Bromoform Toxicity to Marine Microalgae: Studies using the Diatom Chaetoceros lorenzianus as a Model Organism.

Bromoform is the most prominent, relatively long-lived chlorination by-product in condenser effluents from seawater-based power plant cooling systems. There are few reports on the potential toxicity of this trihalomethane to marine phytoplankton. We investigated this using a marine diatom, Chaetoceros lorenzianus as the model organism. The study was conducted by exposing the diatom to bromoform concentrations 0, 50, 100, 150, 250, 500 and 1000 µg/L for exposure time of 3 and 24 h. The mode of action of bromoform was examined using endpoints which include chlorophyll a fluorescence, cell viability by SYTOX® green stain and genotoxicity by comet assay. The relative fluorescence unit and percent viability changed significantly at all concentrations in duration of study. The 24-h IC50 for viability and chlorophyll was estimated to be 255.6 µg/L and 343.5 µg/L, respectively. The tail DNA of 5-20% observed by comet assay indicated low-level DNA damage. Bromoform manages to target cell membrane and internal machinery, DNA and chlorophyll molecule of cell, leading to cause damage at multiple physiological levels. Based on the present data, the current discharge levels of bromoform 50-250 µg/L cause significant impact on the phytoplankton under investigation. However, the impact can be limited under actual field conditions wherein mixing of cooling water with natural water bodies is considered. Nevertheless, more studies are required to understand the toxicological response of organisms to bromoform, so that discharge levels can be continued to be kept within safe levels.

Complete Genome Analysis of Undecylprodigiosin Pigment Biosynthesizing Marine Streptomyces Species Displaying Potential Bioactive Applications.

Marine Streptomyces species are underexplored for their pigment molecules and genes. In this study, we report the genome of the undecylprodigiosin biosynthesizing gene cluster carrying Streptomyces sp. strain BSE6.1, displaying antioxidant, antimicrobial, and staining properties. This Gram-positive obligate aerobic bacterium was isolated from the coastal sediment of the Andaman and Nicobar Islands, India. Pink to reddish pigmented colonies with whitish powdery spores on both agar and broth media are the important morphological characteristics of this bacterium. Growth tolerance to NaCl concentrations was 2 to 7%. The assembled genome of Streptomyces sp. BSE6.1 contains one linear chromosome 8.02 Mb in length with 7157 protein-coding genes, 82 tRNAs, 3 rRNAs and at least 11 gene clusters related to the synthesis of various secondary metabolites, including undecylprodigiosin. This strain carries type I, type II, and type III polyketide synthases (PKS) genes. Type I PKS gene cluster is involved in the biosynthesis of red pigment undecylprodigiosin of BSE6.1, similar to the one found in the S. coelicolor A3(2). This red pigment was reported to have various applications in the food and pharmaceutical industries. The genome of Streptomyces sp. BSE6.1 was submitted to NCBI with a BioProject ID of PRJNA514840 (Sequence Read Archive ID: SRR10849367 and Genome accession ID: CP085300).

Response of green mussels (Perna viridis) subjected to chlorination: investigations by valve movement monitoring.

Perna viridis Linnaeus (1758) is a major foulant in the cooling water systems of electric power stations located on the East coast of India. Though chlorination is considered an effective fouling control measure, the strategy may fail in the case of bivalve mussels, due to the ability of the mussels to close their shells and still survive for extended periods of time. In a given power station, continuous low dose (exomotive) chlorination (0.2 ± 0.1 mg l-1) is practiced to control biofouling. Laboratory experiments were carried out to assess the mortality and valve movement response of Perna viridis exposed to chlorine, using a Mosselmonitor®. All size groups tested showed progressive reduction in valve opening upon chlorination. However, continuous dosing of chlorine concentration as high as 1.0 mg l-1 was required for sustained and complete valve closure response in this mussel. At lower concentration (0.7 mg l-1), the mussels were able to open their shells and feed. Sustained valve closure resulted in physiological stress to the mussels due to reduced feeding, subsequently leading to death. Time to 100% mortality was dependent on the size of the mussels. At 1.0 mg l-1 chlorine residual, smaller size group (30-50 mm) mussels showed 100% mortality in 79.3 h, while larger groups (50-70 mm and 70-90 mm) took 152 h and 243 h, respectively. Frequency of valve opening was high in smaller size group mussels (30-50 mm), compared with larger groups (70-90 mm). Even though the time taken for killing was size-dependent, frequency of valve opening and time period between successive openings were found to be characteristic of individual mussels. The observations provide new insight into the response of bivalve mussels to continuous chlorination in the context of biofouling control and point to the need to adopt pragmatic strategies to prevent mussel spat settlement rather than killing of adult mussels, thereby reducing environmental burden due to chlorine residuals. Usage of target-specific biocidal strategies (intermittent/pulse dosing) or alternative biocides (chlorine dioxide) may help mitigate green mussel fouling in tropical cooling water systems.

Molecular characterization, phylogenetic and in silico sequence analysis data of trehalose biosynthesis genes; otsA and otsB from the deep sea halophilic actinobacteria, Streptomyces qinglanensis NIOT-DSA03.

Trehalose, a non-reducing disaccharide (α-D-glucopyranosyl-(1→1)-α-D-glucopyranoside) is a natural compound, which serves as a protective substance in halophilic bacterial cells. Trehalose biosynthesis genes (otsA and otsB) were PCR amplified from the genomic DNA of deep sea actinobacteria, Streptomyces qinglanensis NIOT-DSA03. The amplified genes were cloned and nucleotide sequences were determined. In silico sequence and phylogenetic analysis of nucleotides and amino acids of otsA and otsB sequences of S. qinglanensis were also determined. The experimental data described in this study will be helpful to develop a recombinant expression system to produce trehalose for biotechnological applications.

A multiplex PCR kit for the detection of three major virulent genes in Enterococcus faecalis.

A multiplex PCR kit that detects three major virulence genes, gelE, hyl and asaI, in Enterococcus faecalis was developed. Analyses of the available sequences of three major virulence genes and designed primers allowed us to develop the three-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting three amplicon bands for gelE, hyl and asaI were even and distinct with product sizes of 213, 273 and 713 bp, respectively. The multiplex PCR procedure was validated with a total of 243 E. faecalis strains that included 02 ATCC strains, 109 isolates from marine samples (sediment, water and sea foods), 22 isolates from cattle fodder, 79 isolates fresh water samples and 31 isolates from nosocomial samples. Specificity of the kit was indicated by amplification of only three major virulent genes gelE, hyl and asaI without any nonspecific bands. Tests for the limit of detection revealed that amplified genes from the sample with a minimum of 104 CFU/g or CFU/mL (10 cells/reaction) of E. faecalis and lower cell load samples, after a 3 h enrichment in NIOT-E. faecalis enrichment medium at 37 °C, a sensitivity level of 10 CFU/g or CFU/mL was achieved.

Applications of Prodigiosin Extracted from Marine Red Pigmented Bacteria Zooshikella sp. and Actinomycete Streptomyces sp.

This study is aimed to determine the distribution, diversity and bioprospecting aspects of marine pigmented bacteria (MPB) isolated from pristine Andaman Islands, India. A total of 180 samples including seawater, sediment, marine plants, invertebrates, and vertebrates were collected and investigated for isolating pigmented bacteria. Results revealed that sediment, invertebrates, and seawater samples were colonized with a greater number of pigmented bacteria pertains to 27.9 × 103 CFU/mL, 24.1 × 103 CFU/mL and 6.7 × 103 CFU/mL respectively. Orange (21.6 × 103 CFU/mL) and red (8.0 × 103 CFU/mL) MPB were predominant than other pigmented bacteria. Fourteen potential MPB were selected based on their intense pigmentation and tested for bioactive nature and food colorant applications. Out of 14, two red pigmented strains BSE6.1 & S2.1 displayed potential multifaceted applications, such as antibacterial, antioxidant, food colorant, and staining properties. Brown pigmented strains CO8 and yellow pigmented strain SQ2.3 have displayed staining properties. Chemical characterization of red pigment using TLC, HP-LC, GC-MS, FT-IR and 1H-NMR analysis revealed prodigiosin as a main chemical constituent. Pure form of prodigiosin compound fractions obtained from both the strains displayed effective antibacterial activity against different human pathogens. MIC and MBC assays revealed that S2.1 requires 300 µg and 150 µg, respectively, and BSE6.1 require 400 µg concentrations of pigment compound for complete inhibition of S. aureus subsp. aureus. On the basis of 16S rRNA sequence analysis, strains S2.1 and BSE6.1 were identified as Zooshikella sp. and Streptomyces sp. and assigned under the GenBank accession numbers: MK680108 and MK951781 respectively.

A novel approach to predict chlorophyll-a in coastal-marine ecosystems using multiple linear regression and principal component scores.

Chlorophyll-a is an established indexing marker for phytoplankton abundance and biomass amongst primary food producers in an aquatic ecosystem. Understanding and modeling the level of Chlorophyll-a as a function of environmental parameters have been found to be very beneficial for the management of the coastal ecosystems. This study developed a mathematical model to predict Chlorophyll-a concentrations based on a data driven modeling approach. The prediction model was developed using principal component analysis (PCA) and multiple linear regression analysis (MLR) approaches. The predictive success (R2) of the model was found to be ~84.8% for first approach and ~83.8% for the second approach. A final model was generated using a combined principal component scores (PCS) and MLR approach that involves fewer parameters and has a predictive ability of 83.6%. The PCS-MLR method helped to identify the relationship amongst dependent as well as predictor variables and eliminated collinearity problems. The final model is quite simple and intuitive and can be used to understand real system operations.

Draft genome sequence of marine sediment-derived red pigmented bacteria Zooshikella sp. strain S2.1 with potential biomedical applications.

The present study is aimed to determine the draft genome of novel species of Zooshikella strain S2.1, a potential red pigmented strain isolated recently from the coastal sediment of Andaman and Nicobar Islands, India. This Gram negative, rod shaped aerobic bacterium produces pink, yellowish-red and dark red with metallic green sheen pigmentation on agar plates. It is able to grow under NaCl concentrations of 1 to 9%. This species has antimicrobial, antioxidant, dye and food colorant applications. Whole genome sequence analysis revealed that strain S2.1 represents a novel species of the genus Zooshikella. Draft genome and 16 s rRNA sequences of this species were deposited in GenBank under the Sequence Read Archive accession number PRJNA514840 and GenBank number MK680108, respectively. Here we report the draft genome of Zooshikella sp. strain S2.1 with ~5.9 Mb of chromosomal content and ~0.34 Mb of extra-chromosomal content.

Ectoine biosynthesis genes from the deep sea halophilic eubacteria, Bacillus clausii NIOT-DSB04: Its molecular and biochemical characterization.

Ectoine, the most prominent osmolyte in nature, is a vital compatible solute present in halophilic bacterium. It protects the cellular biomolecules of the halophilic bacteria and retains their intrinsic function from extreme circumstances. In the current research, ectoine biosynthesis gene cluster (ectABC) in Bacillus clausii NIOT-DSB04 was expressed heterologically in E. coli M15 (pREP4). RP-HPLC resolved several fractions of the purified recombinant product, one of which had been confirmed as ectoine. The recombinant ectoine was further characterized by 1H and 13C Nuclear Magnetic Resonance. The purified recombinant ectoine was also authenticated by FT-IR studies with the existence of ester carbonyl and C-H group. In IPTG induced E. coli M15 transgenic cells, the enzymatic activity of the ectA, B and C genes were found to be higher than that of uninduced cells.

Assessment of trace metal contamination in the marine sediment, seawater, and bivalves of Parangipettai, southeast coast of India.

Industrial and domestic discharge into the coastal environment has driven us to propose an integrated approach to delineate stations contaminated with metals on the Parangipettai coast by collecting sediment and seawater samples monthly at 18 stations from 2015 to 2017. Descriptive statistics revealed that the concentrations of some metals in the sediment and seawater samples were beyond the permissible level. Further, factor analysis showed a sampling adequacy of 0.90 with high positive loading for Ni (0.94), Cd (0.91), Co (0.90), Pb (0.89), and Zn (0.87) in sediment samples. The degree of contamination by metals was evaluated using pollution indices. The results of the contamination index revealed that some stations in the study area were moderately polluted, and those of the ecological index showed that open sea was under low risk while other stations were in the moderate-to-high-risk category. The results obtained are essential to establish the reference condition for a comparative study in similar environments in the tropical regions.

Multifaceted Applications of Microbial Pigments: Current Knowledge, Challenges and Future Directions for Public Health Implications.

Microbial oddities such as versatile pigments are gaining more attention in current research due to their widely perceived applications as natural food colorants, textiles, antimicrobial activities, and cytotoxic activities. This indicates that the future generation will depend on microbial pigments over synthetic colorants for sustainable livelihood. Although several reviews have detailed the comprehensive applications of microbial pigments extensively, knowledge on several aspects of pigmented microbes is apparently missing and not properly reviewed anywhere. Thus, this review has been made to provide overall knowledge on biodiversity, distribution, pathogenicity, and ecological and industrial applications of microbial pigments as well as their challenges and future directions for food, industrial, and biomedical applications. Meticulously, this compendious review treatise on the pigments from bacteria, fungi, yeasts, and microalgae includes reports from the 1970s to 2018. A total of 261 pigment compounds produced by about 500 different microbial species are included, and their bioactive nature is described.

Studies on diversity of Vibrio sp. and the prevalence of hapA, tcpI, st, rtxA&C, acfB, hlyA, ctxA, ompU and toxR genes in environmental strains of Vibrio cholerae from Port Blair bays of South Andaman, India.

Vibrio species are widely distributed in the estuarine and coastal waters that possess the greatest threat to human health worldwide. In this study it is aimed to isolate and observe the abundance of Vibrio sp. and prevalence of biomarker genes and antibiotic resistance profile of V. cholerae isolated from the Port Blair bays of South Andaman. A total of 56 water samples were collected from the seven sampling stations of Port Blair bays in which maximum number of Vibrio sp. population density (1.78 × 104) was recorded in Phoenix Bay. Among the 786 isolates 57.38% of the isolates were confirmed as Vibrio sp., Vibrio cholerae and Vibrio parahaemolyticus. PCR results revealed that the prevalence of biomarker genes was recorded maximum in the isolates from Phoenix Bay and Junglighat Bay samples. Upon further analysis, it was observed that the prevalence of hlyA gene (215 bp), was found to be the most widespread biomarker determinant in 84.17% of isolates. Major virulence determinants; ctxA, ompU and toxR genes were not detected in V. cholerae isolates from Port Blair bays. Maximum antibiotic resistance pattern was observed in Phoenix Bay isolates and maximum number of V. cholerae isolates was resistance to tetracycline (60.76%). Cluster and Principal Component Analysis were employed to understand the diversity and distribution of Vibrio isolates and its biomarker genes. Upon PCA analysis seasonal influence was not much perceived in Vibrio species diversity in Port Blair bays and the lack of significant difference in the detection of species diversity in this study is due to resemblance in geographical conditions and sources of pollution.

Biodiversity and antibacterial potential of cultivable halophilic actinobacteria from the deep sea sediments of active volcanic Barren Island.

Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman & Nicobar Islands. A total of 123 cultivable marine actinobacteria were isolated and identified based on their biochemical and 16S rDNA sequences. The isolates were categorized under 10 genera, of which Streptomyces sp., Dietzia sp. and Brevibacterium sp. are the dominant genera. Of 123 isolates, 73 isolates exhibited appreciable inhibitory activity against both Gram positive and Gram negative bacteria. High frequencies of positive PCR amplification were obtained for PKS-I (39.13%), PKS-II (56.52%) and NRPS (69.57%). The highest levels of biosynthetic systems were observed for NRPS and PKS-II. Majority of the actinobacterial isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance.

Functional characterization of a major compatible solute in Deep Sea halophilic eubacteria of active volcanic Barren Island, Andaman and Nicobar Islands, India.

Ectoine, a cyclic tetrahydropyrimidine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) is a compatible solute, serves as a protective compound in many halophilic eubacterial cells under stress. In this study, the ectoine biosynthesis genes (ectA, B and C) from the genomic DNA of a deep sea eubacteria, Bacillus clausii NIOT-DSB04 was PCR amplified, cloned into the expression vector pQE30 with a 6 × histidine tag and expressed in M15 cells. The lysates of induced cells with diaminobutyric acid aminotransferase and ectoine synthase disclosed two clear expressed bands with molecular masses of 46 kDa and 15 kDa as estimated by SDS-PAGE. The recombinant ectoine synthase activity of the expressed cells was at higher level than that of uninduced cells. In silico sequence and phylogenetic analysis of nucleotides and amino acids revealed that the ectA, B and C sequences of Bacillus clausii NIOT-DSB04 were conserved in many eubacteria.

Seawater quality conditions of the south Andaman Sea (Bay of Bengal, Indian Ocean) in lustrum during 2010s decade.

Andaman and Nicobar islands is one of the major tourism hubs of the World. Most travelers visit these islands for historical attractions, beaches, snorkeling, scuba diving, coral reefs, adventure and recreation. Port Blair is the capital and sole entry/exit point of these islands. The coasts of Port Blair Bay (PBB) and Wandoor Creek (WC) are largely populated due to its services offered to different public/private sectors and for the economic significance. Nevertheless, the global recognition of these islands relies on its healthy ecosystem. Effective management of beaches, bays and their environmental services requires knowledge of coastal water quality. This study assesses the datasets of twenty seawater quality parameters of PBB and WC generated during five years (2011-2015) at eight sites. Multivariate statistical techniques were used for (i) analysis and interpretation of water quality parameters (ii) identification of pollution factors/sources and (iii) understanding spatio-temporal variations valuable for coastal water quality management.

Pluchea lanceolata protects hippocampal neurons from endothelin-1 induced ischemic injury to ameliorate cognitive deficits.

Ischemic brain injury is one of the leading causes of death and disability, where lack of disease modifying treatment strategies make us rely on symptomatic relief. Treatment principles from traditional systems of medicine may fill this gap and its validation in modern medicine perspective is important to bring them to mainstream. Here, we evaluated the neuroprotective efficacy of Ayurvedic medicinal herb Pluchea lanceolata in treating ischemic hippocampal injury. Focal hippocampal ischemia was modeled in Wistar rats through stereotaxic intrahippocampal injection of endothelin-1 (ET-1). Post-surgery, hydroalcoholic extract of the rhizome of Pluchea lanceolata (HAPL) was administered orally, once in a day for 14 consecutive days to ischemic rats. There were two treatment groups based on the HAPL dosage; HAPL200 (200 mg/kg body weight) and HAPL400 (400 mg/kg body weight). Comparisons were made with the ET-1 ischemic rats which received only the vehicle, and the normal surgical control. Ischemic hippocampal injury led to severe cognitive deficits as evaluated by Morris water maze and open field test, along with locomotory dysfunction noted in actophotometer test. HAPL treatment significantly attenuated these behavioural deficits in a dose dependent manner. Loss of pyramidal cells and degenerative phenotype of shrunken hyperdensed soma with pyknotic nuclei in CA1 and CA3 hippocampal neurons in ischemia were reversed after HAPL treatment. We provide first evidence for loss of dendritic architecture in ET-1 induced focal ischemic hippocampal injury using Golgi impregnation, where HAPL could salvage the dendritic branching and intersections. Intriguingly, it enhanced the dentritic arborization beyond what is noted in normal rats. Ability of HAPL to reverse oxidative stress, especially through maintaining glutathione peroxidase levels and lipid peroxidation in ischemic condition evidences that it may exert neuroprotection through its antioxidant properties. Thus, Pluchea lanceolata and its constituents provide potential alternative/adjuvant treatment strategy for ischemic hippocampal stroke.

Evaluation of the environmental quality of Parangipettai, Southeast Coast of India, by using multivariate and geospatial tool.

The anthropogenic pressure in recent years has driven us to investigate the environmental quality at 22 stations in Parangipettai by collecting seawater samples monthly from 2014 to 2015. The sampling stations were grouped into three different environments, namely, Vellar Estuary (VE), Coleroon Estuary (CE), and Open Sea (OS). Factor analysis showed a total variance of 65.63% and exhibited a strong factor loading for atmospheric temperature (0.914), water temperature (0.917), ammonia (0.767), inorganic phosphate (0.897), total phosphorus (0.783), and phytoplankton (0.829). The index value showed water quality was good in OS (74.18), whereas it was moderate in VE (69.73) and CE (68.47). The visual model developed using Geographical Information System (GIS) displayed a spatial pattern of water temperature and phytoplankton dispersion in a distinct manner. The results obtained through multivariate analysis and GIS-based model are imperative to establish reference for a comparative study with other similar ecosystem for better planning and management of tropical seawaters.

Multimarker study of the effects of antifouling biocide on benthic organisms: results using Perna viridis as candidate species.

Toxic effects of continuous low dose application of the antifouling biocide chlorine on marine benthic organisms were monitored using transplanted green mussels (Perna viridis) and a suite of biomarkers. Caged mussels were deployed in chlorinated and non-chlorinated sections of the cooling system of an operating electric power plant. Biomarkers indicative of general stress, oxidative stress (superoxide dismutase and catalase), and DNA integrity, along with expression of stress proteins, were studied to assess the effects. Deterioration in condition index with corresponding increase in DNA strand breaks was indicative of chlorine stress. Superoxide dismutase enzyme did not show any particular trend, but catalase activity was high during the initial days of exposure at the chlorinated site; later, it became almost equal to that at the control site. Similarly, expressions of stress proteins (HSP60, HSP70, HSP22, GSTS1, and CYP4) showed bell-shaped pattern during the period of study. Positive correlation among the endpoints indicated the utility of the multimarker approach to monitor the effects of continuous low dose chlorination on mussels.

Comparative toxicological effects of two antifouling biocides on the marine diatom Chaetoceros lorenzianus: Damage and post-exposure recovery.

Antifouling biocides are commonly used in coastal electric power stations to prevent biofouling in their condenser cooling systems. However, the environmental impact of the chemical biocides is less understood than the thermal stress effects caused by the condenser effluents. In this study, Chaetoceros lorenzianus, a representative marine diatom, was used to analyse the toxicity of two antifouling biocides, chlorine and chlorine dioxide. The diatom cells were subjected to a range of concentrations of the biocides (from 0.05 to 2mg/L, as total residual oxidants, TRO) for contact time of 30min. They were analysed for viability, genotoxicity, chlorophyll a and cell density endpoints. The cells were affected at all concentrations of the biocides (0.05-2mg/L), showing dose-dependent decrease in viability and increase in DNA damage. The treated cells were later incubated in filtered seawater devoid of biocide to check for recovery. The cells were able to recover in terms of overall viability and DNA damage, when they had been initially treated with low concentrations of the biocides (0.5mg/L of Cl2 or 0.2mg/L of ClO2). Chlorophyll a analysis showed irreparable damage at all concentrations, while cell density showed increasing trend of reduction, if treated above 0.5mg/L of Cl2 and 0.2mg/L of ClO2. The data indicated that in C. lorenzianus, cumulative toxic effects and recovery potential of ClO2 up to 0.2mg/L were comparable with those of Cl2, up to 0.5mg/L concentration in terms of the studied endpoints. The results indicate that at the biocide levels currently being used at power stations, recovery of the organism is feasible upon return to ambient environment. Similar studies should be carried out on other planktonic and benthic organisms, which will be helpful in the formulation of future guidelines for discharge of upcoming antifouling biocides such as chlorine dioxide.

Identification of short single disulfide-containing contryphans from the venom of cone snails using de novo mass spectrometry-based sequencing methods.

We identified 12 short single disulfide-containing conopeptides from the venom of Conus coronatus, C. leopardus, C. lividus and C. zonatus. Interestingly, we detected the shortest contryphan sequence thus far characterized which contains only six amino acid residues. We also identified three distinct contryphan sequences of C. lividus without any proline residues and one sequence with an unusual post-translational modification (bromination of tryptophan). Furthermore, we characterized venom peptides of C. zonatus for the first time.