An optimized protocol for routine development of cell culture from adult oyster, Crassostrea madrasensis.

Marine molluscan cell lines, required for virus screening and cultivation, form essential tools for developing health management strategies for these animals in the blue economy. Moreover, they are also crucial to develop cultivated seafood. As there is no valid marine molluscan cell line, primary cell cultures are relied upon for all investigations. A sound protocol for generating primary cell cultures from molluscs is entailed, but existing protocols often involve heavy antibiotic usage and depuration that invariably affect gene expression and cell health. This work presents an easy-to-adopt, time-saving protocol using non-depurated mollusc Crassostrea madrasensis, which requires only initial antibiotic treatment and minimal exposure or no use of antibiotics in the cell culture medium. The important experimental considerations for arriving at this protocol have been elucidated. Accordingly, sodium hypochlorite and neomycin sulfate were chosen for disinfecting tissues. The study is the first to use shrimp cell culture medium (SCCM) as a cell culture medium for molluscan cell culture. Despite being osmoconformers, the oysters exhibited stable intracellular osmotic conditions and pH, which, when provided in vitro, promoted effective cardiomyocyte formation. The cell viability could be enhanced using 10% fetal bovine serum (FBS), but healthy cell culture could also be obtained using SCCM without FBS. The optimized culture conditions allowed for regular beating cardiomyocyte clusters that could be retained for a month. Limited cell proliferation, as shown by the BrdU assay, demands further interventions, such as possibly producing induced pluripotent stem cells. The optimized protocol and culture conditions also align with some requirements for producing cultivated meat from marine molluscs.

Antibacterial activity and modes of action of a novel hepcidin isoform from the shrimp scad, Alepes djedaba (Forsskål, 1775).

Hepcidin, initially identified in human blood ultrafiltrate as cysteine rich Liver Expressed Antimicrobial Peptide (LEAP-1), is a core molecular conduit between iron trafficking and immune response. Though a great share of studies has been focused on the iron regulatory function of hepcidins, investigations on the antimicrobial aspects are relatively less. The present study is aimed at identification of hepcidin from a teleost fish, Alepes djedaba followed by its recombinant expression, testing antibacterial property, stability and evaluation of cytotoxicity. Modes of action on bacterial pathogens were also examined. A novel hepcidin isoform, Ad-Hep belonging to the HAMP1 (Hepcidin antimicrobial peptide 1) group of hepcidins was identified from the shrimp scad, Alepes djedaba. Ad-Hep with 2.9 kDa size was found to be a cysteine rich, cationic peptide (+4) with antiparallel beta sheet conformation, a furin cleavage site (RXXR) and 'ATCUN' motif. It was heterologously expressed in E. coli Rosettagami B(DE3)PLysS cells and the recombinant peptide, rAd-Hep was found to have significant antibacterial activity, especially against Edwardsiella tarda, Vibrio parahaemolyticus and Escherichia coli. Membrane depolarization followed by membrane permeabilization and Reactive Oxygen Species (ROS) production were found to be the modes of action of rAd-Hep on bacterial cells. Ad-Hep was found to be non-haemolytic to hRBC and non-cytotoxic in mammalian cell line. Stability of the peptide at varying temperature, pH and metal salts qualify them for applications in vivo. With significant bactericidal activity coupled with direct killing mechanisms, the rAd-Hep can be a promising drug candidate for therapeutic applications in medicine and fish culture systems.

A novel anti-lipopolysaccharide factor from blue swimmer crab Portunus pelagicus and its cytotoxic effect on the prokaryotic expression host, E. coli on heterologous expression.

BACKGROUND: Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides. RESULTS: This study identified and characterized a novel homolog of ALF (Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four ß sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs. CONCLUSION: Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.

Functional characterization of a histone H2A derived antimicrobial peptide HARRIOTTIN-1 from sicklefin chimaera, Neoharriotta pinnata.

Antimicrobial peptides (AMPs) are gene encoded short peptides which play an important role in the innate immunity of almost all living organisms ranging from bacteria to mammals. Histones play a very important role in defense as precursors to bioactive peptides. The present study is an attempt to decipher the antimicrobial activity of a histone H2A derived peptide, Harriottin-1 from sicklefin chimaera, Neoharriotta pinnata. Analysis in silico predicted the molecule with potent antibacterial and anticancer property. The Harriottin-1 was recombinantly produced and the recombinant peptide rHar-1 demonstrated potent antibacterial activity at 25 µM besides anticancer activity. The study strongly suggests the importance of histone H2A derived peptides as a model for the design and synthesis of potent peptide drugs.

Status in molluscan cell line development in last one decade (2010-2020): impediments and way forward.

Despite the attempts that have started since the 1960s, not even a single cell line of marine molluscs is available. Considering the vast contribution of marine bivalve aquaculture to the world economy, the prevailing viral threats, and the dismaying lack of advancements in molluscan virology, the requirement of a marine molluscan cell line is indispensable. This synthetic review discusses the obstacles in developing a marine molluscan cell line concerning the choice of species, the selection of tissue and decontamination, and cell culture media, with emphasis given on the current decade 2010-2020. Detailed accounts on the experiments on the virus cultivation in vitro and molluscan cell immortalization, with a brief note on the history and applications of the molluscan cell culture, are elucidated to give a holistic picture of the current status and future trends in molluscan cell line development. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00539-x.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization.

BACKGROUND: Increase of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. RESULTS: Open reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. CONCLUSION: Functional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Molecular characterization of a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger (Ruppel, 1836).

BACKGROUND: The concern regarding a post-antibiotic era with increasing drug resistance by pathogens imposes the need to discover alternatives for existing antibiotics. Antimicrobial peptides (AMPs) with their versatile therapeutic properties are a group of promising molecules with curative potentials. These evolutionarily conserved molecules play important roles in the innate immune system of several organisms. The ß-defensins are a group of cysteine rich cationic antimicrobial peptides that play an important role in the innate immune system by their antimicrobial activity against the invading pathogens. The present study deals with a novel ß-defensin isoform from the red-toothed trigger fish, Odonus niger. Total RNA was isolated from the gills, cDNA was synthesized and the ß-defensin isoform obtained by polymerase chain reaction was cloned and subjected to structural and functional characterization in silico. RESULTS: A ß-defensin isoform could be detected from the gill mRNA of red-toothed trigger fish, Odonus niger. The cDNA encoded a 63 amino acid peptide, ß-defensin, with a 20 amino acid signal sequence followed by 43 amino acid cationic mature peptide (On-Def) having a molecular weight of 5.214 kDa and theoretical pI of 8.89. On-Def possessed six highly conserved cysteine residues forming disulfide bonds between C1-C5, C2-C4, and C3-C6, typical of ß-defensins. An anionic pro-region was observed prior to the ß-defensin domain within the mature peptide. Clustal alignment and phylogenetic analyses revealed On-Def as a group 2 ß-defensin. Furthermore, it shared some structural similarities and functional motifs with ß-defensins from other organisms. On-Def was predicted to be non-hemolytic with anti-bacterial, anti-viral, anti-fungal, anti-cancer, and immunomodulatory potential. CONCLUSION: On-Def is the first report of a ß-defensin from the red-toothed trigger fish, Odonus niger. The antimicrobial profile showed the potential for further studies as a suitable candidate for antimicrobial peptide therapeutics.

A novel substituted derivative of sterol from marine actinomycetes Nocardiopsis alba MCCB 110 antagonistic to the aquaculture pathogen Vibrio harveyi.

In an attempt to screen antagonistic microorganisms from marine environment for the management of bacterial pathogens in aquaculture, an isolate of actinomycete MCCB 110 was segregated based on its comparatively higher inhibitory property on Vibrio harveyi (MCCB 111) and profound luminescent inhibition. Based on the culture characteristics, cell wall fatty acid profile and the nucleotide sequence of the 16S rRNA gene (1495 bp), the isolate was identified as Nocardiopsis alba. Solvent extraction of the fermentation broth followed by TLC and HPLC analyses resulted in the isolation of a major fraction active against luminescent Vibrio harveyi. Partial characterization of this bioactive fraction based on spectroscopic data obtained from FT-IR, UV, MS-MS and 1H NMR analyses identified it as a substituted derivative of sterol, and was recognized to differ from those reportedly produced by the same genus. The fraction was not toxic to VERO cell line and shrimp haemocytes up to 1000 ppm tested. The study demonstrated the potential of the putative probiotic Nocardiopsis alba (MCCB 110) and its novel extra-cellular bioactive product in the management of Vibrio harveyi in aquaculture.

Monte Carlo simulation of enriched uranium in the lungs of thorax voxel phantom for assessment of enrichment and its effect on dose.

In-vivo lung monitoring is an important technique for the assessment of internal dose of radiation workers handling actinides. At BARC, counting efficiencies (CEs) of detection systems used for estimation of natural uranium in the lungs are evaluated using realistic thorax physical phantoms or computational voxel phantoms. The quantification of 238U and 235U in lungs is done using CEs determined at 63.3 keV and 185.7 keV photon energies respectively. These CEs can also be used for assessment of enriched uranium in the lungs of the workers. In this study, spectra are generated for HPGe array detectors using Monte Carlo simulations of various enriched uranium compositions distributed in the lungs of thorax voxel phantom. A methodology is developed to predict the 235U enrichment from lung spectrum analysis using the ratio of net counts in 185.7 keV and 63.3 keV energy regions. It is possible to estimate enrichments in the range of 2%-30% using the developed method with less than ±9% error. Finally, effect of 235U enrichment on dose assessment using lung monitoring method is studied.

Molecular and Functional Characterization of an Anti-lipopolysaccharide Factor Mm-ALF from Speckled Shrimp Metapenaeus monoceros.

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides of approximately 100 amino acid residues with a broad spectrum of antimicrobial activity. It is an amphipathic peptide with an N-terminal hydrophobic region and a lipopolysaccharide binding domain (LBD). In the present study, we report an isoform of the anti-lipopolysaccharide factor (Mm-ALF) from the speckled shrimp, Metapenaeus monoceros. A 359 bp cDNA encoded 119 amino acids, and the sequence showed 99.16% similarity to ALF from the shrimp Fenneropenaeus indicus. The mature peptide of 94 amino acids has a net charge of +8, molecular weight 10.62 kDa, and pI 10.11. The mature peptide Mm-ALF was recombinantly expressed in E. coli Rosetta-gami cells, and the peptide was isolated and purified. The rMm-ALF exhibited notable antibacterial activity against Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli, Edwardsiella tarda, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio proteolyticus, Vibrio cholerae and Vibrio fluvialis) bacteria.

Biocompatibility of polyhydroxybutyrate-co-hydroxyvalerate films generated from Bacillus cereus MCCB 281 for medical applications.

Polyhydroxyalkanoates (PHAs) are natural polyesters produced by microorganisms as a source of intracellular energy reserves. These polymers have been extensively studied for tissue engineering and drug delivery applications due to their desirable material properties. Solvent-cast film of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), produced by Bacillus cereus MCCB 281 was characterized to study the surface morphology, roughness, thermal and mechanical properties. PHBV films were slightly hydrophilic with an average surface roughness of 43.66 nm. In vitro cell viability and proliferation studies on PHBV film surface investigated using L929 fibroblasts showed good cell attachment and proliferation. Hemocompatibility of PHBV evaluated by hemolysis assay, in vitro platelet adhesion and coagulation assays demonstrated good blood compatibility for use as blood contact graft materials. Therefore, PHBV produced from the marine bacterium favoured cellular growth of L929 fibroblasts indicating its potential to be used as a biomaterial substrate for cell adhesion in tissue engineering and medical applications.

Marine actinomycetes Nocardiopsis alba MCCB 110 has immunomodulatory property in the tiger shrimp Penaeus monodon.

Shrimp farming constitutes an important source of revenue and employment in many developing countries. However, the shrimp industry has always been plagued with infectious diseases having varied aetiologies. Dominated by non - specific immune mechanism, preventive health care strategy is the most appropriate approach to protect the crop. The present study evaluated the efficacy of an actinomycete, Nocardiopsis alba MCCB 110 in eliciting non - specific immune mechanism in Penaeus monodon having Vibrio harveyi as the challenge organism. Haemocyte count, total protein, phenoloxidase, reactive oxygen intermediates, acid and alkaline phosphatase as well as the gene expression of proPO, peroxinectin, transglutaminase, alpha 2-macroglobulin, astakine, crustin, and penaeidin-3 were evaluated. The results demonstrated that the phenoloxidase, respiratory burst, total protein, acid and alkaline phosphatases were higher in the haemolymph of shrimps fed with Nocardiopsis alba MCCB 110 incorporated feed before and after challenge with Vibrio harveyi, compared to those of placebo fed animals. Up-regulation of six immune genes (alpha 2 macroglobulin, penaeidin -3, transglutaminase, proPO, crustin and peroxinectin) during the post-challenge were recorded. Survival of shrimp among the Nocardiopsis alba administered ones was 83% while it was 50% in placebo fed group. The elevated levels of nonspecific immune gene transcripts and concurrent increase in non specific immunity besides the higher survival rate in the Nocardiopsis alba administered group demonstrated the immunomodulatory property of the marine actinomycete Nocardiopsis alba MCCB 110 in the tiger shrimp Penaeus monodon, and on administering it through diet shrimp could be protected from vibriosis especially of V. harveyi.

Unravelling the menace: detection of antimicrobial resistance in aquaculture.

One of the major problems to be addressed in aquaculture is the prominence of antimicrobial resistance (AMR). The occurrence of bacterial infections in cultured fishes promotes the continuous use of antibiotics in aquaculture, which results in the selection of proliferated antibiotic-resistant bacteria and increases the possibility of transfer to the whole environment through horizontal gene transfer. Hence, the accurate cultivation-dependent and cultivation-independent detection methods are very much crucial for the immediate and proper management of this menace. Antimicrobial resistance determinants carrying mobile genetic transfer elements such as transposons, plasmids, integrons and gene cassettes need to be specifically analysed through molecular detection techniques. The susceptibility of microbes to antibiotics should be tested at regular intervals along with various biochemical assays and conjugation studies so as to determine the extent of spread of AMR. Advanced omic-based and bioinformatic tools can also be incorporated for understanding of genetic diversity. The present review focuses on different detection methods to unearth the complexity of AMR in aquaculture. This monitoring helps the authorities to curb the use of antibiotics, commencement of appropriate management measures and adequate substitute strategies in aquaculture. The long battle of AMR could be overcome by the sincere implementation of One Health approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of antibiotics and increased antimicrobial resistance (AMR) are of major concerns in aquaculture industry. This could result in global health risks through direct consumption of cultured fishes and dissemination of AMR to natural environment through horizontal gene transfer. Hence, timely detection of the antimicrobial-resistant pathogens and continuous monitoring programmes are inevitable. Advanced microbiological, molecular biological and omic-based tools can unravel the menace to a great extent. This will help the authorities to curb the use of antibiotics and implement appropriate management measures to overcome the threat.

Effect of metals of treated electroplating industrial effluents on antioxidant defense system in the microalga Chlorella vulgaris.

The microalga Chlorella vulgaris is one of the prominent and most widely distributed green microalgae found in aquatic environments, often used in toxicity tests due to its sensitivity to various pollutants. To examine the toxicity of metals found in the effluent discharges from an electroplating industry, physicochemical parameters in the microalga C. vulgaris were measured. pH, turbidity, total dissolved solids, color, and the concentrations of metals such as chromium (1.97 mg/L), mercury (104.2 mg/L), and zinc (167.25 mg/L) were found exceeding the permissible limits. Several endpoints such as total protein content, reactive oxygen species (ROS) production, photosynthetic pigment contents, and antioxidant enzymatic activities, including those of superoxide dismutase (SOD) and catalase (CAT), were measured in C. vulgaris in response to treated electroplating industrial effluent (TEPIE). In addition, concentration-dependent morphological changes were also observed in response to TEPIE. Under both acute and chronic TEPIE exposure, increase in the ROS level was observed indicating increased production of ROS in C. vulgaris cells. The total protein and chlorophyll contents were found to be gradually decreasing in an effluent concentration-dependent manner. Moreover, lower concentrations of effluent stimulated the antioxidant enzyme systems. A concentration-dependent increase was observed in both SOD and CAT enzymatic activities. The results indicated toxic impairments by the effluent on the function of C. vulgaris in response to both acute and chronic exposure, indicating an urgent need of proper treatment processes/modification of the existing one of TEPIE, with continuous monitoring of the discharge of the pollutants into the aquatic ecosystems using biological assays.

Neuroendocrine and immunotoxicity of polyaromatic hydrocarbon, chrysene in crustacean post larvae.

Polyaromatic hydrocarbons are a group of chemical pollutants which cause a significant threat to the living organisms in estuaries and marine ecosystems. We report the effect of chrysene, a major PAH pollutant found in Cochin Estuary along the southwest coast of India, on the neuroendocrine and immune gene expression of the post larvae (PL-25) of Penaeus monodon. The PL- 25 of P. monodon were administered with feed coated with increasing concentrations of chrysene (1, 2 and 3 µg/g) for 10 days and the gene expression was studied on 7th, 11th and 15th day. The PL exposed to chrysene showed moulting stress and changes in the levels of moult-inhibiting hormone I (MIH I) indicated by irregular moulting in the experimental tanks. At the molecular level, the higher concentration of chrysene induced two-fold upregulation of neuroendocrine (MIH I) and downregulation of immune (ProPO and crustin) gene on the 7th day of exposure. The expression of MIH I gene reduced on withdrawing the experimental feed (on 11th day), while continued downregulation of ProPO and crustin were observed on the 11th day. The results of the present study indicate that the microgram levels of PAH can impinge the neuroendocrine and immune system of the P. monodon, which may induce morbidity and mortality to the larvae in polluted coastal ecosystems. Therefore, more attention may be given to avoid PAH pollution in the estuaries to maintain a healthy ecosystem and to protect the animals from extinction.

A histone H2A derived antimicrobial peptide, Fi-Histin from the Indian White shrimp, Fenneropenaeus indicus: Molecular and functional characterization.

Antimicrobial peptides (AMPs) derived from histone proteins form an important category of peptide antibiotics. Present study deals with the molecular and functional characterization of a 27-amino acid histone H2A derived AMP from the Indian White shrimp, Fenneropenaeus indicus designated as Fi-Histin. This peptide displayed distinctive features of AMPs such as amphiphilic alpha helical structure and a net charge of +6. The synthetic peptide exhibited significant antimicrobial activity against Gram-negative and Gram-positive bacteria especially against V. vulnificus, P. aeruginosa, V. parahaemolyticus, V. cholera and S. aureus. Disruption of cell membrane and cell content leakage were observed in peptide treated V. vulnificus using scanning electron microscopy. The synthetic peptide Fi-His1-21 exhibited DNA binding activity and found to be non-haemolytic at the tested concentrations. Peptide was also found to possess anticancer activity against NCI-H460 and HEp-2 cell lines with an IC50 of 22.670 ±â€¯13.939 µM and 31.274 ±â€¯24.531 µM respectively. This is the first report of a histone H2A derived peptide from F. indicus with a specific antimicrobial activity and anticancer activity, which could be a new candidate for future applications in aquaculture and medicine.

Community composition of marine and brackish water ammonia-oxidizing consortia developed for aquaculture application.

To mitigate the toxicity of ammonia in aquaculture systems, marine and brackish water ammonia-oxidizing bacterial consortia have been developed and are used for activation of nitrifying bioreactors integrated to recirculating aquaculture systems. To shed more light on to these biological entities, diversity of both the consortia were analyzed based on random cloning of 16S rRNA gene and ammonia-oxidizing bacterial specific amoA gene sequences. The dendrograms of representative clones on the basis of amplified ribosomal DNA restriction analysis generated 22 and 19 clusters for marine and brackish water nitrifying consortia, respectively. Phylogenetic analysis demonstrated the presence of various autotrophic nitrifiers belonging to α-, ß- and γ-Proteobacteria, anaerobic ammonia oxidizers, heterotrophic denitrifiers, Bacteroidetes, and Actinobacteria. Distribution patterns of the organisms within the two consortia were determined using the software Geneious and diversity indices were investigated using Mega 5.0, VITCOMIC and Primer 7. The abundance of ammonia oxidizers was found in the order of 2.21 ± 0.25 × 109 copies/g wet weight of marine consortium and 6.20 ± 0.23 × 107 copies/g of brackish water consortium. Besides, marine ammonia-oxidizing consortium exhibited higher mean population diversity and Shannon Wiener diversity than the brackish water counterparts.

Evaluation of Uncertainties in lung measurements of actinides due to counting statistics.

Counting statistics is an important parameter that can introduce uncertainties in the lung activity measurements of actinides in radiation workers. Evaluation of uncertainties due to counting statistics is practically difficult as it requires monitoring various radiation workers having different levels of lung actinide content, multiple times, each for 50 min of monitoring period. However, different activities in lungs can be simulated by combining uncontaminated male data with LLNL phantom data acquired with 241Am and natural uranium lung sets at various short periods. Therefore, multiple measurements were carried out on realistic thorax LLNL phantom with 241Am and natural uranium lung sets for 15-600 s. The mean counts with the phantom at various time intervals, corresponds to different actinide activities in lungs, assuming they are obtained for 50 min of monitoring interval. Using propagation of error, standard deviations were evaluated for combined phantom and uncontaminated adult male data. The combined standard deviations and mean phantom counts are used to evaluate scattering factors (SFs) for uncertainties due to counting statistics for Phoswich and HPGe array detectors. The SFs due to counting statistics are found to be the function of lung activities of radionuclides as well as energies and yields of the photons emitted by radionuclides. SFs are found to increase with decrease in lung activity. For similar yields photons, SFs are found to be lower for higher energy photons compared to lower energy photons. For photons of similar energies, the SFs are lower when yield is higher compared to lower yield photons.

Biosynthesis and characterization of polyhydroxyalkanoate from marine Bacillus cereus MCCB 281 utilizing glycerol as carbon source.

Polyhydroxyalkanoates (PHAs) are aliphatic polyesters produced by bacteria from renewable resources which serve as a substitute of synthetic plastics. In the present study isolation, screening, identification of PHA producing bacteria from marine water samples and optimization of process variables for increased PHA production were accomplished. The potent isolate identified as Bacillus cereus MCCB 281 synthesized PHA co-polymer with 13 mol% 3-hydroxyvalerate in presence of glycerol. Process parameters optimized using central composite design for enhanced PHA production showed 1.5 fold higher PHA yield. Cell dry weight of 3.72 ±â€¯0.04 g L-1, PHA yield 2.54 ±â€¯0.07 g L-1 and PHA content of 68.27 ±â€¯1.2% (w/w) was achieved in fermenter at the optimized conditions. Purified polymer was characterized by Fourier-transform infrared spectroscopy, Nuclear magnetic resonance spectroscopy, Gas chromatography-Mass spectrometry, X-ray powder diffraction techniques and molecular weight of PHA was found to be 2.56 × 105 Da. PHA nanoparticles with average particle size 179 nm were synthesized for medical applications and biocompatibility analysis was performed with L929 mouse fibroblast cell line. This is the first report of a moderately halophilic B. cereus, which utilizes glycerol as the sole carbon source for PHA co-polymer production.