Actinomyces ruminis sp. nov., an obligately anaerobic bacterium isolated from the rumen of cattle.

An obligately anaerobic, rod-shaped, Gram-stain-positive, non-spore-forming, non-motile bacterial strain; designated as CtC72T was isolated from the rumen of cattle. The 16S rRNA gene sequence similarity of less than 98.65% revealed the strain as a member of the genus Actinomyces, nearest to but distinct from Actinomyces qiguomingii DSM 106201T, Actinomyces ruminicola DSM 27982T, Actinomyces procaprae JCM 33484T, Actinomyces succiniciruminis TISTR 2317, Actinomyces glycerinitolerans TISTR 2318. The low values of digital DNA-DNA hybridization (< 70%) and average nucleotide identity (< 95%) further highlighted the distinctive nature of strain CtC72T from its closest relatives. The strain CtC72T could grow at temperatures between 30 and 50 °C (optimum 40 °C), pH between 6.0 and 9.0 (optimum 7.5-8.0), and NaCl between 0 and 1.5% (optimum 0%). The strain hydrolysed cellulose and xylan and utilised a range of mono-, di-, and oligo-saccharides as a source of carbon and energy. Glucose fermentation resulted in acetic acid and formic acid as major metabolic products, while propionic acid, lactic acid, and ethanol as minor products along with CO2 production. The DNA G + C content of strain CtC72T was 68.40 (mol%, Tm) and 68.05 (%, digital). Major cellular fatty acids (> 10%) were C16:0, C18:1 ω9c, and C18:1 ω9c DMA. Based on these data, we propose that strain CtC72T be classified as a novel species, Actinomyces ruminis sp. nov., under the genus Actinomyces. The type strain is CtC72T (= KCTC 15726T = JCM 32641T = MCC 3500T).

Bioprospecting thermotolerant yeasts from distillery effluent and molasses for high-temperature ethanol production.

AIMS: Isolation, characterization and assessment of inhibitor tolerance of thermotolerant yeasts associated with distillery effluent and molasses, and their use in high-temperature ethanol production from alkali-treated rice straw. METHODS AND RESULTS: A total of 92 thermotolerant yeasts were isolated from seven different distillery effluent and molasses samples. Based on MSP-PCR, 34 yeasts were selected and identified by sequencing the D1/D2 domain of LSU rDNA. These yeasts belonged to eight genera and nine different species. We assessed the inhibitor tolerance of these 34 well-characterized yeasts against various pre-treatment-generated inhibitors (furfural, 5-hydroxymethyl furfural and acetic acid) and also evaluated their ethanol yields at 40, 45 and 50℃. Among selected strains, Pichia kudriavzevii DSA3.2 exhibited the highest ethanol production (24.5 g l-1 ) with an efficiency of 95.7% at 40℃ using 5% glucose. At 45℃, P. kudriavzevii DSA3.2 and Kluyveromyces marxianus MSS6.3 yielded maximum ethanol titres; 22.3 and 23 g l-1 with 87.4% and 90% efficiency, respectively. While using alkali-treated RS at 45℃, K. marxianus MSS6.3 produced 10.5 g l-1 of ethanol with 84.5% fermentation efficiency via separate hydrolysis and fermentation, and 10.9 g l-1 of ethanol with 85% efficiency via simultaneous saccharification and fermentation. Pichia kudriavzevii DSA3.2, DSA3.1 and K. marxianus MSS6.3 also exhibited significant tolerance against multiple inhibitors. CONCLUSIONS: Yeast isolates P. kudriavzevii DSA3.2 and K. marxianus MSS6.3 exhibited significant inhibitor tolerance and proved to be suitable for high-temperature ethanol fermentation. After additional optimization and scale-up experiments, these isolates can be exemplary candidates for industrial-scale ethanol production from lignocellulosic biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study recognizes distillery effluents and molasses as specialized niches for yeasts with a broad substrate range, capable of tolerating multiple inhibitors and yielding high levels of ethanol at elevated temperatures. These yeasts can further be exploited for bioethanol production through SSF/SHF at a larger scale.

Nectar Yeast Community of Tropical Flowering Plants and Assessment of Their Osmotolerance and Xylitol-Producing Potential.

Floral nectar is colonised by microbes, especially yeasts which alter the scent, temperature, and chemical composition of nectar, thereby playing an essential role in pollination. The yeast communities inhabiting the nectar of tropical flowers of India are not well explored. We isolated 48 yeast strains from seven different tropical flowering plants. Post MSP-PCR-based screening, 23 yeast isolates and two yeast-like fungi were identified, which belonged to 16 species of 12 genera viz. Candida (2 species), Aureobasidium (2 species), Metschnikowia (2 species), Meyerozyma (1 species), Saitozyma (1 species), Wickerhamomyces (1 species), Kodamaea (2 species), Pseudozyma (1 species), Starmerella (1 species), Hanseniaspora (1 species), Rhodosporidiobolus (1 species), Moesziomyces (1 species), and two putative novel species. All yeast strains were assessed for their osmotolerance abilities in high salt and sugar concentration. Among all the isolates, C. nivariensis (SRA2.2, SRA1.1 and SRA2.1), M. caribbica (SRA4.8 and SRA4.6), S. flava SRA4.2, and M. reukaufii SRA3.2 showed significant growth in high concentrations of sugar (40-50% glucose), as well as salt (12-15% NaCl). All 25 strains were also screened for their ability to utilise xylose to produce xylitol. Meyerozyma caribbica was the most efficient xylitol producer, wherein three strains of this species (SRA4.6, SRA4.1, and SRA4.8) generated 18.61 to 21.56 g l-1 of xylitol, with 0.465-0.539 g g-1 yields. Through this study, we draw attention towards the tropical floral nectar as a potential niche for the isolation of diverse, osmotolerant, and xylitol-producing yeasts. Such osmotolerant yeasts have potential applications in food industries and biofuel production.

Cultivation of multiple genera of hydrogenotrophic methanogens from different environmental niches.

Six genera of hydrogenotrophic methanogens, namely Methanobrevibacter, Methanobacterium, Methanocorpusculum, Methanothermobacter, Methanoculleus, and Methanospirillum were cultivated from diverse environmental niches like rumen, feces, gut, and sediments using BY medium. We also report a putative novel genus and two novel species of methanogens isolated from termite, Indian star tortoise, and green iguana.

Clostridium punense sp. nov., an obligate anaerobe isolated from healthy human faeces.

An obligately anaerobic, rod-shaped (0.5-1.0 × 2.0-10.0 µm), Gram-stain-positive bacterium, occurring mainly singly or in pairs, and designated BLPYG-8T, was isolated from faeces of a healthy human volunteer aged 56 years. Cells were non-motile. Oval, terminal spores were formed that swell the cells. The strain was affiliated with the genus Clostridium sensu stricto (Clostridium rRNA cluster I) as revealed by 16S rRNA gene sequence analysis. Strain BLPYG-8T showed 97.3 to 97.4 % 16S rRNA gene sequence similarity with Clostridium sulfidigenes DSM 18982T, Clostridium subterminale DSM 6970T and Clostridium thiosulfatireducens DSM 13105T. DNA-DNA hybridization and phenotypic analysis showed that the strain was distinct from its closest relatives, C. sulfidigenes DSM 18982T, C. subterminale DSM 6970T, C. thiosulfatireducens DSM 13105T with 54.2, 53.9 and 53.3 % DNA-DNA relatedness, respectively. Strain BLPYG-8T grew in PYG broth at temperatures between 20 and 40 °C (optimum 37 °C). The strain utilized a range of amino acids as well as carbohydrates as a source of carbon and energy. Glucose fermentation resulted in the formation of volatile fatty acids mainly acetic acid, n-butyric acid and organic acids such as succinic and lactic acid. The DNA G+C content of strain BLPYG-8T was 44.1 mol%. The major fatty acids (>10 %) were C14 : 0, iso-C15 : 0, C16 : 1ω7c and C16 : 0. Phylogenetic analysis and specific phenotypic characteristics and/or DNA G+C content differentiated the strain from its closest relatives. On the basis of these data, strain BLPYG-8T represents a novel species of the genus Clostridium, for which the name Clostridium punense sp. nov. is proposed. The type strain is BLPYG-8T ( = DSM 28650T = CCUG 64195T = MCC 2737T).

Rationally designed peptide conjugated to gold nanoparticles for detection of aflatoxin B1 in point-of-care dot-blot assay.

Aflatoxin B1 (AFB1) is a hepatotoxic and carcinogenic food contaminant. Although on-site paper-based detection is sensitive it depends on expensive antibodies which are difficult to raise against mycotoxins. Here, we rationally designed a high binding octapeptide, N-KSGKSKPR-C peptide for AFB1 detection, by molecular docking, as confirmed by indirect ELISA (Kd 323 nM). Further, conjugation of octapeptide with gold nanoparticles (26 nm) permitted its use as a visual detection agent in rapid, sensitive dot-blot assay (LOD 0.39 µg/kg). The assay displayed negligible cross-reactivity with co-contaminating mycotoxins. AFB1 recovery from spiked wheat sample was comparable by dot-blot (78-91 %) and HPLC (65-87 %). Evaluation of dot-blot using certified reference material and 146 food and feed samples showed high correlation R2 = 0.87 with HPLC. The assay displayed high accuracy (91 %), sensitivity (71 %) and specificity (96.5 %). Therefore, the developed dot-blot assay holds promise for monitoring AFB1 contamination in food and feed.

Changes in human gut flora with age: an Indian familial study.

BACKGROUND: The gut micro flora plays vital role in health status of the host. The majority of microbes residing in the gut have a profound influence on human physiology and nutrition. Different human ethnic groups vary in genetic makeup as well as the environmental conditions they live in. The gut flora changes with genetic makeup and environmental factors and hence it is necessary to understand the composition of gut flora of different ethnic groups. Indian population is different in physiology from western population (YY paradox) and thus the gut flora in Indian population is likely to differ from the extensively studied gut flora in western population. In this study we have investigated the gut flora of two Indian families, each with three individuals belonging to successive generations and living under the same roof. RESULTS: Denaturation gradient gel electrophoresis analysis showed age-dependant variation in gut microflora amongst the individuals within a family. Different bacterial genera were dominant in the individual of varying age in clone library analysis. Obligate anaerobes isolated from individuals within a family showed age related differences in isolation pattern, with 27% (6 out of 22) of the isolates being potential novel species based on 16S rRNA gene sequence. In qPCR a consistent decrease in Firmicutes number and increase in Bacteroidetes number with increasing age was observed in our subjects, this pattern of change in Firmicutes / Bacteroidetes ratio with age is different than previously reported in European population. CONCLUSION: There is change in gut flora with age amongst the individuals within a family. The isolation of high percent of novel bacterial species and the pattern of change in Firmicutes /Bacteroidetes ratio with age suggests that the composition of gut flora in Indian individuals may be different than the western population. Thus, further extensive study is needed to define the gut flora in Indian population.

New Frontiers in Pest Control: Chitosan Nanoparticles-Shielded dsRNA as an Effective Topical RNAi Spray for Gram Podborer Biocontrol.

Chickpea pod borer, Helicoverpa armigera, displays resistance to chemical insecticides and transgenics. The potential nontransformative RNAi approach of specific gene silencing by mRNA breakdown through exogenous double-stranded (dsRNA) delivery to Helicoverpa faces problems of degradation by nucleases and insect gut pH. We demonstrate that chitosan nanoparticles (CNPs) effectively mediate specific dsRNA delivery against Helicoverpa armigerajuvenile hormone methyltransferase (JHAMT) and acetylcholine esterase (ACHE) target genes. Ionotropically synthesized cationic CNPs (100 nm size, +32 mV charge) loaded dsRNA efficiently and protected it effectively from degradation by nucleases and insect gut pH. Tagging CNPs with Calcofluor fluorescence illustrated its efficient uptake in columnar insect gut cells. The potential of CNPs-mediated dsRNA delivery was elucidated with effective silencing of green fluorescent protein transformed Sf9 cells. Furthermore, CNPs-dsRNA complexes were stable for 5 d on leaf surfaces, and their ingestion with leaf effectively silenced H. armigeraJHAMT and ACHE genes to suppress related enzyme activities and caused 100% insect mortality. Further, in planta bioassay with CNPs-dsRNA spray confirmed the RNAi induced insect mortality. Moreover, CNPs-dsRNA fed nontarget insects Spodoptera litura and Drosophila melanogaster were unaffected, and no toxicity was observed for CNPs in cell line studies. Remarkably, only two low dose (0.028 g/ha) topical CNPs-ache-dsRNA sprays on chickpea displayed reduced pod damage with high yields on par with chemical control in the field, which was followed by CNPs-jhamt-dsRNA nanoformulation. These studies can pave the way for the development of topical application of CNPs-dsRNA spray as a safe, specific, innovative insecticide for sustainable crop protection.

Genome sequencing and analysis of a psychrotrophic methanogen Methanosarcina sp. nov. MSH10X1 cultured from methane hydrate deposits of Krishna Godavari Basin of India.

Methanosarcina sp. strain MSH10X1, a psychrotrophic methanogen, was isolated from sub-seafloor methane hydrate deposits of Krishna Godavari Basin on India's east coast. The strain could grow from 5 to 40 °C following all three i.e. methylotrophic, acetoclastic, and hydrogenotrophic modes of methanogenesis utilizing different substrates like methanol, trimethylamine, H2/CO2 (80/20), acetate, valerate, isobutyrate, isopropanol, and isobutanol. The genome sequencing and analysis of this strain revealed a circular chromosome of 3,557,383 bp length having 42.47 mol% G + C content, which consisted of 3110 coding genes, 58 tRNA genes, and 3 rRNA operons. The KEGG analysis highlighted the presence of genes responsible for all three modes of methanogenesis. The presence of genes like mtaB, mtaC, and mttB in the genome provided evidence for possible adaptation of strain MSH10X1 in the deep sea's low-temperature conditions.

Xylanolytic and Ethanologenic Potential of Gut Associated Yeasts from Different Species of Termites from India.

Xylophagous termites are capable of degrading lignocellulose by symbiotic gut microorganisms along with the host's indigenous enzymes. Therefore, the termite gut might be a potential niche to obtain natural yeasts with celluloytic, xylanolytic and ethanologenic traits required for bioethanol production from lignocellulosic biomass. In this study, we cultured 79 yeasts from three different termites viz. Coptotermes heimi, Odontotermes javanicus and Odontotermes obesus. After suitable screening methods, we identified 53 yeasts, which belonged to 10 genera and 16 different species of both ascomycetous and basidiomycetous yeasts. Most yeasts in the present study represent their first-ever isolation from the termite gut. Representative strains of identified yeasts were evaluated for their cellulolytic, xylanolytic, and ethanologenic abilities. None of the isolates showed cellulase activity; 22 showed xylanolytic activity, while six produced substantial quantities of ethanol. Among xylanolytic cultures, Pseudozyma hubeiensis STAG 1.7 and Hannaella pagnoccae STAG 1.14 produced 1.31 and 1.17 IU of xylanase. Among ethanologenic yeasts, the strains belonging to genera Candida and Kodamaea produced high amount of ethanol. Overall, highest ethanol level of 4.42 g/L was produced by Candida tropicalis TS32 using 1% glucose, which increased up to 22.92 g/L at 35 °C, pH 4.5 with 5% glucose. Fermentation of rice straw hydrolysate gave 8.95 g/l of ethanol with a yield of 0.42 g/g using the strain TS32. Our study highlights the gut of wood-feeding termites as a potential source of diverse yeasts that would be useful in the production of xylanase and bioethanol.

Seven new Neocallimastigomycota genera from wild, zoo-housed, and domesticated herbivores greatly expand the taxonomic diversity of the phylum.

We isolated and characterized 65 anaerobic gut fungal (AGF; Neocallimastigomycota) strains from fecal samples of five wild (W, axis deer, white-tailed deer, Boer goat, mouflon, and Nilgiri tahr), one zoo-housed (Z, zebra), and three domesticated (D,  horse, sheep, and goat) herbivores in the US states of Texas (TX) and Oklahoma (OK), Wales (WA), and the Indian states of Kerala (KE) and Haryana (HA). Phylogenetic assessment using the D1-D2 regions of the large subunit (28S) rDNA and internal transcribed spacer 1 (ITS1) identified seven monophyletic clades that are distinct from all currently recognized AGF genera. All strains displayed monocentric thalli and produced exclusively or predominantly monoflagellate zoospores, with the exception of axis deer strains, which produced polyflagellate zoospores. Analysis of amplicon-based AGF diversity surveys indicated that zebra and horse strains are representatives of uncultured AL1 group, whereas domesticated goat and sheep strains are representatives of uncultured AL5 group, previously encountered in fecal and rumen samples of multiple herbivores. The other five lineages, all of which were isolated from wild herbivores, have not been previously encountered in such surveys. Our results significantly expand the genus-level diversity within the Neocallimastigomycota and strongly suggest that wild herbivores represent a yet-untapped reservoir of AGF diversity. We propose seven novel genera and eight novel Neocallimastigomycota species to comprise these strains, for which we propose the names Agriosomyces longus (mouflon and wild Boer goat), Aklioshbomyces papillarum (white-tailed deer), Capellomyces foraminis (wild Boar goat), and C. elongatus (domesticated goat), Ghazallomyces constrictus (axis deer), Joblinomyces apicalis (domesticated goat and sheep), Khoyollomyces ramosus (zebra-horse), and Tahromyces munnarensis (Nilgiri tahr).

Liebetanzomycespolymorphus gen. et sp. nov., a new anaerobic fungus (Neocallimastigomycota) isolated from the rumen of a goat.

An extended incubation strategy to culture slow growing members of anaerobic fungi resulted in the isolation of a novel anaerobic fungus from the rumen of a goat after 15 days. The novel genus, represented by type strain G1SC, showed filamentous monocentric thallus development and produced uniflagellate zoospores, hence, showing morphological similarity to the genera Piromyces, Buwchfawromyces, Oontomyces and Pecoramyces. However, strain G1SC showed genetic similarity to the genus Anaeromyces, which, though produces uniflagellate zoospore, also exhibits polycentric thallus development. Moreover, unlike Anaeromyces, strain G1SC did not show hyphal constrictions, instead produced a branched, determinate and anucleate rhizoidal system. This fungus also displayed extensive sporangial variations, both exogenous and endogenous type of development, short and long sporangiophores and produced septate sporangia. G1SC utilised various complex and simple substrates, including rice straw and wheat straw and produced H2, CO2, formate, acetate, lactate, succinate and ethanol. Phylogenetic analysis, using internal transcribed spacer 1 (ITS1) and D1/D2 domain of large-subunit (LSU) rRNA locus, clearly showed a separate lineage for this strain, near Anaeromyces. The ITS1 based geographical distribution studies indicated detection of environmental sequences similar (93-96%) to this strain from cattle faeces. Based on morphological and molecular characterisation results of strain G1SC, we propose a novel anaerobic fungus Liebetanzomycespolymorphus gen. et sp. nov., in the phylum Neocallimastigomycota.

Alkali pretreatment at ambient temperature: A promising method to enhance biomethanation of rice straw.

Anaerobic digestion is the most cost effective technology for sustainable biogas production from rice straw. Rice straw was subjected to ambient pretreatment with alkali and subsequently digested anaerobically. A dimensional equation was developed to predict the efficacy of alkali treatment in terms of soluble COD. Biomethanation process parameters like temperature, initial pH, particle size, substrate/inoculum ratio, trace element supplementation, C/N ratio and hydraulic retention time were optimized. The highest biogas production under optimized conditions was 514L/kg VS/day (∼59% CH4) from milled rice straw (1mm) pretreated with sodium hydroxide (1% w/v) at ambient temperature for 180min. The digester was operated at 15days HRT at 37°C and neutral pH. C/N ratio was optimized at 25 using urea. Higher biogas yield from rice straw treated with lower concentration of NaOH at ambient temperature may make this process more economical than the previous reports.

Genome sequencing of multidrug resistant novel Clostridium sp. BL8 reveals its potential for pathogenicity.

BACKGROUND: The human gut microbiome is important for maintaining the health status of the host. Clostridia are key members of the human gut microbiome, carrying out several important functions in the gut environment. Hence understanding the role of different Clostridium species isolated from human gut is essential. The present study was aimed at investigating the role of novel Clostridium sp. isolate BL8 in human gut using genome sequencing as a tool. FINDINGS: The genome analysis of Clostridium sp. BL8 showed the presence of several adaptive features like bile resistance, presence of sensory and regulatory systems, presence of oxidative stress managing systems and presence of membrane transport systems. The genome of Clostridium sp. BL8 consists of a wide variety of virulence factors like phospholipase C (alpha toxin), hemolysin, aureolysin and exfoliative toxin A, as well as adhesion factors, proteases, Type IV secretion system and antibiotic resistance genes. In vitro antibiotic sensitivity testing showed that Clostridium sp. BL8 was resistant to 11 different tested antibiotics belonging to 6 different classes. The cell cytotoxicity assay confirmed the cytotoxic effect of Clostridium sp. BL8 cells, which killed 40% of the Vero cells after 4 hrs of incubation. CONCLUSIONS: Clostridium sp. BL8 has adapted for survival in human gut environment, with presence of different adaptive features. The presence of several virulence factors and cell cytotoxic activity indicate that Clostridium sp. BL8 has a potential to cause infections in humans, however further in vivo studies are necessary to ascertain this fact.

Comparative genome analysis of Megasphaera sp. reveals niche specialization and its potential role in the human gut.

With increasing number of novel bacteria being isolated from the human gut ecosystem, there is a greater need to study their role in the gut ecosystem and their effect on the host health. In the present study, we carried out in silico genome-wide analysis of two novel Megasphaera sp. isolates NM10 (DSM25563) and BL7 (DSM25562), isolated from feces of two healthy individuals and validated the key features by in vitro studies. The analysis revealed the general metabolic potential, adaptive features and the potential effects of these isolates on the host. The comparative genome analysis of the two human gut isolates NM10 and BL7 with ruminal isolate Megasphaera elsdenii (DSM20460) highlighted the differential adaptive features for their survival in human gut. The key findings include features like bile resistance, presence of various sensory and regulatory systems, stress response systems, membrane transporters and resistance to antibiotics. Comparison of the "glycobiome" based on the genomes of the ruminal isolate with the human gut isolates NM10 and BL revealed the presence of diverse and unique sets of Carbohydrate-Active enzymes (CAZymes) amongst these isolates, with a higher collection of CAZymes in the human gut isolates. This could be attributed to the difference in host diet and thereby the environment, consequently suggesting host specific adaptation in these isolates. In silico analysis of metabolic potential predicted the ability of these isolates to produce important metabolites like short chain fatty acids (butyrate, acetate, formate, and caproate), vitamins and essential amino acids, which was further validated by in vitro experiments. The ability of these isolates to produce important metabolites advocates for a potential healthy influence on the host. Further in vivo studies including transcriptomic and proteomic analysis will be required for better understanding the role and impact of these Megasphaera sp. isolates NM10 and BL7 on the human host.

Molecular analysis of gut microbiota in obesity among Indian individuals.

Obesity is a consequence of a complex interplay between the host genome and the prevalent obesogenic factors among the modern communities. The role of gut microbiota in the pathogenesis of the disorder was recently discovered; however, 16S-rRNA-based surveys revealed compelling but community-specific data. Considering this, despite unique diets, dietary habits and an uprising trend in obesity, the Indian counterparts are poorly studied. Here, we report a comparative analysis and quantification of dominant gut microbiota of lean, normal, obese and surgically treated obese individuals of Indian origin. Representative gut microbial diversity was assessed by sequencing fecal 16S rRNA libraries for each group (n=5) with a total of over 3000 sequences. We detected no evident trend in the distribution of the predominant bacterial phyla, Bacteroidetes and Firmicutes. At the genus level, the bacteria of genus Bacteroides were prominent among the obese individuals, which was further confirmed by qPCR (P less than 0.05). In addition, a remarkably high archaeal density with elevated fecal SCFA levels was also noted in the obese group. On the contrary, the treated-obese individuals exhibited comparatively reduced Bacteroides and archaeal counts along with reduced fecal SCFAs. In conclusion, the study successfully identified a representative microbial diversity in the Indian subjects and demonstrated the prominence of certain bacterial groups in obese individuals; nevertheless, further studies are essential to understand their role in obesity.