The genome of Symbiodiniaceae-associated Stutzerimonas frequens CAM01 reveals a broad spectrum of antibiotic resistance genes indicating anthropogenic drift in the Palk Bay coral reef of south-eastern India.

An increase in antibiotic pollution in reef areas will lead to the emergence of antibiotic-resistant bacteria, leading to ecological disturbances in the sensitive coral holobiont. This study provides insights into the genome of antibiotics-resistant Stutzerimonas frequens CAM01, isolated from Favites-associated Symbiodiniaceae of a near-shore polluted reef of Palk Bay, India. The draft genome contains 4.67 Mbp in size with 52 contigs. Further genome analysis revealed the presence of four antibiotic-resistant genes, namely, adeF, rsmA, APH (3")-Ib, and APH (6)-Id that provide resistance by encoding resistance-nodulation-cell division (RND) antibiotic efflux pump and aminoglycoside phosphotransferase. The isolate showed resistance against 73% of the antibiotics tested, concurrent with the predicted AMR genes. Four secondary metabolites, namely Aryl polyene, NRPS-independent-siderophore, terpenes, and ectoine were detected in the isolate, which may play a role in virulence and pathogenicity adaptation in microbes. This study provides key insights into the genome of Stutzerimonas frequens CAM01 and highlights the emergence of antibiotic-resistant bacteria in coral reef ecosystems.

Revealing the effect of lipopeptide on improving the probiotics characteristics: Flavor and texture enhancer in the formulated yogurt.

Probiotics are beneficial viable microorganisms that protects from pathogens, enhance immunity and health. In this study, a biosurfactant from marine sponge associated bacteria MS48 was used to effectively enhance the survival of starter cultures and thereby improving their functional properties. The biosurfactant MS48 was characterized as lipopeptide based upon the spectroscopic analysis. The GC-MS analysis showed the moiety as l-glutamic acid methyl ester with the mass of m/z 161.16. Probiotics supplemented with lipopeptide showed better survival in the stress tolerance assays includes acid, bile, heat, salt stress and other assays including auto-aggregation, hydrophobicity, microbial adhesion to solvents (MATS), and simulated gastric juice. Yogurt formulated using lipopeptide showed enhanced flavor components, stability, improved characteristics, EPS production, and lower syneresis than the control. Malonic acid, acidity regulator was detected in the mass spectrum of lipopeptide added yogurt. The texture analysis of the lipopeptide added yogurt showed improved textural and sensorial properties when compared to the control.

Anti-diabetic Potential of a Stigmasterol From the Seaweed Gelidium spinosum and Its Application in the Formulation of Nanoemulsion Conjugate for the Development of Functional Biscuits.

The seaweed Gelidium spinosum was selected for the extraction of phytosterol by the Soxhlet method. The extracted phytosterol was chemically characterized as stigmasterol using Fourier-transform infrared spectrometry and gas chromatography-mass spectrometry analysis. The antioxidant and α-amylase inhibitory activity of stigmasterol has been confirmed by in vitro assays. The in vivo studies demonstrated an anti-diabetic effect in streptozotocin (STZ)-induced hyperglycemic rats. Biochemical analysis showed administration of stigmasterol reduced the blood sugar, urea, and creatinine level. The stigmasterol was then nano-emulsified and incorporated into dough for biscuit formulation. The stigmasterol incorporated biscuit showed higher proximate values, low moisture content, lighter color and the textural property revealed lower hardness. Sensorial results showed acceptability when compared to the control. This study demonstrated the stigmasterol reduced hyperglycemic effects and therefore could be used as a supplement in diets for diabetic patients.

Correction: Characterization of an exopolysaccharide from probiont Enterobacter faecalis MSI12 and its effect on the disruption of Candida albicans biofilm.

[This corrects the article DOI: 10.1039/C5RA10302A.].

Correction to: Microbiome definition re-visited: old concepts and new challenges.

An amendment to this paper has been published and can be accessed via the original article.

Microbiome definition re-visited: old concepts and new challenges.

The field of microbiome research has evolved rapidly over the past few decades and has become a topic of great scientific and public interest. As a result of this rapid growth in interest covering different fields, we are lacking a clear commonly agreed definition of the term "microbiome." Moreover, a consensus on best practices in microbiome research is missing. Recently, a panel of international experts discussed the current gaps in the frame of the European-funded MicrobiomeSupport project. The meeting brought together about 40 leaders from diverse microbiome areas, while more than a hundred experts from all over the world took part in an online survey accompanying the workshop. This article excerpts the outcomes of the workshop and the corresponding online survey embedded in a short historical introduction and future outlook. We propose a definition of microbiome based on the compact, clear, and comprehensive description of the term provided by Whipps et al. in 1988, amended with a set of novel recommendations considering the latest technological developments and research findings. We clearly separate the terms microbiome and microbiota and provide a comprehensive discussion considering the composition of microbiota, the heterogeneity and dynamics of microbiomes in time and space, the stability and resilience of microbial networks, the definition of core microbiomes, and functionally relevant keystone species as well as co-evolutionary principles of microbe-host and inter-species interactions within the microbiome. These broad definitions together with the suggested unifying concepts will help to improve standardization of microbiome studies in the future, and could be the starting point for an integrated assessment of data resulting in a more rapid transfer of knowledge from basic science into practice. Furthermore, microbiome standards are important for solving new challenges associated with anthropogenic-driven changes in the field of planetary health, for which the understanding of microbiomes might play a key role. Video Abstract.

Diversity of a bacterial community associated with Cliona lobata Hancock and Gelliodes pumila (Lendenfeld, 1887) sponges on the South-East coast of India.

Marine sponges are sources of various bioactive metabolites, including several anticancer drugs, produced mainly by sponge-associated microbes. Palk Bay, on the south-east coast of India, is an understudied, highly disturbed reef environment exposed to various anthropogenic and climatic stresses. In recent years, Palk Bay suffered from pollution due to the dumping of untreated domestic sewage, effluents from coastal aquaculture, tourism, salt pans, cultivation of exotic seaweeds, and geogenic heavy-metal pollution, especially arsenic, mercury, cadmium, and lead. Low microbial-abundant sponge species, such as Gelliodes pumila and Cliona lobata, were found to be ubiquitously present in this reef environment. Triplicate samples of each of these sponge species were subjected to Illumina MiSeq sequencing using V3-V4 region-specific primers. In both C. lobata and G. pumila, there was an overwhelming dominance (98 and 99%) of phylum Candidatus Saccharibacteria and Proteobacteria, respectively. The overall number of operational taxonomic units (OTUs) was 68 (40 and 13 OTUs unique to G. pumila and C. lobata, respectively; 15 shared OTUs). Alphaproteobacteria was the most abundant class in both the sponge species. Unclassified species of phylum Candidatus Saccharibacteria from C. lobata and Chelotivorans composti from G. pumila were the most abundant bacterial species. The predominance of Alphaproteobacteria also revealed the occurrence of various xenobiotic-degrading, surfactant-producing bacterial genera in both the sponge species, indirectly indicating the possible polluted reef status of Palk Bay. Studies on sponge microbiomes at various understudied geographical locations might be helpful in predicting the status of reef environments.

Differential bleaching and recovery pattern of southeast Indian coral reef to 2016 global mass bleaching event: Occurrence of stress-tolerant symbiont Durusdinium (Clade D) in corals of Palk Bay.

Information about coral community response to bleaching on Indian reefs is much more limited compared with Indo-Pacific reefs, with no understanding of algal symbionts. We investigated a reef in Palk Bay to understand the coral community response to 2016 bleaching event and to reveal dominant symbiont type association in four common coral genera. Out of 508 colonies surveyed, we found 20.9% (106) mortality in 53.8% (n = 290) of bleached corals. We found differential bleaching and recovery pattern among coral genera. Bleaching was most prevalent in Acropora (86.36%), followed by Porites (65.45%), while moderate to no bleaching was recorded in Favites 5.88%, Symphyllia 51.11% and Favia 55.77%, Platygyra 41.67%, Goniastrea 41.83%. Pre-bleaching and post bleaching samplings revealed changes in dominant symbiont type following bleaching only in Acropora (Cladocopium, Clade C to Durusdinium Clade D) while no such changes were found in other coral genera hosted Clade D. This is the first observation of coral symbiont diversity in the Indian reef.

Culture-dependent and metagenomic analysis of lesser horseshoe bats' gut microbiome revealing unique bacterial diversity and signatures of potential human pathogens.

Bats are highly diverse and ecologically important mammals. They harbor various bacteria, viruses, and fungal communities that are either beneficial or potentially pathogenic. Extensive metagenomic studies in bats are limited, particularly for the gut, and to date, there are no reports on the bacterial diversity of Rhinolophus monoceros from Meghalaya, India. There are limited studies on the isolation of potential harmful or beneficial bacteria and their interactions with the environment through culture-dependent approaches. Therefore, high-throughput screening was used to understand the population structure, genetic diversity, and ecological role of the microorganisms. High-throughput sequencing of the 16S rRNA marker for gene mapping showed that the gut samples constitute a diverse group of bacteria that is dominated by Proteobacteria, followed by Firmicutes. The bacterial genera Corynebacterium and Mycobacterium were also observed in the Illumina dataset. Illumina sequencing revealed eight bacterial phyla composed of 112 genera. The metagenomic analysis of the OTUs from the gut revealed diverse bacterial communities as well as zoonotic and human pathogens. There were differences in the bacterial communities between the two methods used in this study, which could be related to host specificity, diet, and habitat. The culture-dependent technique resulted in the isolation of 35 bacterial isolates, of which Bacillus cereus and B. anthracis are well-known bacterial pathogens that show virulent traits including hemolytic and proteolytic activities. Pseudomonas stutzeri is an opportunistic human pathogen that was also isolated and showed similar traits. Antibiotic sensitivity tests were performed on all 35 isolates, and different antibiotics were used for Gram-positive and -negative bacteria. The result showed that some isolates are resistant to antibiotics such as penicillin G and Cefoxitin. This report on gut bacterial communities could attract interest in the possibility of isolating and characterizing bacteria for the production of antibiotics, enzymes, plant growth promoters, and probiotics. However, the presence of potential pathogenic bacteria that may impose health hazards cannot be ignored and needs to be studied further.

Marine sponge microbial association: Towards disclosing unique symbiotic interactions.

Sponges are sessile benthic filter-feeding animals, which harbor numerous microorganisms. The enormous diversity and abundance of sponge associated bacteria envisages sponges as hot spots of microbial diversity and dynamics. Many theories were proposed on the ecological implications and mechanism of sponge-microbial association, among these, the biosynthesis of sponge derived bioactive molecules by the symbiotic bacteria is now well-indicated. This phenomenon however, is not exhibited by all marine sponges. Based on the available reports, it has been well established that the sponge associated microbial assemblages keep on changing continuously in response to environmental pressure and/or acquisition of microbes from surrounding seawater or associated macroorganisms. In this review, we have discussed nutritional association of sponges with its symbionts, interaction of sponges with other eukaryotic organisms, dynamics of sponge microbiome and sponge-specific microbial symbionts, sponge-coral association etc.

Production and characterisation of glycolipid biosurfactant by Halomonas sp. MB-30 for potential application in enhanced oil recovery.

Biosurfactant-producing Halomonas sp. MB-30 was isolated from a marine sponge Callyspongia diffusa, and its potency in crude oil recovery from sand pack column was investigated. The biosurfactant produced by the strain MB-30 reduced the surface tension to 30 mN m(-1) in both glucose and hydrocarbon-supplemented minimal media. The critical micelle concentration of biosurfactant obtained from glucose-based medium was at 0.25 mg ml(-1) at critical micelle dilution 1:10. The chemical structure of glycolipid biosurfactant was characterised by infrared spectroscopy and proton magnetic resonance spectroscopy. The emulsification activity of MB-30 biosurfactant was tested with different hydrocarbons, and 93.1 % emulsification activity was exhibited with crude oil followed by kerosene (86.6 %). The formed emulsion was stable for up to 1 month. To identify the effectiveness of biosurfactant for enhanced oil recovery in extreme environments, the interactive effect of pH, temperature and salinity on emulsion stability with crude oil and kerosene was evaluated. The stable emulsion was formed at and above pH 7, temperature >80 °C and NaCl concentration up to 10 % in response surface central composite orthogonal design model. The partially purified biosurfactant recovered 62 % of residual crude oil from sand pack column. Thus, the stable emulsifying biosurfactant produced by Halomonas sp. MB-30 could be used for in situ biosurfactant-mediated enhanced oil recovery process and hydrocarbon bioremediation in extreme environments.

Optimization of polyhydroxybutyrate production by marine Bacillus megaterium MSBN04 under solid state culture.

A marine sponge-associated bacterium Bacillus megaterium MSBN04 was used for the production of polyhydroxybutyrate (PHB) under solid state culture (SSC). A central composite design (CCD) was employed to optimize the production medium and to find out the interactive effects of four independent variables, viz. tapioca industry waste, palm jaggery, horse gram flour and trace element solution on PHB production. The maximum yield of PHB 8.637 mg g(-1) of substrate (tapioca industry waste) was achieved from biomass 15.203 mg g(-1) of substrate, using statistically optimized medium. The horse gram flour (nitrogen source) and trace element solution were found to be critical control factors for PHB synthesis. The (1)H NMR analysis revealed that the polymer was a PHB monomer. PHB obtained from this study having high molecular weight (6.7×10(5) Da) with low polydispersity index (PDI) value (1.71) and produced PHB was used to synthesize PHB polymeric nanoparticles using solvent displacement approach. Therefore, B. megaterium MSBN04 is an ideal candidate that can be exploited biotechnologically for the commercial production of PHB under solid state culture.

Synthesis of silver nanoparticles by polysaccharide bioflocculant produced from marine Bacillus subtilis MSBN17.

The polysaccharides are emerging as stabilizing and reducing agents for nanoparticles synthesis, however the commercial polysaccharides are not economically viable. Therefore, the exopolysaccharide from microbial origin such as bioflocculants are promising alternate for the synthesis and stabilization of nanoparticles. In this report, a bioflocculant (MSBF17) was produced from marine sponge-associated Bacillus subtilis MSBN17 under submerged fermentation using the economical substrates. The production was statistically optimized with most significant factors such as palm jaggery, NH(4)NO(2), K(2)HPO(4) and NaCl. The maximum bioflocculant production obtained with statistically optimized medium was 13.42 g/l. Based on the biochemical composition and FT-IR analysis, the flocculant compound was predicted as a polysaccharide derivative. The flocculating activity of the MSBF17 was invariably considerable at high salinity and temperature. It was found that the nano-scale silver can be synthesized in reverse micelles using the bioflocculant as stabilizer. The silver nanoparticles (AgNPs) were characterized by UV-spectroscopy, FT-IR and TEM analysis. The AgNPs were spherical shaped (60 nm) and stable for 5 months. Therefore, the bioflocculant-mediated synthesis of nanomaterials can be considered as environmental benign greener approach.

Isolation identification and biochemical characterization of a novel halo-tolerant lipase from the metagenome of the marine sponge Haliclona simulans.

BACKGROUND: Lipases (EC 3.1.1.3) catalyze the hydrolysis of triacyl glycerol to glycerol and are involved in the synthesis of both short chain and long chain acylglycerols. They are widely used industrially in various applications, such as baking, laundry detergents and as biocatalysts in alternative energy strategies. Marine ecosystems are known to represent a large reservoir of biodiversity with respect to industrially useful enzymes. However the vast majority of microorganisms within these ecosystems are not readily culturable. Functional metagenomic based approaches provide a solution to this problem by facilitating the identification of novel enzymes such as the halo-tolerant lipase identified in this study from a marine sponge metagenome. RESULTS: A metagenomic library was constructed from the marine sponge Haliclona simulans in the pCC1fos vector, containing approximately 48,000 fosmid clones. High throughput plate screening on 1% tributyrin agar resulted in the identification of 58 positive lipase clones. Following sequence analysis of the 10 most highly active fosmid clones the pCC1fos53E1 clone was found to contain a putative lipase gene lpc53E1, encoded by 387 amino acids and with a predicted molecular mass of 41.87 kDa. Sequence analysis of the predicted amino acid sequence of Lpc53E1 revealed that it is a member of the group VIII family of lipases possessing the SXTK motif, related to type C ß-lactamases. Heterologous expression of lpc53E1 in E. coli and the subsequent biochemical characterization of the recombinant protein, showed an enzyme with the highest substrate specificity for long chain fatty acyl esters. Optimal activity was observed with p- nitrophenyl palmitate (C16) at 40°C, in the presence of 5 M NaCl at pH 7; while in addition the recombinant enzyme displayed activity across broad pH (3-12) and temperature (4 -60°C) ranges and high levels of stability in the presence of various solvents at NaCl concentrations as high as 5 M and at temperatures ranging from 10 to 80°C. A maximum lipase activity of 2,700 U/mg was observed with 10 mM p-nitrophenyl palmitate as substrate, in the presence of 5 mM Ca2+ and 5 M NaCl, and a reaction time of 15 min at pH 7 and 40°C; while KM and Vmax values were calculated to be 1.093 mM-1 and 50 µmol/min, respectively. CONCLUSION: We have isolated a novel halo tolerant lipase following a functional screen of a marine sponge fosmid metagenomic library. The activity and stability profile of the recombinant enzyme over a wide range of salinity, pH and temperature; and in the presence of organic solvent and metal ions suggests a utility for this enzyme in a variety of industrial applications.

Biosurfactants as green stabilizers for the biological synthesis of nanoparticles.

Taking into consideration the needs of greener bioprocesses and novel enhancers for synthesis using microbial processes, biosurfactants, and/or biosurfactant producing microbes are emerging as an alternate source for the rapid synthesis of nanoparticles. A microemulsion technique using an oil-water-surfactant mixture was shown to be a promising approach for nanoparticle synthesis. Biosurfactants are natural surfactants derived from microbial origin composed mostly of sugar and fatty acid moieties, they have higher biodegradability, lower toxicity, and excellent biological activities. The biosurfactant mediated process and microbial synthesis of nanoparticles are now emerging as clean, nontoxic, and environmentally acceptable "green chemistry" procedures. The biosurfactant-mediated synthesis is superior to the methods of bacterial- or fungal-mediated nanoparticle synthesis, since biosurfactants reduce the formation of aggregates due to the electrostatic forces of attraction and facilitate a uniform morphology of the nanoparticles. In this review, we highlight the biosurfactant mediated synthesis of nanoparticles with relevant details including a greener bioprocess, sources of biosurfactants, and biological synthesized nanoparticles based on the available literature and laboratory findings.

Synthesis of silver nanoparticles by glycolipid biosurfactant produced from marine Brevibacterium casei MSA19.

The surfactants are emerging as potential nanoparticle stabilizing agents, however, the synthetic surfactants are not economically viable as well as they are not environmentally friendly. Therefore, the biosurfactants are emerging as a green alternate for the synthesis and stabilization of nanoparticles. In this report, a glycolipid biosurfactant was produced from sponge-associated marine Brevibacterium casei MSA19 under solid state fermentation using the agro-industrial and industrial waste as substrate. The production was optimized with factors such as oil seed cake as substrate, glucose as carbon source, beef extract as nitrogen source, FeSO(4).7H(2)O as metal, 2% NaCl, pH 7.0 and 30 degrees C. Based on the biochemical composition, TLC chromatogram, FT-IR and GC-MS analysis, the surface active compound produced by the strain MSA19 was elucidated as a glycolipid derivative. The emulsification index of the biosurfactant produced by B. casei MSA19 was invariably high over the synthetic surfactants such as SDS, Tween20 and Tween80. The purified surfactant concentration in the extract was 18 g/L. It was found that the nano-scale silver can be synthesized in reverse micelles using the glycolipid as stabilizer. The silver nanoparticles synthesized in this study were uniform and stable for 2 months. Therefore, the biosurfactant-mediated nanoparticles synthesis can be considered as "green" stabilizer of nanoparticles.

Production of a new glycolipid biosurfactant from marine Nocardiopsis lucentensis MSA04 in solid-state cultivation.

Considering the need of potential biosurfactant producers and economic production processes using industrial waste, the present study aims to develop solid-state culture (SSC) of a marine actinobacterium for biosurfactant production. A potential biosurfactant producer Nocardiopsis lucentensis MSA04 was isolated from the marine sponge Dendrilla nigra. Among the substrates screened, wheat bran increased the production significantly (E(24) 25%) followed by oil seed cake and industrial waste such as tannery pretreated sludge, treated molasses (distillery waste) and pretreated molasses. Enhanced biosurfactant production was achieved under SSC conditions using kerosene as carbon source, beef extract as nitrogen source and wheat bran as substrate. The maximum production of biosurfactant by MSA04 occurred at a C/N ratio of 0.5 envisaging that a higher amount of nitrogen source is required by the strain compared to that of the carbon source. The kerosene and beef extract interactively increase the production and a stable production was attained with the influence of both factors independently. A significant interactive influence of secondary control factors such as copper sulfate and inoculum size was validated in response surface methods-based experiments. The surface active compound produced by MSA04 was characterized as glycolipid with a hydrophobic non-polar hydrocarbon chain (nonanoic acid methyl ester) and hydrophilic sugar, 3-acetyl 2,5 dimethyl furan. In conclusion, the strain N. lucentensis MSA04 was a potential source of glycolipid biosurfactant, could be used for the development of bioremediation processes in the marine environment.

Sponge-microbial interactions: Ecological implications and bioprospecting avenues.

Sponges are closely associated with microorganisms that occur either intracellularly and extracellularly. Sponges are soft-bodied sessile organisms appear to be defenseless in facing predation. Microbial symbionts supposed to have a functional role in the host defense against pathogens, predation and microfouling processes. Recently, the ubiquitous defense enzyme, phospholipase A2 (PLA2) detected in the sponge associated bacterium envisaged the possible functional role in the ecological succession of host sponge against predatory / fouling pressure in the habitat. In present review, we highlighted the possible functional interactions between associated microbes and host sponges and its potentials in bioprospecting approaches.

Optimization, purification, and characterization of extracellular mesophilic alkaline cellulase from sponge-associated Marinobacter sp. MSI032.

Marinobacter sp. (MSI032) isolated from the marine sponge Dendrilla nigra was optimized for the production of extracellular cellulolytic enzyme (CMCase) by submerged fermentation. Initial experiments showed that the culture medium containing 1% maltose as carbon source and 1% peptone and casein as nitrogen source supported maximal enzyme production at 27 degrees C and at a pH of 9.0. Further optimization carried out showed the maximal enzyme production was supported by the presence of 2% NaCl and 10 mM Zn(2+) ions in the production media. The production of enzyme cellulase occurred at 48 h of incubation which proved the importance of this strain for cellulase production in large scale. Further, the enzyme was purified to 12.5-fold with a 37% yield and a specific activity of 2,548.75 U/mg. The purified enzyme displayed maximum activity at mesophilic temperature (27-35 degrees C) and at a broad pH range with optimal activity at pH 9.0. The purified enzyme was stable even at a higher alkaline pH of 12.0 which is greater than the pH stability that has not been reported in any of the cellulolytic isolates studied so far. Thus, from the present study, it is crucial that, instead of exploring the thermophilic resource that is limited in natural environments, the mesophilic bacteria that occurs commonly in nature can be added up to the database of cellulolytic bacteria. Thus, it is possible that a wide diversity of mesophilic bacteria associated with marine sponges opens up a new doorstep for the degradation of cellulosic waste material for the production of liquid fuels. This is the first report elucidating the prospects of sponge-associated marine bacterium for the production of extracellular alkaline cellulase.

Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3.

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 degrees C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.