Biomineralization has garnered significant attention in the field of wastewater treatment due to its notable cost reduction compared to conventional methods. The reinjection water from oilfields containing an exceedingly high concentration of calcium and ferric ions will pose a major hazard in production. However, the utilization of biomineralization for precipitating these ions has been scarcely investigated due to limited tolerance among halophiles towards such extreme conditions. In this study, free and immobilized halophiles Virgibacillus dokdonensis were used to precipitate these ions and the effects were compared, at the same time, biomineralization mechanisms and mineral characteristics were further explored. The results show that bacterial concentration and carbonic anhydrase activity were higher when additionally adding ferric ion based on calcium ion; the content of protein, polysaccharides, deoxyribonucleic acid and humic substances in the extracellular polymers also increased compared to control. Calcium ions were biomineralized into calcite and vaterite with multiple morphology. Due to iron doping, the crystallinity and thermal stability of calcium carbonate decreased, the content of OC = O, NC = O and CO-PO3 increased, the stable carbon isotope values became much more negative, and β-sheet in minerals disappeared. Higher calcium concentrations facilitated ferric ion precipitation, while ferric ions hindered calcium precipitation. The immobilized bacteria performed better in ferric ion removal, with a precipitation ratio exceeding 90%. Free bacteria performed better in calcium removal, and the precipitation ratio reached a maximum of 56%. This research maybe provides some reference for the co-removal of calcium and ferric ions from the oilfield wastewater.

Virgibacillus spp. stand out as a potent starter culture for accelerating the fermention of fish sauces and shrimp pastes. However, the underlying molecular mechanisms responsible for their adaptation and biotechnological potential remain elusive. Therefore, the present study focuses on phenotypic and genomic analyses of a halophilic bacterium Virgibacillus dokdonensis T4.6, derived from Vietnamese high-salt fermented shrimp paste. The draft genome contained 4,096,868 bp with 3780 predicted coding sequences. Genome mining revealed the presence of 143 genes involved in osmotic adaptation explaining its resistant phenotype to 24% (w/v) NaCl. Among them, 37 genes making up the complete ectoine metabolism pathway, confirmed its ability to produce 4.38 ± 0.29 wt% ectoine under 12.5% NaCl stress. A significant finding was the identification of 39 genes responsible for an entire degradation pathway of the toxic biogenic amine histamine, which was in agreement with its histamine degradation rate of 42.7 ± 2.1% in the HA medium containing 5 mM histamine within 10 days at 37 °C. Furthermore, 114 proteolytic and 19 lipolytic genes were detected which might contribute to its survival as well as the nutrient quality and flavor of shrimp paste. Of note, a putative gene vdo2592 was found as a possible novel lipase/esterase due to its unique Glycine-Aspartate-Serine-Leucine (GDSL) sequence motif. This is the first report to reveal the adaptative strategies and related biotechnological potential of Virgibacillus associated with femented foods. Our findings indicated that V. dokdonensis T4.6 is a promising starter culture for the production of fermented shrimp paste products.

Anthranilic acid (AA) holds significant importance in the chemical industry. It serves as a crucial building block for the amino acid tryptophan by manipulating the tryptophan biosynthesis pathway, it is possible to increase the production of anthranilic acid. In this study, we utilized metabolic engineering approaches to produce anthranilic acid from the halophilic bacterium Virgibacillus salarius MML1918. The halophilic bacteria were grown in an optimized production medium, and mass production of secondary metabolites was made in ATCC medium 1097 Proteose peptone-for halophilic bacteria and subjected to column chromatography followed by sub-column chromatography the single band for the purified compound was confirmed. Further, various spectral analyses were made for the partially purified compounds, and fluorescence microscopy for fungal cell observation was performed. The purified compound was confirmed by single crystal X-ray diffraction (XRD) analysis, and it was identified as 2-amino benzoic acid. The Fourier transform infrared Spectroscopy (FT-IR) spectrum and nuclear magnetic resonance (NMR) spectrum also confirm the structural characteristic of 2-amino benzoic acid. The UV-Vis absorption spectrum of AA shows the maximum absorption at 337.86 nm. The emission spectrum of 2-amino benzoic acid showed the maximum emission at 453 nm. The bio-imaging application of 2-amino benzoic acid was examined with fungal mycelium of Rhizoctonia solani. It was effectively bound and emitted the blue color at the concentration of 200 and 300 µg/mL. The halophilic bacterium (V. salarius), may have unique metabolic pathways and requirements compared to non-halophilic organisms, to produce AA effectively. This could have implications for industrial biotechnology, particularly in manufacturing environments where high salt concentrations are present and also it can be used as bio-imaging agent.

OBJECTIVE: The present study aims to determine the antimicrobial potential of Virgibacillus salairus (MML1918) against human pathogens and its in-vitro and in-silico properties.
RESULTS: In this present study, totally 63 halophilic bacterial cultures were obtained and cultivated in nutrient broth medium containing 8% NaCl and the metabolites, were extracted using ethyl acetate and screened for their antimicrobial property by cell viability assay against 12 pathogenic bacteria and fungi, among 63 halophilic bacteria the Vir. salaries (MML1918) found to be the best producer for secondary metabolites production against clinical pathogens. The optimization of growth for important physiochemical parameters was characterized and applied for different production media and based on its highest activity as 17.5 ± .07 mm zone of inhibition (ZOI) for Bacillus cereus followed by 17.5 ± 00 mm ZOI for Staphylococcus aureus, the production medium ATCC1097 was chosen for mass production. The mass production of secondary metabolites from Vir. salaries MML1918 was carried out in a fermenter under controlled conditions and crude metabolites was extracted and condensed. The antimicrobial activity of crude metabolites showed B. cereus (19.3 ± 0.5 mm ZOI), Staph. aureus, and Candida albicans (18.3 ± 0.5 mm ZOI) as the highest ZOI in production media for halophilic bacteria ATCC1097. Further, the gas chromatography-mass spectrometry analysis showed 24 compounds present in crude metabolites. Among the 24 compounds, four molecules were found to be important based on molecule percentage in crude and structural similarity. The molecular docking studies show that the selected four molecules effectively bind with the active region DNA gyrase B.
CONCLUSIONS: Virgibacillus salarius (MML1918) effectively showed antimicrobial activity against several pathogenic organisms and can be employed as a suitable candidate for producing novel antimicrobial agents.

Virgibacillus salarius 19.PP.SC1.6 is a coral symbiont isolated from Indonesia\'s North Java Sea; it has the ability to produce secondary metabolites that provide survival advantages and biological functions, such as ectoine, which is synthesized by an ectoine gene cluster. Apart from being an osmoprotectant for bacteria, ectoine is also known as a chemical chaperone with numerous biological activities such as maintaining protein stability, which makes ectoine in high demand in the market industry and makes it beneficial to investigate V. salarius ectoine. However, there has been no research on genome-based secondary metabolite and ectoine gene cluster characterization from Indonesian marine V. salarius. In this study, we performed a genomic analysis and ectoine identification of V. salarius. A high-quality draft genome with total size of 4.45 Mb and 4426 coding sequence (CDS) was characterized and then mapped into the Cluster of Orthologous Groups (COG) category. The genus Virgibacillus has an \"open\" pangenome type with total of 18 genomic islands inside the V. salarius 19.PP.SC1.6 genome. There were seven clusters of secondary metabolite-producing genes found, with a total of 80 genes classified as NRPS, PKS (type III), terpenes, and ectoine biosynthetic related genes. The ectoine gene cluster forms one operon consists of ectABC gene with 2190 bp gene cluster length, and is successfully characterized. The presence of ectoine in V. salarius was confirmed using UPLC-MS/MS operated in Multiple Reaction Monitoring (MRM) mode, which indicates that V. salarius has an intact ectoine gene clusters and is capable of producing ectoine as compatible solutes.

Carotenoids have shown beneficial applications in cosmetology, pharmacology, and medicine. However, environmental stress in the marine environment can trigger the production of unique secondary metabolites, such as carotenoids. These compounds can also be sustainably produced by symbiotic bacteria. We hypothesized that the soft corals in tropical regions may produce diverse biological secondary metabolites, including carotenoids, both by the host organism and their bacterial symbiont. The unique carotenoids may provide promising biological activity such as antioxidant, UV photoprotector, and antibacterial activities. To this end, we isolated and characterized the carotenoids isolated from the bacterial symbiont of Sinularia sp., a soft coral from Panjang Island, North Java Sea, strain 19. PP.Sc.13. Bacterial identification was performed using DNA barcoding of the 16S rRNA region. Identification of carotenoids was carried out using a spectrophotometer, High-Performance Liquid Chromatography (HPLC), and attenuated total reflection fourier-transformed infrared (ATR-FTIR) spectroscopy. The antioxidant activity was estimated using the diphenylpicrylhydrazyl (DPPH) method, while the Sun Protection Factor (SPF) and % transmission of erythema and pigmentation were determined based on colorimetric methods. The antibacterial activity assay was carried out using the agar diffusion method against two multidrug-resistant bacteria. The bacterial symbiont was identified as Virgibacillus sp. and the carotenoids isolated from this symbiont exhibited significant antioxidant activity and extra sun protection effect, thus categorized as UVA sunblock. Furthermore, the isolated carotenoids exhibited antibacterial activities against Methicillin Resistant-Staphylococcus aureus (MRSA) and Multidrug-resistant (MDR) Escherichia coli. This study provides evidence of the carotenoids produced by the soft coral bacterial symbiont Virgibacillus sp., which may be used as an antioxidant, sun protection, and antibacterial agent. Further investigation of the de novo biological production of carotenoids by Virgibacillus sp. is warranted.

Traditionally produced fish sauce can contain significant amounts of histamine. In some instances, the histamine concentration may be well above the limit recommended by the Codex Alimentarius Commission. The aim of this study was to discover new bacterial strains capable of growing under the stressful environmental conditions of fish sauce fermentation and metabolizing histamine. In this study, 28 bacterial strains were isolated from Vietnamese fish sauce products based on their ability to grow at high salt concentrations (23% NaCl) and tested for their ability to degrade histamine. Strain TT8.5 showed the highest histamine-degradation (45.1 ± 0.2% of initially 5 mM histamine within 7 days) and was identified as Virgibacillus campisalis TT8.5. Its histamine-degrading activity was shown to be localized intracellularly and the enzyme is a putative histamine dehydrogenase. The strain exhibited optimal growth and histamine-degrading activity at 37°C, pH 7%, and 5% NaCl in halophilic archaea (HA) histamine broth. It also showed pronounced histamine-degrading activity in HA histamine broth when cultivated at temperatures of up to 40 °C as well as in the presence of up to 23% NaCl. After treatment with immobilized cells, 17.6-26.9% of the initial histamine in various fish sauce products were reduced within 24 h of incubation, while no significant changes in other parameters of fish sauce quality were observed after this treatment. Our results indicate that V. campisalis TT8.5 is of potential interest to be applied in histamine degradation of traditional fish sauce.

Calcium carbonate, one of the most abundant minerals in the geological records is considered as primary source of the carbon reservoir. The role of microorganisms in the biotic precipitation of calcium carbonate has been extensively investigated, especially at extreme life conditions. In Qatar, Sabkhas which are microbial ecosystems housing biomineralizing bacteria, have been carefully studied as unique sites of microbial dolomite formation. Dolomite (CaMg(CO3)2 is an important carbonate mineral forming oil reservoir rocks; however, dolomite is rarely formed in modern environments. The enzyme carbonic anhydrase is present in many living organisms, performs interconversion between CO2 and the bicarbonate ion. Thus, carbonic anhydrase is expected to accelerate both carbonate rock dissolution and CO2 uptake at the same time, serving as carbonite source to carbonites-forming bacteria. This study gathered cross-linked data on the potential role of the carbonic anhydrase excreted by mineral-forming bacteria, isolated from two different extreme environments in Qatar. Dohat Faishakh Sabkha, is a hypersaline coastal Sabkha, from where various strains of the bacterium Virgibacillus were isolated. Virgibacillus can -not only-mediate carbonate mineral formation, but also contributes to magnesium incorporation into the carbonate minerals, leading to the formation of high magnesium calcite. The latter is considered as precursor for dolomite formation. In addition, bacterial strains isolated from marine sediments, surrounding coral reef in Qatar sea, would provide additional knowledge on the role of carbonic anhydrase in mineral formation. Here, the quantification of the two mostly described activities of carbonic anhydrase; esterase and hydration reactions were performed. Mineral-forming strains were shown to exhibit high activities as opposed to the non-forming minerals, which confirms the relation between the presence of active carbonic anhydrase combined with elevated metabolic activity and the biomineralizing potential of the bacterial strains. The highest specific intracellular carbonic anhydrase activity; as both esterase and hydration (i.e., 66 ± 3 and 583000 ± 39000 WAU/108 cells respectively), was evidenced in mineral-forming strains as opposed to non-mineral forming strains (i.e., 6 ±. 0.5 and 1223 ± 61 WAU/108cells) respectively. These findings would contribute to the understanding of the mechanism of microbially mediated carbonate precipitation. This role may be both in capturing CO2 as source of carbonate, and partial solubilization of the formed minerals allowing incorporation of Mg instead of calcium, before catalyzing again the formation of more deposition of carbonates.

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) under hypersaline environments has received increasing attention, whereas the study of anaerobic PAH biodegradation under hypersaline environments is still lacking. Here, we found a pure culture designated PheN4, which was affiliated with Virgibacillus halodenitrificans and could degrade phenanthrene with nitrate as the terminal electron acceptor and a wide range of salinities (from 0.3% to 20%) under anaerobic environments. The optimal salinity for biodegradation of phenanthrene by PheN4 was 5%, which could degrade 93.5% of 0.62 ± 0.04 mM phenanthrene within 10 days with the initial inoculum of 0.01 gVSS/L. Meanwhile, an increased microbial amount could efficiently promote the phenanthrene biodegradation rate. The metabolic processes of anaerobic phenanthrene biodegradation under hypersaline conditions by PheN4 were proposed based on intermediates and genome analyses. Phenanthrene was initially activated via methylation to form 2-methylphenanthrene. Next, fumarate addition and β-oxidation or direct oxidation of the methyl group, ring reduction and ring cleavage were identified as the midstream and downstream steps. In addition, PheN4 could utilize benzene, naphthalene, and anthracene as carbon sources, but Benz[a]anthracene, pyrene, and Benzo[a]pyrene could not be biodegraded by PheN4. This study could provide some guidance for the bioremediation of PAH pollutants in anaerobic and hypersaline zones.

Halophilic phage are a type of virus that exist in salty environments within halophilic archaeal or bacterial hosts. However, relatively few reports on halophilic bacteriophages exist, and our overall understanding of halophilic bacteriophages is quite limited. We used SYBR Green I fluorescent staining to detect the abundance of viruses in Yuncheng Saline Lake, China. Using the double-layer plate method, a lytic phage that could infect halophilic bacterium Salinivibrio sp. YM-43 was isolated and named YXM43. We studied host range, optimal host, morphological characteristics, nucleic acid type, protein composition, and other biological characteristics of the virus. Results reveal a high abundance of this halophilic virus in Yuncheng Saline Lake. The newly isolated bacteriophage YXM43 has a narrow host range, with the most suitable host being Virgibacillus sp. SK39. After purification and enrichment, YXM43 is observed as a spherical particle with a diameter of approximately 30 nm, with no tail. No lipid envelope can be seen in YXM43. The capsid protein of the virus can be separated into seven proteins with molecular weights ranging from 62.0 to 13.0 kDa. YXM43 is a DNA virus with a genome approximately 23 kb. The virus is tolerant of low salinity, and its activity is highest at a temperature of 60 °C and a pH of 10. YXM43 is temperature and pH tolerant, and can adapt to environmental change, even withstanding chloroform treatment. The results indicate that bacteriophage YXM43 is a novel halophilic bacteriophage with broad tolerance to environmental change.