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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.

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.

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.

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.

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.

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.

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.

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)).