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Background: A major cause of economic losses in the medical implant sector has been bacterial biofilms due to their ability to persist on various surfaces and their tolerance against endogenous defences, antibiotics, or other anti-microbial agents. The quest for potential sources causing inhibition or disruption of bacterial biofilms has been taken up to alleviate the loss. Plantderived extracts such as essential oils, bioactive compounds and other solvent extracts are regularly being used instead of antibiotics and other synthetic compounds as they are safer, economical, and in many instances, have an elaborate history of traditional medicinal usage. Objectives: As a plant that has been traditionally used over the centuries, the Moringa oleifera Lam., or more commonly the drumstick tree, is being tapped for myriad pharmaceutical applications. The protein-rich leaf of this tree has not only proved to be of great nutritional value but also divulged numerous potential therapeutic applications. Methods: While reports of proteinaceous components are rare, here we report the efficacy of the aqueous extract of the leaf of M. oleifera and a 62 kDa protein component in the disruption of staphylococcal biofilms, which are largely implicated in nosocomial infections. Results: The application of the M. oleifera leaf extract protein had a marked effect on the biofilm growth or formation by Staphylococcus aureus. Conclusion: While the crude extract itself showed considerable disruption of biofilm formation, the application of the purified protein obtained after a two-step process led to a significant increase in the anti-biofilm activity.

Polycyclic aromatic hydrocarbons (PAHs) are released into the environment via several natural and anthropogenic sources leading to long-term consequences that severely affect the environment, ecosy...

The bacterial diversity associated with eroding sponges belonging to the Cliona viridis species complex is scarcely known. Cliona thomasi described from the West Coast of India is a new introduction to the viridis species complex. In this study, we determined the bacterial diversity associated with C. thomasi using next-generation sequencing. The results revealed the dominance of Proteobacteria followed by Cyanobacteria, Actinobacteria and Firmicutes. Among Proteobacteria, the Alphaproteobacteria were found to be the most dominant class. Furthermore, at the genus level, Rhodothalassium were highly abundant followed by Endozoicomonas in sponge samples. The beta-diversity and species richness measures showed remarkably lower diversity in Cliona thomasi than the ambient environment. The determined lower bacterial diversity in C. thomasi than the environmental samples, thus, categorized it as a low microbial abundance (LMA). Functional annotation of the C. thomasi-associated bacterial community indicates their possible role in photo-autotrophy, aerobic nitrification, coupling of sulphate reduction and sulphide oxidization. The present study unveils the bacterial diversity in bioeroding C. thomasi, which is a crucial step to determine the functions of the sponge holobiont in coral reef ecosystem.

Copper is one of the essential trace dietary minerals for all living organisms, but is potentially toxic at higher concentrations, mainly due to the redox reactions in its transition state. Tolerance of microbes towards copper is primarily attributed to chelation and biosorption. In this study, marine-derived filamentous fungi were evaluated for their ability to remove Cu(ii) from a culture medium. Further, the cellular response of a select isolate to salinity stress (0, 35 and 100 PSU) and Cu(ii) stress (0, 100, and 500 ppm) was studied using the peptide mass fingerprinting technique, which revealed expression of 919 proteins, of which 55 proteins were commonly expressed across all conditions. Housekeeping proteins such as citrate synthase, pyruvate carboxylase, ribosomal proteins, ATP synthases, and more were expressed across all conditions. Reactive oxygen species scavenging proteins such as glutaredoxin, mitochondrial peroxiredoxins and thioredoxins were expressed under Cu(ii) and salinity stresses individually as well as in combination. Up-regulation of glutaredoxin under Cu(ii) stress with fold change values of 18.3 and 13.9 under 100 ppm and 500 ppm of Cu(ii) indicated active scavenging of free radicals to combat oxidative damage. The common mechanisms reported were enzymatic scavenging of free radicals, activation of DNA damage and repair proteins and probable intracellular metal chelation. This indicated multiple stress mechanisms employed by the isolate to combat the singular and synergistic effects of Cu(ii) and salinity stress.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the effectiveness of interventions. Asymptomatic breakthrough infections have been a major problem during the ongoing surge of Delta variant globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines used in the higher-income regions. Here, we show for the first time how statistical and machine learning (ML) approaches can discriminate SARS-CoV-2 infection from immune response to an inactivated whole virion vaccine (BBV152, Covaxin, India), thereby permitting real-world vaccine effectiveness assessments from cohort-based serosurveys in Asia and Africa where such vaccines are commonly used. Briefly, we accessed serial data on Anti-S and Anti-NC antibody concentration values, along with age, sex, number of doses, and number of days since the last vaccine dose for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine (SVM) model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, 724 were classified as infected. Since the vaccine contains wild-type virus and the antibodies induced will neutralize wild type much better than Delta variant, we determined the relative ability of a random subset of such samples to neutralize Delta versus wild type strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, Delta variant, was neutralized more effectively than the wild type, which cannot happen without infection. The fraction rose to 71.8% (28 of 39) in subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period.

The deep sea is the largest environment on Earth, comprising important resources of commercial interest. It is composed of a wide variety of ecosystems, which is home to often unique organisms that are yet to be described. The deep-sea is one of the least studied environments, where research is strongly linked to technological access and advances. With the recent advances in the next-generation sequencing and bioinformatics tools, there is an enhanced understanding of microbial diversity and ecological functions in deep sea. Multidisciplinary programs are being undertaken to investigate into microbial communities in diverse marine environments. As compared to other Oceans, the deeper parts of Indian Ocean are still poorly sampled and studied for bacterial, and more so fungal diversity. The studies reporting usage of modern sequencing tools to describe uncultured microbial diversity have seen a rise in numbers in the last decade. In this review, we summarize the important findings of research works carried on bacterial and fungal diversity from the abyssal regions of the Indian Ocean and provide our views on possible future paths.

Background:Microbial remediation is an ecologically safe alternative to controlling environmental pollution caused by toxic aromatic compounds including azo dyes. Marine bacteria show excellent potential as agents of bioremediation. However, a lack of understanding of the entailing mechanisms of microbial degradation often restricts its wide-scale and effective application.Objective:To understand the changes in a bacterial proteome profile during azo dye decolorization.Methods:In this study, we tested a Gram-negative bacterium, Shewanella seohaensis NIODMS14 isolated from an estuarine environment and grown in three different azo dyes (Reactive Black 5 (RB5), Reactive Green 19 (RG19) and Reactive Red 120 (RR120)). The unlabeled bacterial protein samples extracted during the process of dye decolorization were subject to mass spectrometry. Relative protein quantification was determined by comparing the resultant MS/MS spectra for each protein.Results:Maximum dye decolorization of 98.31% for RB5, 91.49% for RG19 and 97.07% for RR120 at a concentration of 100 mg L-1 was observed. The liquid chromatography-mass spectrometry - Quadrupole Time of Flight (LCMS-QToF) analysis revealed that as many as 29 proteins were up-regulated by 7 hours of growth and 17 by 24 hours of growth. Notably, these were common across the decolorized solutions of all three azo dyes. In cultures challenged with the azo dyes, the major class of upregulated proteins was cellular oxidoreductases and an alkyl hydroperoxide reductase (SwissProt ID: A9KY42).Conclusion:The findings of this study on the bacterial proteome profiling during the azo dye decolorization process are used to highlight the up-regulation of important proteins that are involved in energy metabolism and oxido-reduction pathways. This has important implications in understanding the mechanism of azo dye decolorization by Shewanella seohaensis.

Data science has been an invaluable part of the COVID-19 pandemic response with multiple applications, ranging from tracking viral evolution to understanding the vaccine effectiveness. Asymptomatic breakthrough infections have been a major problem in assessing vaccine effectiveness in populations globally. Serological discrimination of vaccine response from infection has so far been limited to Spike protein vaccines since whole virion vaccines generate antibodies against all the viral proteins. Here, we show how a statistical and machine learning (ML) based approach can be used to discriminate between SARS-CoV-2 infection and immune response to an inactivated whole virion vaccine (BBV152, Covaxin). For this, we assessed serial data on antibodies against Spike and Nucleocapsid antigens, along with age, sex, number of doses taken, and days since last dose, for 1823 Covaxin recipients. An ensemble ML model, incorporating a consensus clustering approach alongside the support vector machine model, was built on 1063 samples where reliable qualifying data existed, and then applied to the entire dataset. Of 1448 self-reported negative subjects, our ensemble ML model classified 724 to be infected. For method validation, we determined the relative ability of a random subset of samples to neutralize Delta versus wild-type strain using a surrogate neutralization assay. We worked on the premise that antibodies generated by a whole virion vaccine would neutralize wild type more efficiently than delta strain. In 100 of 156 samples, where ML prediction differed from self-reported uninfected status, neutralization against Delta strain was more effective, indicating infection. We found 71.8% subjects predicted to be infected during the surge, which is concordant with the percentage of sequences classified as Delta (75.6%-80.2%) over the same period. Our approach will help in real-world vaccine effectiveness assessments where whole virion vaccines are commonly used.

Background: India has been amongst the most affected nations during the SARS CoV2 pandemic, with sparse data on country wide spread of asymptomatic infections and antibody persistence. This longitudinal cohort study was aimed to evaluate SARS CoV2 seropositivity rate as a marker of infection and evaluate temporal persistence of antibodies with neutralization capability and to infer possible risk factors for infection. Methods: Council of Scientific and Industrial Research, India (CSIR) with its more than 40 laboratories and centers in urban and semi urban settings spread across the country piloted the pan country surveillance. 10427 adult individuals working in CSIR laboratories and their family members based on voluntary participation were assessed for antibody presence and stability was analyzed over 6 months utilizing qualitative Elecysys SARS CoV2 specific antibody kit and GENScript cPass SARS CoV2 Neutralization Antibody Detection Kit. Along with demographic information, possible risk factors were evaluated through self to be filled online forms with data acquired on blood group type, occupation type, addiction and habits including smoking and alcohol, diet preferences, medical history and transport type utilized. Symptom history and information on possible contact and compliance with COVID 19 universal precautions was also obtained. Findings:1058 individuals (10.14%) had antibodies against SARS CoV2. A follow up on 346 seropositive individuals after three months revealed stable to higher antibody levels against SARS CoV2 but declining plasma activity for neutralizing SARS CoV2 receptor binding domain and ACE2 interaction. A repeat sampling of 35 individuals, at six months, revealed declining antibody levels while the neutralizing activity remained stable compared to three months. Majority of seropositive individuals (75%) did not recall even one of nine symptoms since March 2020. Fever was the most common symptom with one fourth reporting loss of taste or smell. Significantly associated risks for seropositivity (Odds Ratio, 95% CI, p value) were observed with usage of public transport (1.79, 1.43 to 2∙24, 2.81561x10-6), occupational responsibilities such as security, housekeeping personnel etc. (2.23, 1.92 to 2.59, 6.43969x10-26), non smokers (1.52, 1.16 to 1.99, 0.02) and non vegetarianism (1.67, 1.41 to 1.99, 3.03821x10-8). An iterative regression analysis was confirmatory and led to only modest changes to estimates. Predilections for seropositivity was noted with specific ABO blood groups; O was associated with a lower risk. Interpretation: In a first of its kind study from India, we report the seropositivity in a country wide cohort and identify variable susceptible associations for contacting infection. Serology and Neutralizing Antibody response provides much sought for general insights on the immune response to the virus among Indians and will be an important resource for designing vaccination strategies. Funding: Council of Scientific and Industrial Research, India (CSIR)

We report here the production of an alkaline serine protease by Aspergillus flavus isolated at 5600-m depth from deep-sea sediments of the Central Indian Basin. When grown on defatted groundnut oil meal at 30 °C for 48–72 h, this fungal isolate produced 2000–2500 ACU mL−1 of alkaline protease. The purified protease had activity optima at pH 10.0 and 45 °C. It was a thiol-independent serine protease, identified as an alkaline serine protease ALP1 with a molecular mass of 42.57 kDa. The thermostability and activity of the enzyme increased at 60 °C, in the presence of additives such as sucrose, Tween 20, sorbitol, Ca2+ and glycerol and was not adversely affected by H2O2 indicating its potential as a detergent additive.

We report here the production of an alkaline serine protease by Aspergillus flavus isolated at 5600-m depth from deep-sea sediments of the Central Indian Basin. When grown on defatted groundnut oil meal at 30 °C for 48–72 h, this fungal isolate produced 2000–2500 ACU mL(−1) of alkaline protease. The purified protease had activity optima at pH 10.0 and 45 °C. It was a thiol-independent serine protease, identified as an alkaline serine protease ALP1 with a molecular mass of 42.57 kDa. The thermostability and activity of the enzyme increased at 60 °C, in the presence of additives such as sucrose, Tween 20, sorbitol, Ca(2+) and glycerol and was not adversely affected by H(2)O(2) indicating its potential as a detergent additive. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02520-x) contains supplementary material, which is available to authorized users.

Cellular response against different heavy metal stress differs with the metal. Arsenic and chromium are heavy metals and toxic to living systems. The concentration of these metals in seawater is very low. However, due to their solubility in nature, they actively enter cells via various transport mechanisms and cause damage to the cells. Brevibacterium casei #NIOSBA88, a marine-derived, gram-positive isolate was multi-metal tolerant. Proteomic analysis of this isolate in response to arsenic and chromium resulted in the identification of total 2549 proteins, out of which 880 proteins were found to be commonly expressed at 750 mgL(−1) arsenic and 100 mgL(−1) chromium and in absence of both the metals. In contrast, 533, 212, and 270 proteins were found to be unique in the absence of any metal, 750 mgL(−1) of arsenic and 100 mgL(−1) of chromium respectively. Proteins such as antibiotic biosynthesis monooxygenase, ArsR family transcriptional regulator, cytochrome C oxidase subunit II, and thioredoxin reductase were exclusively expressed only in response to arsenic and chromium. Other proteins like superoxide dismutase, lipid hydroperoxide reductase, and thioredoxin-disulfide reductase were found to be upregulated in response to both the metals. Most of the proteins involved in the normal cell functioning were found to be downregulated. Major metabolic functions affected include amino acid metabolism, carbohydrate metabolism, translation, and energy metabolism. Peptide mass fingerprinting of Brevibacterium casei #NIOSBA88 exposed to arsenic and chromium respectively revealed the deleterious effect of these metals on the bacterium and its strategy to overcome the stress. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s42770-020-00353-7) contains supplementary material, which is available to authorized users.

The subsurface waters of the northern Arabian Sea display a pronounced oxygen minimum layer associated with high nitrite maxima which provide an ideal niche for organisms that can respire nitrate. Culture-based studies elaborate the physiological characteristics of the organisms and their metabolic activities in biogeochemical cycles. In this study, the bacterial diversity and nitrate utilizing activity of the culturable heterotrophic bacteria inhabiting the water column oxygen minimum zone of Arabian Sea were investigated. Nitrate-reducing bacteria were isolated from the water column in the central Arabian Sea. Genotypic characterization of the isolates using 16 S rDNA gene sequencing grouped them into three phylogenetic groups i.e. Proteobacteria, Firmicutes, and Actinobacteria. Out of the 56 isolated bacteria, 45 strains belonged to Proteobacteria, 6 to Firmicutes and 5 to Actinobacteria. The nitrate reducing ability of the isolates was tested using Griess test. Thirty-six species belonging to genera Alcanivorax, Alteromonas, Halomonas, Pseudoalteromonas, Marinobacter, Bacillus, and Vibrio were positive for NO2-/NO3- reduction. Our results imply that cultivable bacteria capable of utilizing NO3 - available in the system are present in the Arabian Sea oxygen minimum zone and the conditions existing therein must be favorable for their growth and functionality.

Filamentous fungi are ubiquitous eukaryotes having chitin as a major constituent of the cell wall. Chitin is tough to lyse due to which the intracellular fungal proteins are not readily accessible. The problem is further enhanced when the biomass to be analyzed for protein studies is too little due to the extreme experimental parameters under consideration such as increased or lowered pH, temperature, hydrostatic pressure, nutrients, etc. The method described here is capable of obtaining proteins from minuscule quantities of biomass (~5 mg lyophilized biomass). In this study, different lysing conditions and varied composition of extraction buffers were tried to obtain maximum protein of high quality. Lysis with zirconium beads in a combination buffer system (Tris–MgCl(2) buffer, urea buffer I and urea buffer II) was best for extracting proteins from the fungal isolates used. The protocol described here provides for a simple and quick method for extraction of high-quality proteins from very less biomass that could be extended to other tough to lyse biological material also. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12088-018-0752-y) contains supplementary material, which is available to authorized users.

Abiotic factors can cause substantial limitation of growth of microbes. A combination of salinity stress along with chromium (Cr+6), one of the carcinogen, can pose an immediate threat to any living system. To understand how salinity (0, 35 and 100 PSU) and Cr(VI) stress (0, 100 and 500 ppm), affects cells at the molecular level, the cellular response of Purpureocillium lilacinum to the individual as well the combination of both the stresses were studied by peptide mass fingerprinting technique. The study reports 1412 proteins, of which 105 proteins were found to be present across all conditions. The most prevalent functional class expressed was genetic information processing. Proteins involved in free radical scavenging were up-regulated in response to the oxidative stress generated due to both the applied stresses while expression of metal chelators, transporters systems, indicated towards multiple stress tolerance mechanisms to combat synergistic effects of salt and Cr stress.

Chromium (Cr) released from industrial units such as tanneries, textile and electroplating industries is detrimental to the surrounding ecosystems and human health. The focus of the present study was to check the Cr(VI) removal efficiency by marine-derived fungi from liquid broth. Amongst the three Cr(VI) tolerant isolates, #NIOSN-SK56-S19 (Aspergillus sydowii) showed Cr-removal efficiency of 0.01 mg Cr mg(−1) biomass resulting in 26% abatement of total Cr with just 2.8 mg of biomass produced during the growth in 300 ppm Cr(VI). Scanning Electron Microscopy revealed aggregation of mycelial biomass with exopolysaccharide, while Electron Dispersive Spectroscopy showed the presence of Cr(2)O(3) inside the biomass indicating presence of active Cr(VI) removal mechanisms. This was further supported when the Cr(VI) removal was monitored using DPC (1,5-diphenylcarbazide) method. The results of this study point to the potential of marine-derived fungal isolates for Cr(VI) removal. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12088-018-0719-z) contains supplementary material, which is available to authorized users.

Tarballs, the remnants of crude oil which change into semi-solid phase due to various weathering processes in the sea, are rich in hydrocarbons, including toxic and almost non-degradable hydrocarbons. Certain microorganisms such as fungi are known to utilize hydrocarbons present in tarballs as sole source of carbon for nutrition. Previous studies have reported 53 fungal taxa associated with tarballs. There is apparently no gene sequence-data available for the published taxa so as to verify the fungal identification using modern taxonomic tools. The objective of the present study is to isolate fungi from tarballs collected from Candolim beach in Goa, India and investigate their phylogenetic diversity based on 5.8S rRNA gene and the flanking internal transcribed spacer regions (ITS) sequence analysis. In the ITS-based NJ tree, eight tarball-associated fungal isolates clustered with 3 clades of Dothideomycetes and 2 clades of Saccharomycetes. To the best of our knowledge, this is the first study that has employed ITS-based phylogeny to characterize the fungal diversity associated with tarballs. Further studies are warranted to investigate the role of the tarball-associated fungi in degradation of recalcitrant hydrocarbons present in tarballs and the role of tarballs as carriers of human pathogenic fungi.

Chromium in its hexavalent state is a water-soluble and toxic element to living organisms present in the environment. However, some organisms are resistant and reduce the toxic forms of Cr(vi) to less toxic or non-toxic forms. A global proteomic analysis of Staphylococcus sp. #NIOSBK35 under different chromate concentrations (0, 100, 200 and 300 mg L−1) at different time points in its growth stages (6, 9, 12, 18, 24 and 36 h) resulted in the identification of 878 proteins. Of all the proteins expressed, 13 proteins [23 rDNA (uracil-5-) methyltransferase RumA, multidrug ABC transporter ATP binding protein, dihydroxy acid dehydratase, polysaccharide biosynthesis protein, etc.] were expressed only in the presence of chromium. 14 proteins were up-regulated in response to chromium(vi), namely, alkyl hydroperoxide reductase, ATP-dependent Zn metallopeptidase, hsp90- like protein, NAD (P)-dependent oxidoreductase, etc. Most of the proteins involved in normal cell functioning like 1-pyrroline-5-carboxylate dehydrogenase, ribosomal proteins (30S ribosomal protein S11, 30S ribosomal protein S2, and 50S ribosomal protein L32), aconitate hydratase, DNA primase, serine-tRNA ligase, phosphoenolpyruvate-protein phosphotransferase, enolase, sulfur transferase FdhD, etc. were found to be down-regulated. On grouping these proteins into their COG (cluster of orthologous groups) functional categories, they were found to be involved in translation, carbohydrate metabolism, stress proteins, amino acid transport and membrane transport mechanisms. The proteomic response given by Staphylococcus sp. #NIOSBK35 did not show expression of Cr-specific proteins, indicating a different mechanism of Cr-tolerance as the organism was able to survive and grow at high concentrations of Cr(vi).

Halophilic bacteria are well known to produce highly salt-tolerant enzymes that have unusual applications in biotechnology. Production of halophilic proteins is generally not expected in mesohaline microorganisms. Ulkenia sp. AH-2, a mesohaline, marine straminipilan thraustochytrid isolated from waters of a mangrove ecosystem, produces halophilic alpha-amylases. Four enzyme fractions, viz.., A, B, C, and D, were obtained upon ammonium sulfate fractionation and gel filtration. These had a broad salinity tolerance ranging from 0 to 4 M NaCl, with an optimum at 3 M NaCl. Pools A, C, and D each resolved as a single band on PAGE and zymogram analysis, and the purified proteins were designated Amy a, Amy c, and Amy h. The major activity resided in “pool B,” consisting of several amylases which could not be further resolved into pure fractions. Together, these had an optimum at 2 M NaCl. All the enzymes were stable to storage for 2 to 24 h at 4 °C in a range of salt concentrations and even showed enhanced activity following such incubations. True to halophilic enzymes, the complex of “pool B” amylases showed improved activity in the presence of a wide range of organic solvents at 20% concentration. These enzymes are of particular interest by virtue of their constitutive nature as well as production under culture conditions that do not require salinity beyond that of seawater.

There are regions in the world oceans where oxygen saturation is at its lowest, evident at depths between shelf to upper bathyal zone. These regions are known as Oxygen Minimum Zones (OMZs), which reportedly support phylogenetically diverse microbes. In this study, we aimed to characterize prokaryotic diversity in the water samples collected from 43, 200 and 1000 m depth of the Bay of Bengal Time Series location (BoBTS—18.0027°N, 89.0174°E) in the OMZ region. Illumina sequencing generated 3,921,854 reads of 16S rRNA gene amplicons, which corresponded to 5778 operational taxonomic units. The distribution of bacteria at class level varied with depth and oxygen concentration. α-Proteobacteria was found in abundance in 43 m and 1000 m depth water samples. γ-Proteobacteria was prominently detected in oxygen-depleted depths of 200 m and 1000 m. AB16 (Marine Group A, originally SAR406) was restricted at dissolved oxygen concentration of 1.5 μM at 200 m. Archaeal members were observed in low abundance (2%), with a high occurrence of phylum Euryarchaeota at 43 m, while Crenarchaeota was detected only at 200 m depth. Select bacterial cultures were screened for their ability to reduce nitrate in vitro, to obtain insights into their possible role in the nitrogen cycle. A total of 156 bacterial isolates clustered majorly with Alcanivorax, Bacillus, Erythrobacter, Halomonas, Idiomarina and Marinobacter. Among them, 11 bacterial genera showed positive nitrate reduction in the Griess test. A large percentage (63.55%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxonomic levels, suggesting a greater undiscovered prokaryotic diversity in this oxygen depleted region. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12088-019-00786-1) contains supplementary material, which is available to authorized users.

Liquid Chromatography–Mass Spectrometry-Quadrupole Time of Flight (LC/MS QToF) protein profiling of marine-derived Staphyloccous gallinarum NIOMR8 was carried out to evaluate proteins conferring chromate (Cr(6+)) resistance and possible metabolic pathways that were altered as a result. Expressional (up or down-regulation) responses to varying Cr(6+) (0, 50, 100, 150, and 200 µg mL(− 1)) concentrations varied, with as many as 346 proteins identified. Most number of proteins—their numbers in parentheses—were up-regulated when grown in medium with 50 µg mL(− 1) (162) and, down-regulated in medium with 100 (281) or 200 µg mL(− 1) Cr(6+) (280). Among these, eight proteins were commonly up-regulated, while 58 were commonly down-regulated across all conditions of Cr(6+). Expression of protein moieties in metabolic pathways like translation (38), transcription (14), replication (18) and repair (4), metabolism of carbohydrates (26), amino acids (27), nucleotides (17), and membrane transport (21) was evidenced. Up-regulation patterns suggest that reduction of molecular oxygen (5), DNA repair (4) and peptide misfolding (7) were the potential protective mechanisms employed to counter Cr(6+) stress. Additionally, proteins associated with biofilm and cell wall biogenesis highlight their hypothetical involvement in toxicity tolerance. Results also indicate that at higher concentrations of Cr(6+), down-regulation of functional proteins impedes normal cellular functions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-018-1522-6) contains supplementary material, which is available to authorized users.

Sedimentary bacteria play a role in polymetallic nodule formation and growth. There are, however, limited reports on bacterial diversity in nodule-rich areas of the Central Indian Ocean Basin (CIOB). In this study, bacterial abundance in thirteen sediment cores collected from the CIOB was enumerated, followed by phylogenetic characterisation and, screening of select heterotrophic bacteria for extracellular enzyme activities. Total bacterial counts (TBC) were in the order of 107 cells g−1; there was a significant difference (p > 0.05) among the cores but not within the sub-sections of the cores. The retrievable heterotrophic counts ranged from non-detectable to 5.33 × 105 g−1; the heterotrophic bacteria clustered within the phyla Firmicutes, Proteobacteria and Actinobacteria. Bacillus was the most abundant genus. The extracellular enzyme activities were in the order: amylase > lipase > protease > phosphatase > Dnase > urease. Major findings are compared with previous studies from the CIOB and other areas.

Peptide mass fingerprinting of Gram-positive marine-derived Staphylococcus cohnii #NIOSBK35 gave us an insight into the proteins involved in conferring arsenic resistance as well as the probable metabolic pathways affected under metal stress. Analysis of the protein profiles obtained from LC/MS QToF (Liquid Chromatography-Mass Spectrometry-Quadrupole Time of Flight) resulted in the identification of 689 proteins. Further grouping of these proteins based on the arsenic concentration (0, 250, 500 and 850 ppm) and the time points (6, 9, 12, 18, 24 and 36 h) in growth phase showed that a total of 13 proteins were up-regulated, while 178 proteins were down-regulated across all the concentrations and time points. Arsenic specific proteins like arsenical pump-driving ATPase, ArsR family transcriptional regulator and arsenic operon resistance repressor were found to be highly up-regulated throughout all the conditions indicating their possible involvement in the tolerance to arsenic. MBL fold metallo-hydrolase, a known stress protein, was the only protein that was up-regulated at all time points across all arsenic concentrations. Metabolic pathways like translation, carbohydrate metabolism, amino acid metabolism, membrane transport, metabolism of cofactors and vitamins, replication and repair, nucleotide metabolism along with stress proteins and hypothetical proteins were found to be significantly expressed. Our results also suggest that arsenic stress at higher levels is negatively affecting the expression of many normal functional proteins required for cell survival.

The fate of the enormous amount of reactive nitrogen released to the environment by human activities in India is unknown. Here we show occurrence of seasonal stratification and generally low concentrations of dissolved inorganic combined nitrogen, and high molecular nitrogen (N2) to argon ratio, thus suggesting seasonal loss to N2 in anoxic hypolimnia of several dam-reservoirs. However, 15N-experiments yielded low rates of denitrification, anaerobic ammonium oxidation and dissimilatory nitrate reduction to ammonium—except in the presence of methane (CH4) that caused ~12-fold increase in denitrification. While nitrite-dependent anaerobic methanotrophs belonging to the NC10 phylum were present, previously considered aerobic methanotrophs were far more abundant (up to 13.9%) in anoxic hypolimnion. Methane accumulation in anoxic freshwater systems seems to facilitate rapid loss of reactive nitrogen, with generally low production of nitrous oxide (N2O), through widespread coupling between methanotrophy and denitrification, potentially mitigating eutrophication and emissions of CH4 and N2O to the atmosphere., The fate of anthropogenic nitrogen (N) remains understudied in South Asian water bodies despite its impact on water chemistry and quality. Here the authors show that N loss in Indian freshwater reservoirs is tightly coupled to methanotrophy, which has helped curb eutrophication and greenhouse gas emissions.

Ten fungal isolates with an ability to degrade crude oil were isolated from select marine substrates, such as mangrove sediments, Arabian Sea sediments, and tarballs. Out of the ten isolates, six belonged to Aspergillus, two to Fusarium and one each to Penicillium and Acremonium as identified using ITS rDNA sequencing. The selected ten fungal isolates were found to degrade the long-chain n-alkanes as opposed to short-chain n-alkanes from the crude oil. Mangrove fungus #NIOSN-M126 (Penicillium citrinum) was found to be highly efficient in biodegradation of crude oil, reducing the total crude oil content by 77% and the individual n-alkane fraction by an average of 95.37%, indicating it to be a potential candidate for the development into a bioremediation agent.

Wind-blown dry fungal spores and mycelial fragments from the nearest landmass or terrestrial run-off may find their way to the deep sea by hitching a ride on other sinking detrital particles. Once in the deep, they are affected by elevated hydrostatic pressure, low temperature and low nutrients. We have examined effects of these on germination of spores from a few deep-sea Aspergillus isolates. Spores from most of the fungi germinated under elevated hydrostatic pressure at 30 °C. The ambient temperature of the deep sea, ∼4–5 °C, was found to inhibit spore germination totally. Sediment extracts prepared in seawater promoted spore germination as did additions of dimethylsulfoxide and sucrose, but only at 30 °C/200 bar pressure and not at 4–5 °C. Heat shock of 15 min at 50 °C helped in breaking the dormancy of the spores and induced germination at 5 °C at 1 bar pressure but not at 200 bar. More than 90% of the spores from several deep-sea Aspergillus isolates and the terrestrial isolate of Aspergillus terreus lost viability within 16–17 days of incubation at 5 °C/1 bar. About 2–3% remained viable for more than 3 months at 5 °C/1 bar. Mycelial fragments showed growth and biomass production under elevated pressure at 5 °C. These results indicate that building biomass under deep-sea conditions from spores is not a viable option for the deep-sea Aspergillus isolates. Mycelial fragments, on the other hand, are more likely to grow.

Fungi from coastal environments have been widely studied with respect to the production of secondary metabolites and biotechnologically useful lignocellulolytic enzymes. A few studies on mycology of deep-sea sediments, however, have been carried out. This paper reports a study on alkaline, cold-tolerant proteases from deep-sea fungi. A total of 221 deep-sea isolates of fungi from 5000 m in the Central Indian Basin were screened for the enzyme. Many of these grew and produced alkaline protease at 5 and 30 °C and 1 bar pressure. Aspergillus ustus (NIOCC #20) producing the highest amounts of the enzyme was selected for further studies. The growth yield was substantial at 30 and 5 °C at 1 bar and elevated hydrostatic pressures. The fungus produced alkaline, cold-tolerant protease when grown at 30 °C and 1 bar pressure. The enzyme was active at combinations of 30, 5 °C and 50 and 300 bar pressure. However, protease production was negligible when the fungus was grown at 5 °C, under 1 bar or elevated hydrostatic pressures. The enzyme produced at 30 °C and 1 bar pressure was further characterized. The fungus produced a maximum of 1639 ACU mL −1 of protease by day 7. The enzyme, with molecular mass of 32 kDa and p I values of 6.6 and 6.9 showed several interesting properties. It had a broad pH range of 6–10, with an optimum at pH 9. The optimum temperature for protease activity was 45 °C and approximately 10% of the activity was retained at 2 °C. The enzyme was totally inhibited in the presence of 2 mM PMSF suggesting it to be a serine protease. It was active in the presence of several commercial detergents at 2 g L −1 concentration and in the presence of 0.5 M NaCl, equivalent to 29 parts per thousand salinity. In the presence of stabilizing agents such as glycerol, CaCl 2 its thermostability at 60 °C was enhanced. Heavy metal ions Cu, Hg, Fe, Ni and Zn did not inhibit the enzyme activity considerably. This study indicates that fungi from deep-sea sediments could be a useful source of proteases.

Although a great amount of information is available on bacteria inhabiting deep-sea sediments, the occurrence of fungi in this environment has been poorly studied and documented. We report here the occurrence of fungi in deep-sea sediments from ∼5000 m depth in the Central Indian Basin (9–16°S and 73–76°E). A total of 181 cultures of fungi, most of which belong to terrestrial sporulating species, were isolated by a variety of isolation techniques. Species of Aspergillus and non-sporulating fungi were the most common. Several yeasts were also isolated. Maximum species diversity was observed in 0–2 cm sections of the sediment cores. Direct staining of the sediments with Calcofluor, a fluorescent optical brightener, revealed the presence of fungal hyphae in the sediments. Immunofluorescence using polyclonal antibodies raised against a deep-sea isolate of Aspergillus terreus (# A 4634) confirmed its presence in the form of hyphae in the sub-section from which it was isolated. A total of 25 representative species of fungi produced substantial biomass at 200 bar pressure at 30° as well as at 5 °C. Many fungi showed abnormal morphology at 200 bar/5 °C. A comparison of terrestrial isolates with several deep-sea isolates indicated that the former could grow at 200 bar pressure when growth was initiated with mycelial inocula. However, spores of a deep-sea isolate A. terreus (# A 4634), but not the terrestrial ones, showed germination at 200 bar pressure and 30 °C. Our results suggest that terrestrial species of fungi transported to the deep sea are initially stressed but may gradually adapt themselves for growth under these conditions.

This chapter focuses on issues related to collection and isolation of deep-sea fungi, direct detection in deep-sea sediments, diversity and biomass, growth and physiology, adaptations, and their biotechnological applications. The presence of fungi in oceanic waters and the deep sea has been sporadically reported in the past. Their presence in shells collected from deep-sea waters at a depth of 4,610 m was the first report on deep-sea fungi. Immunofluorescence has been widely used to detect specific fungi in terrestrial and in a few marine substrates. The authors used this for detecting one of the commonly isolated fungi, Aspergillus terreus (isolate A 4634), from deep-sea sediments of the Central Indian Basin. The deep-sea fungi when grown under elevated pressure synthesized extracellular protease, albeit in very low quantities in comparison with that produced under 0.1 MPa. Aspergillus ustus (NIOCC20) isolated by the authors from deep-sea sediments produced cold-active alkaline serine protease, whereas A. ustus obtained from a terrestrial habitat did not. This might be due to differences in strains but may also indicate the adaptation that deep-sea fungi have undergone for their survival. Study of the cold shock or stress proteins or genes produced in response to hydrostatic pressure shock in fungi using proteomics and microarray technology will help to understand the response in eukaryotic organisms to pressure. New techniques for retrieval of sediment samples with in situ pressure, isolation, and culture of the vast diversity of organisms from the deep sea will open new vistas in deep-sea biology.

BACKGROUND: Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. METHODOLOGY/PRINCIPAL FINDINGS: B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. CONCLUSION/SIGNIFICANCE: We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.

The relationship of the marine heterokont stramenopilan protists, the aplanochytrids, with the zooplankton was studied in coastal waters. The aplanochytrids were fed to the zooplankton specimens and observed for grazing by two different approaches: [1] using fluorescently-labeled prey approach and [2] using internal transcribed spacer-based molecular probe and in situ hybridization approach. The aplanochytrid cells were detected in the guts as well as fecal pellets of the zooplankton, thus serving as prey to them. Aplanochytrids were consistently isolated from zooplankton specimens. The isolates did not produce a wide array of enzymes, implicating that they may not play a major role in degradation of zooplankton exoskeleton. They were found to produce only protease considerably and sometimes lipase too. The amplified rDNA restriction analysis showed similar patterns, suggesting that most of the isolates might be same strains of Aplanochytrium spp. The existence of aplanochytrids with the zooplankton in marine waters points towards their probable association either as predator–prey or as commensalistic rather than saprophytic type of association.

The occurrence and distribution of an actinobacteria group of bacteria capable of dissolving insoluble phosphates were investigated in this study in marine environments, especially in sediments of Chorao Island, Goa Province, India. A total of 200 bacterial isolates of actinobacteria was isolated. All isolates were screened for phosphate-solubilizing activity on Pikovskaya's agar. Thirteen different isolates exhibiting maximum formation of halos (zone of solubilization) around the bacterial colonies were selected for quantitative estimations of P-solubilization. Quantitative estimations for P-solubilization were analyzed for up to 10 days at intervals of 24 h. Maximum solubilization from 89.3 ± 3.1 to 164.1 ± 4.1 μg ml(-1) was observed after 6 days of incubation in six of all isolates, while the isolate NII-1020 showed maximum P-solubilization. The increase in solubilization coincided with the drop in pH. Many of these species showed wide range of tolerance to temperature, pH, and salt concentrations. Further, 16S rRNA gene sequence analyses were carried to identify the bacterial groups which are actively solubilized phosphate in vitro. Gene sequencing results reveal that all isolates were clustered into six different actinobacterial genera: Streptomyces, Microbacterium, Angustibacter, Kocuria, Isoptericola, and Agromyces. The presence of phosphate-solubilizing microorganisms and their ability to solubilize phosphate were indicative of the important role played by bacteria in the biogeochemical cycle of phosphorus and the plant growth in coastal ecosystems.

A Gram-positive, non-motile, rod-shaped bacterium strain NIO-1018(T) isolated from a mangrove sediment sample of the Chorao Island, Goa, India, was subjected to a detailed polyphasic taxonomic study. The strain designated as NIO-1018(T) matched with most of the phenotypic and chemotaxonomic properties of the genus Agromyces and represents a novel species. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain NIO-1018(T) fell within the cluster comprising species of the genus Agromyces, clustering with Agromyces soli (98.1 %), Agromyces flavus (97.9 %), Agromyces aurantiacus (97.7 %) and Agromyces ulmi (97.3 %). The predominant menaquinone was MK-12, and the major cellular fatty acids were anteiso-C(15:0), iso-C(16:0), anteiso-C(17:0) and iso-C(15:0). The polar lipids consisted of diphosphatidylglycerol and phosphatidylglycerol. The genomic DNA G+C content of strain NIO-1018(T) was 71.8 mol%. The combination of phylogenetic analysis, DNA-DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain NIO-1018(T) represents a novel species of the genus Agromyces, for which the name Agromyces indicus sp. nov. is proposed. The type strain is NIO-1018 (=JCM 17573(T) = CCTCC AB2011122(T)).

Filamentous fungi are the most widely used eukaryotes in industrial and pharmaceutical applications. Their biotechnological uses include the production of enzymes, vitamins, polysaccharides, pigments, lipids and others. Marine fungi are a still relatively unexplored group in biotechnology. Taxonomic and habitat diversity form the basis for exploration of marine fungal biotechnology. This review covers what is known of the potential applications of obligate and marine-derived fungi obtained from coastal to the oceanic and shallow water to the deep-sea habitats. Recent studies indicate that marine fungi are potential candidates for novel enzymes, bioremediation, biosurfactants, polysaccharides, polyunsaturated fatty acids and secondary metabolites. Future studies that focus on culturing rare and novel marine fungi, combined with knowledge of their physiology and biochemistry will provide a firm basis for marine mycotechnology.

Filamentous fungi are the most widely used eukaryotes in industrial and pharmaceutical applications. Their biotechnological uses include the production of enzymes, vitamins, polysaccharides, pigments, lipids and others. Marine fungi are a still relatively unexplored group in biotechnology. Taxonomic and habitat diversity form the basis for exploration of marine fungal biotechnology. This review covers what is known of the potential applications of obligate and marine-derived fungi obtained from coastal to the oceanic and shallow water to the deep-sea habitats. Recent studies indicate that marine fungi are potential candidates for novel enzymes, bioremediation, biosurfactants, polysaccharides, polyunsaturated fatty acids and secondary metabolites. Future studies that focus on culturing rare and novel marine fungi, combined with knowledge of their physiology and biochemistry will provide a firm basis for marine mycotechnology.

The few studies that have been carried out on deep-sea fungi in recent years either by culturing or molecular signatures, have provided evidence about their presence. A majority of culture-dependent and culture-independent fungi recovered showed homology to species reported in the terrestrial environment indicating their possible arrival in deep sea either with wind or terrestrial runoffs. However, presence of novel fungal sequences with less than 97 % similarity to previously identified fungal sequences in public databases has also been reported. Several filamentous fungi and yeasts recovered from deep-sea sediments of the Central Indian Basin from a depth of ~5000 m showed growth under hydrostatic pressures of 20-40 MPa and 5 o C. Some of these fungi showed unusual morphology during the initial culturing and also when grown at 20 MPa/5 o C. Direct detection of fungal hyphae in deep-sea sediments is a daunting task as they are present in low abundance. We demonstrated the presence of fungal hyphae in deep-sea sediments by direct staining of the sediments with Calcofluor, a fluorescent optical brightener. This review presents data on the occurrence, diversity and adaptations of fungi to various deep-sea habitats, with a special emphasis on their possible ecological role under these extreme environmental conditions.

Whereas fungi in terrestrial soils have been well studied, little is known of them in deep-sea sediments. Recent studies have demonstrated the presence of fungal hyphae in such sediments but in low abundance. We present evidence in this study that one of the apparent reasons for the poor detection of fungi in deep-sea sediments is their cryptic presence in macroaggregates. Fungal biomass carbon from different core sections of deep-sea sediments from approximately 5000 m depth in the Central Indian Ocean was estimated based on direct microscopic detection of fungal mycelia. Treatment of sediment samples with ethylenediamine tetra-acetic acid (EDTA) enabled more frequent detection and significantly higher biomass than in samples without such treatment. Treatment with EDTA resulted in detecting various stages of breakdown of aggregates in the sediments, gradually revealing the presence of fungal hyphae within them. Experimental studies of a deep-sea, as well as three terrestrial isolates of fungi, showed that all could grow at 200 bar and 5 degrees C in a nutrient medium and in deep-sea sediment extract. Hyphae of fungi grown in sediment extract under the above conditions showed various stages of accretion of particles around them, leading to the formation of aggregates. Such aggregates showed the presence of humic material, carbohydrate, and proteins. We suggest that fungi in deep-sea sediments may be involved in humic aggregate formation by processes very similar to those in terrestrial sediments. The importance of such a process in carbon sequestration and food web in the deep sea needs to be examined.