is serving as an International Depositary Authority (IDA) under the Budapest Treaty and Designated National Repository under Ministry of Environment and Forests.

To know more about scientific activities of NCMR , visit:

Happy National Science Day 2021!

A novel species isolated from Queen Maud Land, Antarctica

Scanning electron micrograph of Marisediminicola senii

Antarctica is Earth’s southernmost continent, which is beautifully covered by snow and ice. It is coldest continent with extreme environmental conditions. Antarctica was once thought to be frozen and lifeless, and is now known for its biodiversity where microbial life is thriving. Its important to understand the microbial diversity on this continent as microorganisms play an important role in the functioning of Antarctic ecosystems. A type strain was isolated from glacier sediment sample collected from the Queen Maud Land, Antarctica, during the 38th Indian Scientific Expedition to Antarctica in 2019. Research group of Dr. Avinash Sharma at NCMR-NCCS Pune studied the characteristics of the isolated strain.

Dr. Avinash Sharma collecting sample at Antarctica

Distinguishing characteristics based on the polyphasic analysis indicated the strain as a novel species of genus Marisediminicola for which the name Marisediminicola senii sp. nov., is proposed. The strain is named as Marisediminicola senii in the honor of late Mr. Subhajit Sen, a researcher from India who lost his life in an accident during the 37th Indian Scientific Expedition to Antarctica in 2017. Mr. Sen’s research in Antarctica was focused on fabric analysis of glacial deposits.

Marisediminicola senii sp. nov., with accession number MCC 4327 (Type strain) is now stored at NCMR along with other approximately two lakh microbes. The National Centre for Microbial Resource (NCMR) has an authorization to preserve and catalogue the diversity of microorganisms collected from various ecological niches in India and to make them available for research use.


Microbes sustaining climate change by oxidizing ammonia and sulfur in an Arctic Fjord

Microorganisms play an important role in the ecological balancing of extreme ecosystems. Over the last few years, polar regions have been affected by global warming and extinction of native species. The archaeal and bacterial communities have a very significant and interchangeable role in the nitrogen and sulfur cycling as they perform biological oxidation of ammonia and sulfur. A study was conducted to understand the microbial communities present at different oceanic depths of Krossfjorden with the help of high throughput sequencing methods. The aim of the study was also to decipher the role of microbial communities in the oceanic biogeochemical cycling with focus on ammonia and sulfur cycling.

The sediment samples were collected in triplicates from Krossfjorden (Norway) during the arctic summer. DNA extraction and further amplicon sequencing and analysis was done. The data suggested that bacterial communities are prevalent at the middle and lower sediments while archaeal communities are mainly present at the middle sediment. Thaumarchaeota population was predominant followed by Crenarchaeota, Euryarchaeota, Woesearchaeota and Marine Hydrothermal Vent Group. The study indicated major microbial biomass comprising of Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, Chloroflexi and Lentisphaerae, along with Marinicella, Desulfobulbus, Lutimonas, Sulfurovum and clade SEEP-SRB4 as major members of surface sediments. Interestingly, Bacteroidetes, Firmicutes, Verrucomicrobia and Lentisphaerae were found to be dominant members at lower depth (~180 m), while Proteobacteria, Actinobacteria and Planctomycetes showed more profusion at depth of ~250 m which can be related to its maximum activity at relatively higher depths in the Arctic. Similarly, Fusobacteria, Chloroflexi and Acidobacteria outnumbered other bacterial phyla at the depth of ~300 m. The genera Psychrilyobacter, Psychromonas, Marinifilum were observed in this study are likely to be involved in the hydrolysis and fermentation of spirulina forming volatile fatty acids, mainly acetate which is later utilized by sulfate-reducing bacteria. Sequences related to sulfate-reducing bacteria like Desulfobacteraceae and Desulfobulbaceae were detected in this study which are known for the acetate mineralization. Besides that, the abundant proportion of sulfur oxidizers Sulfurovum, Sulfurimonas from Epsilonproteobacteria was observed which could grow chemolithoautotrophically, which implies its ability to survive in nutrient-deprived conditions.

The study also indicated that archaeal communities across all depths of the fjord were found to engage in ammonia cycling. The bacterial communities showed divergence in the gene abundance of ammonia and sulfur cycling along the different depths. Members of Thaumarchaeota from the domain archaea have the ability to oxidize ammonia and are present ubiquitously in soil, ocean and extreme environments. Members
of Desulfobulbus that reduce both iron and sulfur were observed in this study known for the potential to reduce iron oxide. Sulfurovum and Sulfurimonas belong to the Epsilonproteobacteria and are known for their important role in sulfur cycling in marine and other aquatic environments . The predominance of Sulfurovum with a significant proportion of Sulfurimonas at this site may play the crucial role in the sulfur cycle as Sulfurovum is known to grow chemolithoautotrophically using hydrogen, sulfur, and thiosulfate as an electron donor while oxygen, nitrate, thiosulfate, and sulfur as an electron acceptor.

The study provided a detailed insight into the microbial community composition at Krossfjorden and understanding their metabolic fate. The study also tried to understand the potential of the microbial community to oxidize ammonia and Sulfur at different sites of Arctic fjord by targeted metagenomics.


Description of a novel taxon associated with Sugarcane Grassy Shoot (SCGS) disease

Phytoplasma is a group of extremely small bacteria (mollicutes). They don’t have a cell wall and any particular shape (pleomorphic). Phytoplasma was first identified by a Japanese scientist Yoji Doi as ‘mycoplasma-like-organisms’ in 1967. They are bacterial parasites of plants and insects. Phytoplasmas reside in plant’s phloem tissue while insects serve as vectors for the transmission of infection from plant to plant. Once disease caused by phytoplasma is established, entire fields of crops might be wiped out. Sugarcane is the world’s fourth largest and commercially important crop. Sugarcane Grassy Shoot disease is related to Rice Yellow Dwarf (RYD) phytoplasma which occurs in sugarcane growing countries throughout the world.

The major characteristic of SCGS disease are stunting, profuse tillering, side shoots, chlorotic stripes and bleached white leaf blades. The common symptoms of SCGS in sugarcane plant are narrowing and partially or almost chlorotic leaf lamina, excessive tillering and witches’ broom symptoms. Severely infected younger plants appear yellowish. The phytoplasma infection often leads to stunted growth, reduction in leaf size, and excessive proliferation of shoots.

Complete leaf chlorosis in SCGS disease &
Grassy appearance of phytoplasma-infected sugarcane plant

It’s important to study the genome of phytoplasma to understand how this tiny microbe causes infection in plants and gets transmitted through insect vectors. Phytoplasma DNA is difficult to isolate and then sequence it further, as researchers have not yet been active in this organism’s laboratory cultivation. Recently, the researchers at NCMR Pune successfully isolated and sequenced sugarcane phytoplasma. In this study, researchers demonstrated the phylogenetic position of 16SrXI-B group phytoplasmas by characterizing the phytoplasma strain associated with Sugarcane Grassy Shoot (SCGS) disease based on comparative genome features and phylogenetic analyses with its closely related phytoplasma taxa and proposed a novel ‘Ca. Phytoplasma’ taxon. This study is the first description of phytoplasma from India and the first description of phytoplasma species based on genome sequences.


Understanding microbial salt-stress biology through “omic” approaches

Halotolerant microorganisms are capable of growing in the absence as well as in the presence of relatively high salt concentrations. The biology of the salt affected habitats is studied using high throughput “omic” approaches consisting of metagenomics, transcriptomics, metatranscriptomics, metabolomics, and proteomics. The study of Metagenome-assembled genomes of uncultured halophilic microbes has uncovered the genomic basis of salt stress tolerance in “yet to culture” microorganisms. Also, functional metagenomic approaches have been used to decipher the novel genes from uncultured microbes and their possible role in microbial salt-stress tolerance. A recently published collaborative review helped in understanding microbial salt-stress biology and it also summarized the key molecular processes contributing to microbial salt-stress response.

Microorganisms employ two different strategies to adjust to hypersaline conditions: ‘salt-out’ strategy and ‘salt-in’ strategy. ‘salt-out’ strategy is adopted by halotolerant microbes while ‘salt-in’ strategy is common in halophiles. The review article described the important genes governing microbial halotolerance. The description of many genes, gene clusters or operons that are reported to play an important role in microbial salt-stress response are mostly related to compatible osmolyte biosynthesis. Ectoine biosynthesis gene cluster is considered as a potential marker gene for halotolerance in bacteria. A brief and comprehensive description of genes related to compatible osmolyte biosynthesis, along with their encoded enzymes is presented in the review.

Ectoine biosynthesis: an osmoadaptive response to salt stress is reported in bacteria and is conventionally believed to be absent in Archaea, and Eukarya. The annotation of the genome sequences of ammonia oxidizing archaea has first time lead to the observation about the presence of genes for ectoine biosynthesis even in ammonia-oxidizing archaea. Later, ectoine biosynthetic genes were observed in methanogen genome as well. But the mere presence of the ectoine biosynthesis genes in the genomes of archaea and methanogen does not imply that these genes are functional and ectoine synthesis do exist in Archaea. Further genomic and molecular studies uncovered the uncommon instance of ectoine biosynthesis in Archaea.

Microbes adopting ‘high salt-in’ strategy, display different genomic features. The ‘salt-in’ strategy is well documented in haloarchaea and extremely halophilic bacteria like Salinibacter ruber. The genome of obligate halophiles adopting ‘salt-in’ strategy possess distinct genomic and molecular features supporting a halophilic lifestyle. Most of the halophiles have highly acidic proteome. The halophiles relying on ‘salt-in’ strategy have evolved unique molecular features that differentiate them from microbes adopting ‘salt-out’ strategy for halotolerance.

Genome-resolved metagenomics offer a great opportunity to explore the niche adaptation and metabolic potential of uncultivated microbes. The MAGs obtained can serve as the blueprint for understanding the physiology and salt-stress adaptation strategies of uncultured microbes. The metagenomic genome reconstruction has been applied to recover the genomes of uncultured halophiles.

Using functional metagenomic approach, molecular basis of halotolerance in uncultured microbes is studied. The halotolerant metagenomic clones are studied for the identification and characterization of genes present in metagenomic DNA insert which actually renders the salt tolerance to such metagenomic clones. The techniques like transposon mutagenesis are also used to confirm the role of identified genes in halotolerance. A number of salt-tolerant genes are identified using functional metagenomics. But there are few limitations to metagenomics approach. The major limitation is because many genes/ORFs imparting salt stress tolerance to metagenomic clones, tend to annotate as the hypothetical protein and some even do not show similarity to any other known protein in the database.

The review also covered the detailed transcriptomic studies in microbial halotolerance research like microarray-based gene expression studies for salt-stress response and next-generation sequencing-based transcriptomic studies. The review also provide proteomic insights into microbial halotolerance, metabolic basis of microbial salt-tolerance.

“Omic'”-based microbial halotolerance research suffer few shortcomings like scarce information about halophiles confronting low salt conditions, halophiles warranting reassessment of ‘salt-in’ and ‘salt-out’ delimitations etc. The review concludes that the “Omic” landscape of microbial salt stress tolerance is as vast as the salt afflicted habitats on the earth landscape. It is important to undertake detailed and comprehensive studies that can generate in-depth data at different “Omic” levels on a single biological sample, in order to generate the systems biology view of microbial salt-stress tolerance


A novel species isolated from a bacterial consortia of contaminated soil enriched for the remediation of e-waste

The genus Pseudomonas is widespread and has been reported to occur in diverse ecological niches. Members of the genus Pseudomonas are metabolically versatile and harbor various biotechnologically important properties. Pseudomonas sesami is reported to have plant growth-promoting activity. Strains of Pseudomonas produce thermotolerant proteolytic and lipolytic enzymes which result in food spoilage. Pseudomonas putida, Pseudomonas furukawaii, Pseudomonas knackmussii and many other pseudomonads have been reported for the remediation of xenobiotic compounds. In the present study, researchers at NCMR-NCCS Pune report the detailed characterization of a Pseudomonas strain, which was isolated from soil samples from Lalkuan, Nainital, Uttarakhand, India. The strain was found to be part of the bacterial consortia obtained for developing remediation of e-waste.

The strain was Gram-stain-negative, rod-shaped, aerobic, oxidase-positive and catalase-positive. Colonies are L-form with entire margins, creamy color, umbonate elevation and non-mucoid. Cell can tolerate up to 3% salinity. Based on 16S rRNA gene sequence the strain belongs to the genus Pseudomonas and showed highest sequence similarity to Pseudomonas furukawaii followed by Pseudomonas aeruginosa and Pseudomonas resinovorans. The G+C content in the genome was 64.24mol%. The phylogenetic analysis revealed that the strain forms a distinct clade in the family Pseudomonadaceae. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The phenotypic, chemotaxonomic and genetic analysis, including overall genome relatedness index values, indicated that the strain represents a novel species of the genus Pseudomonas, for which the name Pseudomonas lalkuanensis sp. nov. is proposed.

The cells of the strain were motile as observed by using the hanging drop method. The oxidase and catalase activities were investigated using an oxidase disc and observing bubble production. The phylogenetic analysis showed that strain formed a separate clade with P. resinovorans keeping P. furukawaii and P. aeruginosa in an outer clade with strong bootstrap support. This phylogenetic analysis reveals that the strain is phylogenetically distinct from P. furukawaii and P. resinovorans. The orthoANI and dDDH values of the strain were clearly below the thresholds for the proposal of novel prokaryotic species indicating that the strain belongs to a novel species of the genus Pseudomonas.


Bacterial communities associated with the biofilm formation in Pangong Tso Lake

Pangong Tso Lake is situated in the Himalayan Plateau on both sides of India/China border. This high-altitude lake has an oligotrophic environment with extremes of temperature and exposure to UV radiation. The water of the Pangong Tso is generally very clear. The sediments, including the pebbles and small rocks, did not show any biofilm or microbial mats formation in the past.  However, human activities have increased tremendously near this lake, which might lead to disturbance of this lake ecosystem.  The presence of biofilms in a small area near the shore of the Pangong Tso next to the Maan village was observed by researchers.

Pangong Tso lake

Researchers at NCMR-NCCS Pune were curious to understand the bacterial communities associated with the Pangong Tso lake sediment, water and biofilms. Researchers also studied the metabolic potential of the bacterial community. They used amplicon sequencing of the particular region of 16S rRNA gene and other different tools for this study.  Based on the previous findings on biofilm bacterial communities, researchers hypothesized that the biofilm bacterial communities at Pangong Tso Lake consist of phototrophs and chemotrophs. They also hypothesized that the diversity of the biofilms community is different from suspended water and sediments, where biofilm formation was not observed.

Researchers collected sediment and microbial biofilms sample from the Pangong Tso Lake. Analysis of physio-chemical parameters of water was done. The Calcium and Magnesium chloride contents of water were analyzed .The dissolved Chloride content of water, Sulphate concentration, Nitrate nitrogen and Ammonium nitrogen estimation was done using different techniques. DNA extraction was done from water, sediment and microbial biofilm samples. Different bioinformatics and statistic tools were used in the study. The metabolic potential of the microbial community was predicted using functional prediction tool Tax4Fun and the relative abundance of highly abundant genes involved in different functions were compared across the biofilm, sediment and the water sample.

Overall a total of 6,682,012 raw sequences were generated in the study. Proteobacteria was the most dominant and diverse phylum followed by Bacteroidetes, Acidobacteria, Planctomycetes, Actinobacteria, Firmicutes, Verrucomicrobia, Chlorofexi and Gemmatimonadetes. Significant differences were observed in the microbial diversity of water with sediment and microbial biofilm samples. The water sample was least diverse in comparison to the microbial biofilm and sediment samples. Among the top 50 bacterial genera, which constitutes about 50% of the entire microbiome, Loktanella was highly abundant in the water sample, Rhizobium in sediment samples, and Planktosalinus and Aliidomarina were in biofilm samples. The relative abundance of Proteobacteria was the highest in the water. A sharp decline was observed in the relative abundance of Proteobacteria in sediment and biofilm samples.

Loktanella constitutes nearly half of the total bacterial communities in the water sample, while Loktanella represented less than 1% in the biofilm and sediment samples. Differences were observed in the relative abundance of bacterial taxa across the biofilm and the sediment samples at the phylum and genus based on the Welch t test. Bacterial phyla Verrucomicrobia, Deinococcus-Thermus and Cyanobacteria were explicitly enriched in the biofilm samples. The abundance of Planktosalinus, Aliidiomarina, Halomonas, Predibacter, Paracoccus, and Hyphomonas was significantly high in the microbial mat, whereas Enterobacter and Mesorhizobium were highly abundant in the sediment samples. In addition to this higher abundance of Flavobacterium, Pseudomonas, Luteolibacter, Dyadobacter, Chryseobacterium, Halomonas, Stenotrophomonas, Hyphomonas, Enterobacter, Peredibacter, Acinetobacter, Arenibacter and Exiguobacterium was also recorded across the samples.

A total of 49 pathways were highly abundant, with more than 0.5% mean relative abundance. The pathways related to different functions like peptidases, porphyrin and chlorophyll metabolism, glycoxylate and dicarboxylate metabolism, chaperones and folding catalysts, DNA repair and recombination proteins, pyruvate metabolism, nitrogen metabolism, propanoate metabolism, cysteine and methionine metabolism, butanoate metabolism, transcription machinery, prokaryotic defense system, alanine, aspartate and glutamate metabolism, and homologous recombination were highly abundant in the biofilm samples.

The less diverse bacterial communities in microbial biofilm in comparison to sediments indicated the enrichment of a specific group of bacteria. Stratification of Cyanobacteria (primary producer), sulfate-reducing/ oxidizing bacteria and anoxygenic phototrophic bacteria in the hypersaline microbial mat, took place according to the micro-gradient of oxygen, sulfide, and light which selectively allows the specific bacteria to colonize. The higher abundance of Cyanobacteria in the biofilm samples in comparison to sediment and water sample supported the hypothesis on the establishment of primary producers in the biofilm samples. Sediment samples were the most diverse in comparison to water and microbial biofilm samples, which represents both rare and abundant taxa in the sample. The less diverse bacterial communities in microbial biofilm in comparison to sediments indicated the enrichment of a specific group of bacteria.

To conclude, significant differences were observed in the bacterial diversity in the lake water, sediment, and microbial biofilm samples. Enrichment of specific phyla like Verrucomicrobia, Deinococcus-Thermus, and Cyanobacteria in the microbial biofilm samples indicated the development of saprophytic and photosynthetic communities, which is an important succession event in this high-altitude lake. The predictive analysis of potential functions of these communities also supported the observation as the genes involved in porphyrin and chlorophyll metabolism, glyoxylate and dicarboxylate metabolism, DNA repair and recombination proteins were enriched in the microbial biofilm samples.


Characterization of hyper tolerant bacteria for arsenic oxidation

Groundwater arsenic pollution causes many deaths worldwide. Arsenic levels in ground water are increasing day by day due to human activities, leading to higher threats of arsenic exposure. Arsenic release in drinking water resources causes major health problems.

Researchers at SPPU Pune, NCMR-NCCS Pune and NCL Pune collaboratively attempted to bio-prospect the microorganisms causing arsenic transformation. They used culture-dependent and independent approaches to study the microorganisms from Lonar Lake. Growth and Arsenic oxidation potential of microorganisms at increasing concentration was studied. The study also tried to understand possible pathway of Arsenic oxidation by studying the genes, transcripts and proteins involved.

Soil samples were collected from alkaline Lonar Crater Lake in Maharashtra. DNA was extracted from soil sample using soil DNA extraction kit. Unculturable and culturable diversity of soil sample was studied. Hyper-tolerance towards arsenic was studied. Growth and As(III) oxidation profile, ArsB amplification, enzyme inhibitor assay, As (III) oxidase assay, resting cells assay, Liquid chromatography mass spectrometry analysis , relative quantification of transcripts, microcosm studies and statistical analysis was done.

Bacterial community in the sample set comprised of total 21 phyla. Proteobacteria was predominantly found in the samples followed by Bacteroidetes. The bacterial member Pelagibacteraceae was detected predominantly in the sample followed by Microbacteriaceae, Flavobacteriaceae, Flammeovirgaceae, Vibrionaceae and Rhodobacteraceae with more than 5% abundance in all the samples. The bacterial diversity from Lonar Lake soil exhibited the presence of 10 As(III) oxidizing, 2 As(V) reducing, and 5 arsenic tolerant bacteria. Total 10 different genera were obtained viz. Bacillus, Lysinibacillus, Halomonas, Noviherbaspirillum, Roseomonas, Zobellela, Allidiomarina, Indibacter, Nocardioides, and Oceanimonas.

Arsenic hyper-tolerant Firmicute Bacillus firmus L-148 was isolated from arsenic limiting Lonar lake soil, which tolerated more than 3 M arsenic and could oxidize 75 mM arsenite [As (III)] in 14 days. It oxidized As (III) in presence of heavy metals. B. firmus L-148 was studied at the biochemical, protein, genomic and transcript level for understanding its arsenic oxidizing machinery. This study can be explored for bioremediation of arsenic contaminated water.

The tolerance towards arsenic in bacteria may be due to many reasons like expression of certain genes to combat the deleterious toxic effect. Expression of ArsA can contribute to high tolerance apart from the presence of more than one arsenic transforming operons.  Once it gets delivered, arsenic shows actual level of oxidation . The means of transport of Arsenic was through water pipes and oxidation of samples in the waste water.

Though the potential cultures in this study were isolated from trivial arsenic content environment, they tolerated moderate to high concentration of As(III) and As(V). These findings clearly demonstrated that arsenic tolerance level of bacteria is not correlated to the arsenic content of the environment in which they thrive.


Stress tolerance in bacterial strains of the genus Rhodanobacter isolated from a mixed waste contaminated subsurface

Oak Ridge Integrated Field Research Challenge (ORIFRC) site is characterized by low pH and consists of high nitrate, organics and heavy metals. ORIFRC field laboratory comprises variety of contaminants (uranium, technetium, nitrate, volatile organic carbon species etc.) which are of interest to US Department of Energy. Rhodanobacter is a dominant bacterial species found at this site and ideal for remediation of such mixed contaminated sites.

A collaborative study was conducted by researchers at Florida State University, NCMR-NCCS Pune, University of Illinois, Georgia Institute of Technology and Symbiosis School of Biological Sciences to understand the physiologic basis of stress tolerance in members of the genus Rhodanobacter. The study was conducted in order to understand how bacterial strains of the genus Rhodanobacter survive and dominate in the mixed waste contaminated habitats of the ORIFRC site. To address this, a systematic analysis of relevant phenotypic properties of strains of the genus Rhodanobacter was studied.

Eight strains of Rhodanobacter were isolated from high and low contaminated zones and used for pH and nitrate utilization studies. NaCl, nitrate, nitrite and heavy metal tolerance capacity was studied for the two selected strains of R. denitrificans. Based on metals known to be present at the ORIFRC site, Rhodanobacter strains were tested for tolerance to zinc, cadmium, cobalt, nickel, copper and uranium. To determine the effect of incubation time on growth of R. denitrificans at high metal concentrations, studies were carried out with nickel and uranium since these two metals were found to be present at very high concentrations at the test site.

The results supported the growth potential of Rhodanobacter in acidic subsurface groundwater conditions and confirmed that under suitable cultivation conditions, isolated R. denitrificans strains can tolerate acidic pH consistent with ORIFRC site pH values. It was also observed that organisms adapted to stress better under conditions of high organic content. The ability of Rhodanobacter strains to grow at extremely low pH and under high nitrate and heavy metals concentrations is responsible for their dominance at the contaminated subsurface of the ORIFRC site. The data indicated that both the strains are well adapted to the eco physiological conditions of the contaminated ORIFRC site.

As bacteria from the genus Rhodanobacter are denitrifiers, their activity in the ORIFRC site subsurface is also linked to carbon and nitrogen cycling and may play a critical role in the bioremediation of uranium. Based on prior findings and the results of the current study, researchers postulated that low pH tolerance and high level of stress tolerance for a range of metals along with denitrification potential gives a selective advantage to members of the genus Rhodanobacter. Bacteria from the genus Rhodanobacter are facultative anaerobes, and this physiologic capability makes them ideal candidates for robust growth in the contaminated subsurface of the ORIFRC site. Due to their enhanced stress tolerance abilities, Rhodanobacter spp. survives at low pH and in the presence of elevated concentrations of heavy metals, nitrate and nitrite.