Physiological and biochemical response in green mussel Perna viridis subjected to continuous chlorination: Perspective on cooling water discharge criteria.

Heavy infestation by Perna viridis has been observed in the sub-seabed seawater intake tunnel and CWS of a tropical coastal power station in-spite of continuous low dose chlorination regime (0.2 ± 0.1 mg L-1) (CLDC), indicating periodical settlement and growth. Continuous arrival of mussels (colonized in the sub seabed tunnel intake section) at the pump house indicated that the mussels were able to tolerate and survive in a chlorinated environment, for varying time periods and were dislodged when they become weak and subsequent death, leading to flushing out of the system. In the present study, effect of continuous chlorination [0.2 mg L-1 (in-plant use); 0.5 mg L-1 (shock dose) & 1.0 mg L-1 (high levels)] was evaluated on mussels to assess; (a) time taken for mortality, (b) action of chlorine on physiological, genetic, metabolic and neuronal processes. 100% mortality of mussels was observed after 15 (0.2 mg L-1); 9 (0.5 mg L-1) and 6 days (1.0 mg L-1) respectively. Extended valve closure due to chlorination resulted in stress, impairing the respiratory and feeding behavior leading to deterioration in mussel health. Pseudofaeces excretion reduced to 68% (0.2 mg L-1); 10% (0.5 mg L-1) and 89% (1.0 mg L-1) compared to controls. Genotoxicity was observed with increase in % tail DNA fraction in all treatments such as 86% (0.2 mg L-1); 76% (0.5 mg L-1) and 85% (1.0 mg L-1). Reactive Oxygen Species (ROS) stress biomarkers increased drastically/peaked within the first 3 days of continuous chlorination with subsequent quenching by antioxidant enzymes. Gill produced highest generation of ROS; 38% (0.2 mg L-1); 97% (0.5 mg L-1); 98% (1.0 mg L-1). Additionally, it was shown that 84% (0.2 mg L-1), 72% (0.5 mg L-1), and 80.4% (1.0 mg L-1) of the neurotransmitter acetylcholinesterase activity was inhibited by chlorine at the nerve synapse. The cumulative impact of ROS generation, neuronal toxicity, and disrupted functions weakens the overall health of green mussels resulting in mortality.

Bacterial community structure of early-stage biofilms is dictated by temporal succession rather than substrate types in the southern coastal seawater of India.

Bacterial communities colonized on submerged substrata are recognized as a key factor in the formation of complex biofouling phenomenon in the marine environment. Despite massive maritime activities and a large industrial sector in the nearshore of the Laccadive Sea, studies describing pioneer bacterial colonizers and community succession during the early-stage biofilm are scarce. We investigated the biofilm-forming bacterial community succession on three substrata viz. stainless steel, high-density polyethylene, and titanium over 15 days of immersion in the seawater intake area of a power plant, located in the southern coastal region of India. The bacterial community composition of biofilms and peripheral seawater were analyzed by Illumina MiSeq sequenced 16S rRNA gene amplicons. The obtained metataxonomic results indicated a profound influence of temporal succession over substrate type on the early-stage biofilm-forming microbiota. Bacterial communities showed vivid temporal dynamics that involved variations in abundant bacterial groups. The proportion of dominant phyla viz. Proteobacteria decreased over biofilm succession days, while Bacteroidetes increased, suggesting their role as initial and late colonizers, respectively. A rapid fluctuation in the proportion of two bacterial orders viz. Alteromonadales and Vibrionales were observed throughout the successional stages. LEfSe analysis identified specific bacterial groups at all stages of biofilm development, whereas no substrata type-specific groups were observed. Furthermore, the results of PCoA and UPGMA hierarchical clustering demonstrated that the biofilm-forming community varied considerably from the planktonic community. Phylum Proteobacteria preponderated the biofilm-forming community, while the Bacteroidetes, Cyanobacteria, and Actinobacteria dominated the planktonic community. Overall, our results refute the common assumption that substrate material has a decisive impact on biofilm formation; rather, it portrayed that the temporal succession overshadowed the influence of the substrate material. Our findings provide a scientific understanding of the factors shaping initial biofilm development in the marine environment and will help in designing efficient site-specific anti-biofouling strategies.

Copper oxide nanoparticles as an effective anti-biofilm agent against a copper tolerant marine bacterium, Staphylococcus lentus.

Biofilm formation on antifouling coatings is a serious concern in seawater cooling systems and the maritime industry. A prolific biofilm forming strain (Staphylococcus lentus), possessing high tolerance (>1,000 µg ml-1) to dissolved copper ions (Cu++) was isolated from titanium coupons exposed in the coastal waters of Kalpakkam, east coast of India. S. lentus formed increased biofilm (p < 0.05) at 100 µg ml-1 of Cu++ ions, when compared with the untreated control. To combat biofilm formation of this strain, the efficacy of copper oxide nanoparticles synthesized from copper nitrate by varying the concentrations of hexamine and cetyl trimethyl ammonium bromide (CTAB), was investigated. Complete (100%) inhibition of biofilm formation was observed with plain CuO NP (0.5 M hexamine, uncapped) at 1,000 µg ml-1. Capping with CTAB, influenced the morphology and the purity of the synthesized CuO NPs but did not alter their surface charge. Capping reduced metal ion release from CuO NPs and their antibacterial and anti-biofilm property against S. lentus. Overall, uncapped CuO NPs were effective in controlling biofilm formation of S. lentus. Concurrent release of copper ions and contact mediated physical damage by CuO NPs offer a promising approach to tackle metal tolerant biofilm bacteria.

Genomic insights of Vibrio harveyi RT-6 strain, from infected "Whiteleg shrimp" (Litopenaeus vannamei) using Illumina platform.

The pathogenicity of "Vibriosis" in shrimps imposes prominent menace to the sustainable growth of mariculture economy. Often the disease outbreak is associated speciously with Vibrio harveyi and its closely related species. The present study investigated the complete genome of the strain V. harveyi RT-6 to explore the molecular mechanism of pathogenesis. The genome of V. harveyi possesses a single chromosome of 6,374,398 bp in size, G + C content (44.7%) and 5730 protein coding genes. The reads of 1.3 Gb were retained from Illumina Hiseq 2500 sequencing method, assembled into 5912 predicted genes, 114 tRNAs genes, and 11 rRNAs genes. Unigenes were annotated by matching against Clusters of Orthologous Groups of proteins (COG)-5730, Gene ontology (GO)-1088, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases-3401. Furthermore, 13 insertion sequences-(IS), virulence factors and prophage regions were also identified. A total of 94 pathogenic genes and 36 virulence factor genes were mainly identified using Virulence Factors Database (VFDB). Out of the 36 virulence factors, 23 genes responsible for encoding flagella-based motility protein were exclusively predicted to take part in pathogenic mechanism. The Whole Genome Sequencing (WGS) of the strain RT-6 (accession number: SRR5410471) highlighted the underlying genes and specifically accountable functional genes that were responsible for pathogenic infections in shrimps.

PYRROLO isolated from marine sponge associated bacterium Halobacillus kuroshimensis SNSAB01 - Antifouling study based on molecular docking, diatom adhesion and mussel byssal thread inhibition.

In the present study, an attempt has been made to explore the antifouling potential of bioactive compound isolated from sponge associated bacterium Halobacillus kuroshimensis SNSAB01. The crude extract of SNSAB01 strongly inhibited the growth of fouling bacterial strains with least minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The bioactive compound was characterized through FT-IR, HPLC, GCMS and NMR predicted as 'pyrrolo". From the mass spectral library, structure was elucidated as pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro. The in silico studies provided encouraging docking scores with two interactions by GLN 200 and GLU 304. The extract inhibited 89% diatom adhesion at 350 µg/ml concentration against Amphora sp. An EC50 value of 150 µg/ml for 50% inhibition of byssal thread of Perna viridis and LC50 was found to be 500 µg/ml. The LC50/EC50 ratio of 3.0 indicated nontoxic to nature. The result suggested that pyrrolo[1,2-a]pyrazine-1,4-dione can be used for antifouling coating.

Chlorine dioxide as an alternative antifouling biocide for cooling water systems: Toxicity to larval barnacle Amphibalanus reticulatus (Utinomi).

Chlorine dioxide (ClO2) is seen as an effective alternative to chlorine, which is widely used as an antifouling biocide. However, data on its efficacy against marine macrofoulants is scanty. In this study, acute toxicity of ClO2 to larval forms of the fouling barnacle Amphibalanus reticulatus was investigated. ClO2 treatment at 0.1mg/L for 20min elicited 45-63% reduction in naupliar metamorphosis, 70% inhibition of cyprid settlement and 80% inhibition of metamorphosis to juveniles. Increase in concentration to 0.2mg/L did not result in any significant difference in the settlement inhibition or metamorphosis. Treatment with 0.2mg/L of ClO2 elicited substantial reduction in the settlement of barnacle larvae compared to control. The study indicates the possibility of using ClO2 as an alternative antifouling biocide in power plant cooling water systems. However, more work needs to be done on the environmental effects of such switchover, which we are currently undertaking.

Chlorination induced damage and recovery in marine diatoms: Assay by SYTOX® Green staining.

Phytoplankton entrained into cooling water systems of coastal power stations are subjected to acute chemical stress due to biocides (chlorine) used for biofouling control. They are subsequently released into the environment, where they may survive/recover or succumb. Experiments were conducted to evaluate the susceptibility of a centric (Chaetoceros lorenzianus) and pennate (Navicula sp.) diatom to in-plant administered concentrations of chlorine (0.2-0.5mg/L, TRO). Viability of cells exposed to chlorine was assessed by SYTOX® Green fluorimetry and was compared with other conventional end points like total cell counts, chlorophyll a content and cellular autofluorescence. Results showed a concentration-dependant reduction in viability, chlorophyll a and autofluorescence. C. lorenzianus cells were more susceptible to chlorine compared to Navicula sp. SYTOX® Green staining appears to be a sensitive method to assess chlorine-induced damages. The data show that in-use levels of chlorination can potentially impact entrained organisms; however, they can recover when returned to coastal waters.

Acidogenic fermentation of proteinaceous solid waste and characterization of different bioconversion stages and extracellular products.

The purpose of this study was to investigate hydrolysis of animal fleshing (ANFL), a predominant tannery solid waste and to characterize the acetogenic fermentation products of anaerobic digestion. The acidogenic digestibility of the tannery solid wastes were evaluated up to 120 h using batch anaerobic digestion tests performed under mesophilic condition at 37 degrees C. The degradation of ANFL starts with non-fibrillar proteins and proceeds with fibrillar proteins. The release of aliphatic amino acid in the early stages of hydrolysis (24 h) and followed by aromatic amino acids (24-72 h) were evidenced by HPLC analysis. The maximum production of propionic and valeric acid were observed in 72 h followed by rapid increase in acetic acid in 96 h using GC-MS. Breakdown of ANFL and formations of other metabolites were evidenced by FT-IR and (1)H-NMR spectroscopy.