Fungal diversity associated with Goa's tarballs: Insights from ITS region amplicon sequencing.

This study explores the fungal diversity associated with tarballs, weathered crude oil deposits, on Goa's tourist beaches. Despite tarball pollution being a longstanding issue in Goa state in India, comprehensive studies on associated fungi are scarce. Our research based on amplicon sequence analysis of fungal ITS region fills this gap, revealing a dominance of Aspergillus, particularly Aspergillus penicillioides, associated with tarballs from Vagator and Morjim beaches. Other notable species, including Aspergillus sydowii, Aspergillus carbonarius, and Trichoderma species, were identified, all with potential public health and ecosystem implications. A FUNGuild analysis was conducted to investigate the potential ecological roles of these fungi, revealing a diverse range of roles, including nutrient cycling, disease propagation, and symbiotic relationships. The study underscores the need for further research and monitoring, given the potential health risks and contribution of tarball-associated fungi to the bioremediation of crude oil-contaminated beaches.

Halotolerant Bacteria from Genus Nesterenkonia sandarakina VSA9 as a Potential Polyhydroxyalkanoate Producer.

Nesterenkonia sandarakina VSA9 pigmented bacteria isolated from Sargassum is being reported to produce polyhydroxyalkanoates (PHA) deduced through detecting the presence of pha C gene using the molecular method. The PHA synthase gene was of type I which has been concluded from the phylogenetic tree and multiple sequence analysis. The amino acid analysis of pha C gene confirms the involvement of the lipase box having a sequence of G-Y-C-I-G-G with cysteine as the active center of the PHA synthase. Homology modeling predicted the 3D protein structure which is similar to the PHA synthase of Chromobacterium sp. USM2. The solvent extract of N. sandarakina VSA9 showed the presence of Carotenoid compound with maximum wavelength at 475 nm. The study's findings could have far-reaching implications, contributing to advancements in the biotechnology, industrial processes, and sustainable practices. The simultaneous production of carotenoids and PHAs by N. sandarakina VSA9 presents exciting opportunities for the development of innovative and environmentally friendly applications.

Jellyfish Rhizostoma pulmo collected off Goa Coast (India) as a rich source of tryptophan containing collagen and its enhanced antioxidant potential.

The extraction of Rhizostoma pulmo discarded off the Goa coast, India resulted in obtaining remarkably higher yield of 47% (based on lyophilized weight) type I collagen. The amino acid composition showed presence of glycine, hydroxyproline and proline and other amino acids in consistent with mammalian collagens. Interestingly, it also possessed essential amino acid tryptophan that makes this collagen superior than other commercially available collagen products. The dose and time response anti-oxidant activity (DPPH assay) of jellyfish collagen showed increase in the percentage activity with its efficiency better than marine fish collagen. In the dose response, IC50 was found to be 11.0 mg/ml. The percentage DPPH activity gradually increased from 47.58 to 81.11% with time 1-8 h, respectively at concentration of 7 mg/ml of jellyfish collagen. It was noteworthy to observe that the anti-oxidant activity remained 80% even after 24 h of analysis. The EDX analysis showed presence of minerals like Cl, Na, Mg, K, Cu, Fe, Zn etc. essential for healthy bones. The mass assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) data showed several precursor peaks of different peptides which has been presented here for the first time. The finding showed higher production of tryptophan containing anti-oxidant collagen that will certainly enhance its benefit in neurotransmission and cognitive function.

Variability of biochemical compounds in surface sediments along the eastern margin of the Arabian Sea.

Different fractions of organic matter in surface sediments from three transects along the eastern margin of the Arabian Sea (AS) were quantified to determine the sources of organic matter, and also to study its impact on microbial community structure. From the extensive analyses of different biochemical parameters, it was evident that the distribution of total carbohydrate (TCHO), total neutral carbohydrate (TNCHO), proteins, lipids, and uronic acids (URA) concentrations and yield (% TCHO-C/TOC) are affected by organic matter (OM) sources and microbial degradation of sedimentary OM. Monosaccharide compositions from surface sediment was quantified to assess the sources and diagenetic fate of carbohydrates, suggesting that the deoxysugars (rhamnose plus fucose) had significant inverse relationship (r = 0.928, n = 13, p < 0.001) with hexoses (mannose plus galactose plus glucose) and positive relationship (r = 0.828, n = 13, p < 0.001) with pentoses (ribose plus arabinose plus xylose). This shows that marine microorganisms are the source of carbohydrates and there is no influence of terrestrial OM along the eastern margin of AS. During the degradation of algal material, the hexoses seem to be preferentially used by heterotrophic organisms in this region. Arabinose plus galactose (glucose free wt %) values between 28 and 64 wt% indicate that OM was derived from phytoplankton, zooplankton, and non-woody tissues. In the principal component analysis, rhamnose, fucose, and ribose form one cluster of positive loadings while glucose, galactose, and mannose form another cluster of negative loadings which suggest that during OM sinking process, hexoses were removed resulting in increase in bacterial biomass and microbial sugars. Results indicate sediment OM to be derived from marine microbial source along the eastern margin of AS.

Microplastics a Novel Substratum for Polyhydroxyalkanoate (PHA)-Producing Bacteria in Aquatic Environments.

Polyhydroxyalkanoates (PHA) being biological polymers have attracted great attention. PHA have similar properties to that of synthetic plastic and are biodegradable. To discourage plastic pollution in the environment alternative solutions to the plastic pollution has to be readily available. High cost in production of PHA limits the production of these polymers at industrial scale. Bacteria are screened for PHA from diverse niches to meet the current requirements of cheap PHA production at industrial level. The microbial biofilm formed on the surface of microplastic could be a potential source in providing bacteria of economic importance. This paper is an attempt to search microplastic niche for potential PHA producers. PHA production variation was observed with different parameters such as type of carbon source, nitrogen source concentration and also time of incubation. Bacillus sp. CM27 showed maximum PHA yield up to 32.1% among other isolates at 48 h with 2% glucose as carbon source. Optimization of media leads to increase in PHA yield (37.69%) of CDW in Bacillus sp. CM27. Amino acid sequence of Bacillus sp.CM27 showed the presence of PhaC box with sequence, G-Y-C-M-G-G having cysteine in the middle of the box. The extracted polymer was confirmed by FTIR spectroscopy.

Xylanolytic enzyme consortia from Bacillus sp. NIORKP76 for improved biobleaching of kraft pulp.

A cellulase-free xylanolytic enzyme consortia consisting of a xylanase, arabinofuranosidase, and acetyl xylan esterase produced by Bacillus sp. NIORKP76 isolate under solid-state fermentation was assessed for its bio-bleaching ability on kraft pulp. In the biobleaching analysis, the xylanase dose of 5 Ug-1 dry pulp denoted the optimum bleaching of pulp at 40 °C and pH 8.0 after 2 h of treatment. The reduction in kappa number of pre-treated hardwood pulp using xylanolytic enzyme consortium (XEC) was found to be ~ 55%, while solo xylanase could reduce the kappa number to 44-46%. In the case of chemical bagasse pulp, a reduction of ~ 27.5% and 19-20% was seen in kappa number using XEC and solo xylanase, respectively. Enzyme-treated pulp (HW and CB) showed a 50% reduction in hypochlorite consumption during the chlorine treatment. The current study results reveal the significant potential of xylanolytic enzyme consortium from Bacillus sp. NIORKP76 on the environmentally friendly bio-bleaching process.

Comparative assessment of amino acids composition in two types of marine fish silage.

Fish silage is a brown liquefied product achieved by the action of enzymes when finely grounded whole/parts of either single or mixed fish types are subjected to acidification. This study made a comparative assessment of biochemical and nutritive properties, especially the amino acid composition in supernatant phase of formic acid silages prepared from two fish types, Indian mackerel (Rastrelliger kanagurta) and false travely (Lactarius lactarius) representing fat fish (FF, fat content > 5%) and lean fish (LF, fat content < 5%), respectively during 35 days of fermentation (DoF). Significantly higher content of total amino acid (TAA) and free amino acids (FAA) were recorded in FFS (TAA, 41.2 ± 0.03 mg/g; FAA, 31.3 ± 0.003 mg/g) compared to LFS (TAA, 35.8 ± 0.07 mg/g; FAA, 18.26 ± 0.003 mg/g; FAA, 31.3 ± 0.003 mg/g) (p < 0.05). At the end of 35 DoF, the concentrations of amino acids such as asparagine, histidine, isoleucine, valine, cysteine, serine, lysine and arginine were significantly higher in FFS as compared to LFS. The relative amino acid composition of FFS and LFS varied in accordance with DoF and the relationship was found to be highly significant (ANOVA, p < 0.00001). High concentrations of L-amino acids such as leucine, glutamic acid and arginine were recorded in both FFS and LFS. In conclusion, the analysis suggested that a fermentation period of 25-30 days showed a significant effect on the composition of amino acids in both types of ensilage compared to other fermentation periods (p < 0.05). Considering the role of amino acids in enhancing the plant growth and proliferation, the findings of the present study are quite useful.

Morphological and molecular evidence for first records and range extension of the Japanese seahorse, Hippocampus mohnikei (Bleeker 1853) in a bay-estuarine system of Goa, central west coast of India.

Accurate information of taxonomy and geographic range of seahorse species (genus Hippocampus) is the first step in preparing threat assessments and designing effective conservation measures. Here, we report first records and a range extension of the Japanese seahorse, Hippocampus mohnikei (Bleeker, 1853) from the Mandovi estuarine ecosystem of Goa, central west coast of India (CWCI) based on morphological and molecular analyses. The morphometric and meristic traits, particularly short snout (29-35% head length), double cheek spine, low coronet, long tail (51.2-57.9% of standard length), 11 trunk rings, 37-39 tail rings, 15-16 dorsal and 12-14 fin rays observed in four collected specimens matched with the reported key diagnostic morphological criteria of vouchered specimens of H. mohnikei. The seahorse mitochondrial cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b) genes were partially sequenced for conclusive genetic identification of the species under study. Molecular analysis showed that all four individuals clustered together suggesting a monophyletic lineage. Using the maximum similarity with GenBank database, maximum likelihood network and subsequent morphological analysis, the identity of the collected seahorse species was reconfirmed as H. mohnikei. With this new report, the geographic range of H. mohnikei extended significantly to the west from its previously known range. This new sighting of H. mohnikei could indicate a long-distance dispersal facilitated by the prevailing oceanic circulation in the Indo-Pacific region or increased habitat suitability in bay-estuarine systems of Goa, CWCI. Comparison of the pair-wise genetic distances (Kimura 2-parameter) based on COI and Cyt b sequences revealed that the specimens examined in this study are genetically closer to H. mohnikei populations from Vietnam and Thailand than they are to those in Japan and China. To test the hypothesis whether H. mohnikei are vagrants or previously unreported established population, long-term inter-annual sampling and analyses are warranted.

High abundance of Vibrio in tarball-contaminated seawater from Vagator beach, Goa, India.

Tarballs are semi-solid remnants of crude oil and they are formed in marine environment after oil-spill incidents. They are composed of diverse hydrocarbons; some of which are recalcitrant in nature. Recent studies based on amplicon sequencing of 16S rRNA gene suggested that tarballs support hundreds of bacterial genera and provided insights into their role as hydrocarbon degraders and potential human pathogens. In this study, bacterial composition of tarball-contaminated seawater from Vagator beach, Goa, India was characterized by amplicon sequencing of V3-V4 regions of 16S rRNA gene. The DNA data revealed an unusual surge of Vibrio in sea-water contaminated with tarballs in May 2018 (16.16% OTUs), compared to tarball-free seawater samples collected in March 2018 (no detectable OTUs) and September 2018 (0.17% OTUs). Further studies are required to investigate if Vibrio species form biofilms on tarballs which may act as good reservoirs for their survival and transmission success.

Metagenomic analysis of tarball-associated bacteria from Goa, India.

The beaches of Goa state in India are frequently polluted with tarballs, specifically during pre-monsoon and monsoon seasons. Tarballs contain hydrocarbons, including polycyclic aromatic hydrocarbons, which pose significant environmental risks. Microbes associated with tarballs reportedly possess capabilities to degrade toxic hydrocarbons present in tarballs. In this study, bacterial diversity associated with tarballs from Vagator and Morjim beaches of north Goa was analysed based on V3-V4 regions of 16S rRNA gene sequenced using Illumina Miseq Platform. The Proteobacterial members were dominant in both Vagator (≥85.5%) and Morjim (≥94.0%) samples. Many of the identified taxa have been previously reported as hydrocarbon degraders (e.g. Halomonas, Marinobacter) or possible human pathogens (e.g. Acinetobacter, Klebsiella, Rhodococcus, Staphylococcus, Vibrio). This is the first study reported on a metagenomic analysis of bacteria associated with tarballs from Goa.

Bacterial Community Composition Markedly Altered by Coastal Hypoxia.

Monsoonal upwelling along south and central west coast of India leads to intense biological productivity. As a consequence of excess organic matter production following upwelling during June-October and low dissolved oxygen in the upwelled waters, denitrification occurs in the near shore waters. Implicitly, these processes ought to bring alterations in microbial communities. Therefore, diversity and community structure of bacteria from subsurface layers of a tropical region experiencing intense seasonal lows of oxygen were analyzed through sequencing of 16S rRNA gene clones. The overall diversity was more during hypoxic period of Fall intermonsoon (FIM, October) compared either to Spring intermonsoon (SIM, March) or summer monsoon (SuM, June) months. As many as 14 lineages of bacterial domains: Gammaproteobacteria (37%), Alphaproteobacteria (21%), Cyanobacteria (20%), Deltaproteobacteria (3%), Firmicutes (3%), Betaproteobacteria (2%), Acidobacteria (2%), Actinobacteria (7%), Marinimicrobia (2%), Bacteroidetes (1%), Verrucomicrobia (1%), Planctomycetes (0.4%), Chloroflexi (0.2%) and Omnitrophica bacterium (0.2%), were recognized from our coastal location. Notably, sequences of Planctomycetes, Chloroflexi and Omnitrophica bacterium were found exclusively during FIM. A generally higher representation of sequences of Betaproteobacteria during SuM and of Actinobacteria and Firmicutes during SIM was discernible. This study is thus useful to recognize that microbial community might undergo strong temporal shifts in the monsoon affected tropical coastal ecosystems.

Seawater intrusion and resuspension of surface sediment control mercury (Hg) distribution and its bioavailability in water column of a monsoonal estuarine system.

There is limited knowledge regarding seawater intrusion on Hg distribution in monsoon fed tropical estuarine systems during dry season (February to May). This study examined the influence of resuspension of estuarine sediment and intrusion of Hg bound SPM (HgSPM) (by the tide) from the outside of an estuary in controlling distribution, mobility, and bioavailability of Hg within the estuarine systems during the dry season. This investigation was carried out in the Mandovi estuary, a monsoon fed tropical estuary from the central west coast of India. Total Hg concentrations in the water column showed an increasing trend from upstream to downstream of the estuary. The concentration of Hg in the water column of the estuary was much below the concentration recommended by the EPA for aquatic life ambient water quality. Dissolved Hg (HgDissolved) was found to associate mainly with the higher molecular weight fraction of dissolved organic matter (DOM). A significant portion of the total Hg in the water column was present as HgSPM. The average bio-accumulated Hg concentration in edible oyster was high (collected from the estuary) during the dry season compared to the wet season (June to September). This study reveals that resuspension of Hg associated finer sediment particles and intrusion of HgSPM from the outside of the estuary may increase bioavailability of Hg in the Mandovi estuarine systems during the dry season. It is suggested that Hg bioaccumulation in commercially important biological species from different tropical estuarine system may increase during the dry season. The outcome of this research can be useful for policy making and to take proper decision to reduce and control Hg/toxic metals pollution (if any) in tropical estuarine system.

Inter-annual variability of phytoplankton assemblage and Tetraspora gelatinosa bloom from anthropogenically affected harbour, Veraval, India.

Veraval, one of Asia's largest fishing harbours, situated on the south-west coast of Gujarat, India, has transformed into an industrial hub dominated by fish processing units, rayon manufacturing industry, and transportation facilities. The study investigated the high abundance of Tetraspora gelatinosa along with the augmented level of ammonia in the harbour. The high concentration of ammonia was associated with the accumulation of sewage, industrial, and fishery wastes in the harbour. Low-energy expenditure associated with assimilation of ammonia made it a principal nitrogen source for Tetraspora gelatinosa growth. Even though ammonia is the preferred nitrogen source by phytoplankton, elevated concentration causes toxicity to the cells. Augmented level of ammonia and high TSS hampered the efficiency of PS II, thereby impeding the chlorophyll a degradation and oxygen evolution. Built of the organic load from fish processing industries as well as domestic waste along with a reduction in photosynthetic oxygen evolution has made the harbour hypoxic (DO < 1.6 mg L-1)/anoxic (DO = 0.0 mg L-1). Shannon-Wiener diversity index as a pollution index suggested that the inner harbour area was highly polluted as the diversity ranged from 0.01 to 1.57. Whereas, the outer harbour (Near-shore and off-shore) with less anthropogenic effect recorded high diversity (av. 2.17) suggesting a healthy environment.

Nitrate Reductase Gene Expression in Idiomarina Strain cos21 Obtained from Oxygen Minimum Zone of Arabian Sea.

This study analyses the induction and repression of nitrate reduction activity in a batch culture of Idiomarina strain cos21. On a change from aerobic to anaerobic respiration, the culture entered a stationary phase. The onset of this phase showed 3.75 fold increase in mRNA levels for the nitrate reductase enzyme. mRNA accumulated very rapidly during a short period, after which its overall concentration declined to reach a lower value. The level of nitrite reductase protein reached a maximum value at 36 h of growth when the oxygen concentration dropped below 10 µM. The data set provided here confer new insights into the understanding of the physiological response of Idiomarina strain cos21 to change in oxygen concentration allowing the bacterium to survive and adapt to a new environment by dissimilatory reduction of nitrate to nitrite, which serves to provide energy as the bacteria adapt to anaerobiosis. Main strategy used here is to induce, measure, and track the expression of microbial genes, while they grow in culture conditions to better mimic interaction in a natural environment. This study will help us with a better understanding of the nitrate reduction process in the oxygen minimum zone.

Recombinant Xylanase from Bacillus tequilensis BT21: Biochemical Characterisation and Its Application in the Production of Xylobiose from Agricultural Residues.

Bacterial strain Bacillus tequilensis BT21 isolated from marine sediments was found to produce extracellular xylanase. The xynBT21 gene encoding xylanase enzyme was cloned and expressed in Escherichia coli. The gene encoded a protein consisting of 213 amino acid residues with calculated molecular mass of 23.3 kDa. Purified recombinant xylanase had optimum activity at 60 °C and pH=6. The enzyme was highly stable in alkaline pH, at pH=7 it remained 100% active for 24 h, while its activity increased at pH=8 and 9 during incubation. B. tequilensis BT21 xylanase had alkaline pI of 9.4 and belongs to glycosyl hydrolase family 11. The mode of action of XynBT21 on beechwood xylan and xylooligosaccharides was studied. It hydrolysed xylooligosaccharides and beechwood xylan yielding mainly xylobiose (X2) with a small amount of xylose (X1), indicating that XynBT21 was probably an endo-acting xylanase. Enzymatic hydrolysis using wheat bran as a substrate revealed that xylanase reported here has the potential to produce xylobiose from wheat bran. Xylooligosaccharides, especially xylobiose, have strong bifidogenic properties and are increasingly used as a prebiotic. This is the first report that describes this novel xylanase enzyme from marine B. tequilensis BT21 used for the release of xylobiose from wheat bran.

Enzymatic saccharification of seaweeds into fermentable sugars by xylanase from marine Bacillus sp. strain BT21.

Enzymatic hydrolysis of seaweed biomass was studied using xylanase produced from marine bacteria Bacillus sp. strain BT21 through solid-state fermentation of wheat bran. Three types of seaweeds, Ahnfeltia plicata, Padina tetrastromatica and Ulva lactuca, were selected as representatives of red, brown, and green seaweeds, respectively. Seaweed biomass was pretreated with hot water. The efficiency of pretreated biomass to release reducing sugar by the action of xylanase as well as the type of monosaccharide released during enzyme saccharification of seaweed biomass was studied. It was seen that pretreated biomass of seaweed A. plicata, U. lactuca, and P. tetrastroma, at 121 °C for 45 min, followed by incubation with 50 IU xylanase released reducing sugars of 233 ± 5.3, 100 ± 6.1 and 73.3 ± 4.1 µg/mg of seaweed biomass, respectively. Gas chromatography analysis illustrated the release of xylose, glucose, and mannose during the treatment process. Hot water pre-treatment process enhanced enzymatic conversion of biomass into sugars. This study revealed the important role of xylanase in saccharification of seaweed, a promising feedstock for third-generation bioethanol production.

Xylanolytic enzyme systems in Arthrobacter sp. MTCC 5214 and Lactobacillus sp.

The production of extracellular xylanolytic enzymes such as xylanase, α-l-arabinofuranosidase (α-l-AFase), and acetyl xylan esterase (Axe) by marine Arthrobacter sp. and Lactobacillus sp. was investigated using different carbon sources. Induction and repression of all these enzymes differed with carbon source and also with the organism. Wheat bran was the best carbon source for the production of xylanase and α-l-AFase, whereas both isolates showed maximum Axe production when grown on oat bran as a carbon source. Preferential utilization of a carbon source for enzyme production can give us better insights into regulatory mechanism in these marine bacteria. Elution profile as well as zymogram analysis indicated the possibility of bifunctional α-l-AFase-Axes in both marine bacteria.

A novel halotolerant xylanase from marine isolate Bacillus subtilis cho40: gene cloning and sequencing.

Although several xylanases have been studied, only few xylanases from marine micro-organisms have been reported. We report here a novel halotolerant xylanase from marine bacterium Bacillus subtilis cho40 isolated from Chorao island of mandovi estuary Goa, India. Extracellular xylanase was produced by using agricultural residue such as wheat bran as carbon source under solid-state fermentation (SSF). The optimal pH and temperature of xylanase were reported to be 6.0 and 60°C, respectively. Xyn40 was highly salt-tolerant, and showed highest activity at 0.5M NaCl. Xylanase activity was greatly induced (140%) when pre-incubated with 0.5M NaCl for 4h. The xylanase gene, xyn40, from marine bacterium B. subtilis cho40 was cloned, and expressed in Escherichia coli. The xylanase gene was 645 bp long and had a 215 amino acid ORF protein with a molecular mass of 22.9 kDa. It had all features of xylanase enzyme and showed homology to xylanases reported from B. subtilis. It differs from the earlier reported xylanase sequences by the presence of more serine residues compared to threonine and also by the presence of polar (hydrophilic) amino acids in higher abundance (61%) than non-polar amino acids (39%). The novel xylanase, reported in this study is a halotolerant enzyme from marine isolate and can play a very important role in bioethanol production from marine seaweeds.

Bifunctional xylanases and their potential use in biotechnology.

Plant cell walls are comprised of cellulose, hemicellulose and other polymers that are intertwined. This complex structure acts as a barrier to degradation by single enzyme. Thus, a cocktail consisting of bi and multifunctional xylanases and xylan debranching enzymes is most desired combination for the efficient utilization of these complex materials. Xylanases have prospective applications in the food, animal feed, and paper and pulp industries. Furthermore, in order to enhance feed nutrient digestibility and to improve wheat flour quality xylanase along with other glycohydrolases are often used. For these applications, a bifunctional enzyme is undoubtedly much more valuable as compared to monofunctional enzyme. The natural diversity of enzymes provides some candidates with evolved bifunctional activity. Nevertheless most resulted from the in vitro fusion of individual enzymes. Here we present bifunctional xylanases, their evolution, occurrence, molecular biology and potential uses in biotechnology.