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Abstract Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.

IntroductionThis work investigates whether rhizosphere microorganisms that colonize halophyte plants thriving in saline habitats can tolerate salinity and provide beneficial effects to their hosts, protecting them from environmental stresses, such as aromatic compound (AC) pollution.MethodsTo address this question, we conducted a series of experiments. First, we evaluated the effects of phenol, tyrosine, 4-hydroxybenzoic acid, and 2,4-dichlorophenoxyacetic (2,4-D) acids on the soil rhizosphere microbial community associated with the halophyte Allenrolfea vaginata. We then determined the ability of bacterial isolates from these microbial communities to utilize these ACs as carbon sources. Finally, we assessed their ability to promote plant growth under saline conditions.ResultsOur study revealed that each AC had a different impact on the structure and alpha and beta diversity of the halophyte bacterial (but not archaeal) communities. Notably, 2,4-D and phenol, to a lesser degree, had the most substantial decreasing effects. The removal of ACs by the rhizosphere community varied from 15% (2,4-D) to 100% (the other three ACs), depending on the concentration. Halomonas isolates were the most abundant and diverse strains capable of degrading the ACs, with strains of Marinobacter, Alkalihalobacillus, Thalassobacillus, Oceanobacillus, and the archaea Haladaptatus also exhibiting catabolic properties. Moreover, our study found that halophile strains Halomonas sp. LV-8T and Marinobacter sp. LV-48T enhanced the growth and protection of Arabidopsis thaliana plants by 30% to 55% under salt-stress conditions.DiscussionThese results suggest that moderate halophile microbial communities may protect halophytes from salinity and potential adverse effects of aromatic compounds through depurative processes.

Extremophiles possess unique cellular and molecular mechanisms to assist, tolerate, and sustain their lives in extreme habitats. These habitats are dominated by one or more extreme physical or chemical parameters that shape existing microbial communities and their cellular and genomic features. The diversity of extremophiles reflects a long list of adaptations over millions of years. Growing research on extremophiles has considerably uncovered and increased our understanding of life and its limits on our planet. Many extremophiles have been greatly explored for their application in various industrial processes. In this review, we focused on the characteristics that microorganisms have acquired to optimally thrive in extreme environments. We have discussed cellular and molecular mechanisms involved in stability at respective extreme conditions like thermophiles, psychrophiles, acidophiles, barophiles, etc., which highlight evolutionary aspects and the significance of extremophiles for the benefit of mankind. [ABSTRACT FROM AUTHOR]

A significant portion of the earth has a salty environment, and the literature on bacterial survival mechanisms in salty environments is limited. During molecular evolution, halophiles increase acidic amino acid residues on their protein surfaces which leads to a negatively charged surface potential that helps them to maintain the protein integrity and protect them from denaturation by competing with salt ions. Through protein family analysis, we have investigated the molecular-level adaptive features of DNA polymerase III’s catalytic subunit (alpha) and its structure–function relationship. This study throws light on the novel understanding of halophilic bacterial replication and the molecular basis of salt adaptation. Comparisons of the amino acid contents and electronegativity of halophilic and mesophilic bacterial proteins revealed adaptations that allow halophilic bacteria to thrive in high salt concentrations. A significantly lower isoelectric point of halophilic bacterial proteins indicates the acidic nature. Also, an abundance of disordered regions in halophiles suggests the requirement of the salt ions that play a crucial role in their stable protein folding. Despite having similar topology, mesophilic and halophilic proteins, a set of very prominent molecular modifications was observed in the alpha subunit of halophiles. [ABSTRACT FROM AUTHOR]

Microorganisms inhabiting saline environments have been known for decades as producers of many valuable bioproducts. These substances include antimicrobial peptides (AMPs), the most recognizable of which are halocins produced by halophilic Archaea. As agents with a different modes of action from that of most conventionally used antibiotics, usually associated with an increase in the permeability of the cell membrane as a result of a formation of channels and pores, AMPs are a currently promising object of research focused on the investigation of antibiotics with non-standard modes of action. The aim of this study was to investigate antimicrobial activity against multidrug-resistant human pathogens of three peptides, which were synthetised based on sequences identified in metagenomes from saline environments. The investigations were performed against Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Subsequently, the cytotoxicity and haemolytic properties of the tested peptides were verified. An in silico analysis of the interaction of the tested peptides with molecular targets for reference antibiotics was also carried out in order to verify whether or not they can act in a similar way. The P1 peptide manifested the growth inhibition of E. faecalis at a MIC50 of 32 µg/mL and the P3 peptide at a MIC50 of 32 µg/mL was shown to inhibit the growth of both E. faecalis and S. aureus. Furthermore, the P1 and P3 peptides were shown to have no cytotoxic or haemolytic activity against human cells. [ABSTRACT FROM AUTHOR]

Microbial mats are biologically diverse communities that are analogs to some of the earliest ecosystems on Earth. In this study, we describe a unique transiently hypersaline microbial mat uncovered in a shallow pond within the Cuatro Cienegas Basin (CCB) in northern México. The CCB is an endemism-rich site that harbors living stromatolites that have been studied to understand the conditions of the Precambrian Earth. These microbial mats form elastic domes filled with biogenic gas, and the mats have a relatively large and stable subpopulation of archaea. For this reason, this site has been termed archaean domes (AD). The AD microbial community was analyzed by metagenomics over three seasons. The mat exhibited a highly diverse prokaryotic community dominated by bacteria. Bacterial sequences are represented in 37 phyla, mainly Proteobacteria, Firmicutes, and Actinobacteria, that together comprised >50% of the sequences from the mat. Archaea represented up to 5% of the retrieved sequences, with up to 230 different archaeal species that belong to 5 phyla (Euryarchaeota, Crenarchaeota, Thaumarchaeota, Korarchaeota, and Nanoarchaeota). The archaeal taxa showed low variation despite fluctuations in water and nutrient availability. In addition, predicted functions highlight stress responses to extreme conditions present in the AD, including salinity, pH, and water/drought fluctuation. The observed complexity of the AD mat thriving in high pH and fluctuating water and salt conditions within the CCB provides an extant model of great value for evolutionary studies, as well as a suitable analog to the early Earth and Mars. [ABSTRACT FROM AUTHOR]

Sergei Winogradsky illuminated revolutionary concepts and produced a tool to visualize complex microbial communities and their metabolisms over time: columns displaying aquatic consortia with variety of niches. We worked with museums in Utah to create Winogradsky columns that would highlight aesthetic properties of the Great Salt Lake (GSL) ecosystem, which has a salinity gradient from the freshwater wetlands to salt saturation. One column, constructed using haloarchaea-rich hypersaline brine and oolitic sand of the lake's north arm, was enriched with nutrients, and resulted in the desired pink hue over time. After a seven-year maturation period, we examined the microbial taxa present in the water through 16S/18S rRNA and Internal Transcribed Spacer (ITS) gene sequencing. A pigment analysis revealed an abundance of bacteriochlorophyll a. The presence of this pigment coupled with the DNA sequencing results, suggest that the haloarchaea that dominate the GSL brine, were not responsible for the pink coloration, but instead Gammaproteobacteria, especially Halorhodospira species. Among the eukaryotes, the lack of phytoplankton and the abundance of fungi were noteworthy observations. These data likely relate to the reduction of oxygen in a non-aerated sealed system over time. Our second exhibit had the goal of educating museum goers about the varying salinities of Great Salt Lake. Here we employed three distinct columns of water and sediment from this salinity gradient. Observations of these columns overtime gave us information about invertebrate communities in addition to the microbial consortia. Both installations taught us about comparing an artificial environment in a museum setting to the natural ecosystem. Taken together, we present the data collected and lessons learned from using Winogradsky columns in public spaces for teaching about an important saline lake. [ABSTRACT FROM AUTHOR]

The recent increase of antibiotic resistance in pathogenic microorganisms stimulated interest in the search for new antimicrobial compounds and their producers. Among the latter, halophilic microorganisms are considered to be the most promising group, since actinomycetes, producers of the majority of the known clinically important classes of antibiotics, are widely represented in this group. The present work reports the results of the search with three different approaches for new antimicrobial compounds in halophilic and halotolerant microorganisms inhabiting three different types of extreme ecosystems. Metagenomic analysis of microbial communities of subsurface thermal mineral waters revealed biosynthetic clusters of putative antimicrobial compounds, which belong to bacteria and archaea of uncultured lineages. Enrichment cultures with antimicrobial activity were obtained from the cold, deep oceanic sediments. Analysis of their phylogenetic diversity resulted in identification of the potential producers of antimicrobial compounds as the members of class Desulfuromonadia. Finally, antimicrobial activity of a new species of soil natronophilic streptomycetes, Streptomyces sp. ACA25, was characterized. It was active only against gram-positive bacteria. The genome of this organism was sequenced, and the pathways for biosynthesis of polypeptide, polyketide, and beta-lactam antibiotics were identified. Active fractions of the ACA25 culture, containing antimicrobial compounds of polyketide and beta-lactam type, were obtained. The active polyketide was identified as rosamicin, an antibiotic of the macrolide structural group. However, the fact that it did not inhibit bacterial translation highlighted structural differences between the new polyketide and rosamicin itself. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHA) are biopolyesters regarded as an attractive alternative to petroleum-derived plastics. Nitrogen limitation and phosphate limitation in glucose cultivations were evaluated for poly(3-hydroxybutyrate) (P(3HB)) production by Halomonas elongata 1H9T, a moderate halophilic strain. Co-production of P(3HB) and gluconic acid was observed in fed-batch glucose cultivations under nitrogen limiting conditions. A maximum P(3HB) accumulation of 53.0% (w/w) and a maximum co-production of 133 g/L of gluconic acid were attained. Fed-batch glucose cultivation under phosphate limiting conditions resulted in a P(3HB) accumulation of only 33.3% (w/w) and no gluconic acid production. As gluconic acid is a valuable organic acid with extensive applications in several industries, this work presents an interesting approach for the future development of an industrial process aiming at the co-production of an intracellular biopolymer, P(3HB), and a value-added extracellular product, gluconic acid. [ABSTRACT FROM AUTHOR]

Exopolysaccharides (EPSs) are polysaccharides produced by microbes in the form of slime or as a capsular material. Their chemical composition and structure lend themselves to fine-tuned properties and the possibility of metabolic engineering and applications in various industries. Among the archaebacteria, halophiles are a group of bacteria that are well known to produce EPS for forming biofilm so that they can withstand adverse conditions like salinity and osmotic pressure. In this work, seven bacterial strains were isolated from the Ennore Salt pan, and the highest EPS-producing strain SSVRVG7 (Paenibacillus alvei) identified by 16s rRNA sequencing showed highest antibacterial activity. The physicochemical parameters that have an effect on EPS production for the isolate SSVRVG7—carbon source (fructose), cheaper carbon source (glucose), nitrogen source (peptone), NaCl concentration (15%), and pH (8)—were optimized to enhance the EPS production and the EPS produced was characterized using ultra violet-visible spectroscopy, Fourier transfer infrared spectroscopy, thin layer chromatography, scanning electron microscopy (SEM), and proton nuclear magnetic resonance. The antibacterial activity of the strain showed the highest activity against S. faecalis. The characterized EPS was then applied for nanofiber synthesis by electrospinning at different polymer concentrations. The produced electrospun fibers were characterized morphologically by SEM. [ABSTRACT FROM AUTHOR]

Oxidative stress adaptation strategies are important to cell function and are linked to cardiac, neurodegenerative disease, and cancer. Representatives of the Archaea domain are used as model organisms based on their extreme tolerance to oxidants and close evolutionary relationship with eukaryotes. A study of the halophilic archaeon Haloferax volcanii reveals lysine acetylation to be associated with oxidative stress responses. The strong oxidant hypochlorite: (i) stimulates an increase in lysine acetyltransferase HvPat2 to HvPat1 abundance ratios and (ii) selects for lysine deacetylase sir2 mutants. Here we report the dynamic occupancy of the lysine acetylome of glycerol-grown H. volcanii as it shifts in profile in response to hypochlorite. These findings are revealed by the: (1) quantitative multiplex proteomics of the SILAC-compatible parent and Δsir2 mutant strains and (2) label-free proteomics of H26 'wild type' cells. The results show that lysine acetylation is associated with key biological processes including DNA topology, central metabolism, cobalamin biosynthesis, and translation. Lysine acetylation targets are found conserved across species. Moreover, lysine residues modified by acetylation and ubiquitin-like sampylation are identified suggesting post-translational modification (PTM) crosstalk. Overall, the results of this study expand the current knowledge of lysine acetylation in Archaea, with the long-term goal to provide a balanced evolutionary perspective of PTM systems in living organisms. [ABSTRACT FROM AUTHOR]

Saline rhizospheric areas are untouched environments for isolating actinomycetes with the potential of valuable salt tolerant enzyme production. In this study, we isolated and evaluated a total number of of 26 actinomycete strains from plant rhizosphere of the periphery of Dasht-e-Kavir desert. Isolated actinomycetes produced different enzymes. Among them 50, 46, 39, 27, 10 and 7 % of the isolates produced amylase, lipase, protease, gelatinase, lecithinase and urease, respectively. The most frequently produced enzymes among the isolates were amylase, lipase and protease. Combined hydrolytic activity was also detected in some actinomycete strains. Among the isolates, strains Q1, Q4 and Q11 with the most diverse enzymes production, were identified and their 16s rRNA analysis showed that they are mostly similar to the Streptomyces scopiformis, Streptomyces argenteolus and Streptomyces manipurensis, respectively. Finally, due to the enzymatic diversity obtained and the valubility of the halophilic bacterial enzymes in industry, it seems that actinomycetes isolated from this saline habitat are potentially suitable for biotechnological applications.

Actinomycetes are Gram-positive filamentous bacteria well known for the production of bioactive compounds. Recently, many halophilic habitats have been explored for isolation of actinomycetes that exhibit biotechnological potentials. In this investigation, a saline habitat of Rajasthan, Sambhar Salt Lake (SSL) was selected to study the actinomycetes population and Carboxy Methyl Cellulase (CMCase) production by native isolates. A total of sixteen actinomycete isolates, halotolerant and moderately halophilic, were obtained using culture-dependent methods and characterized morphologically and biochemically. They were identified as members of Streptomyces, Nocardiopsis, Pseudonocardia, Saccharospolyspora, and Microbispora. Streptomyces was the most dominating genus, followed by Nocardiopsis. Agar plate assay was used for screening the isolates for CMCase production. Thirteen were found to produce the enzyme, apparent by hydrolysis observed on media plates. The highest relative activity of 22.04 was shown by isolate SSL 14 identified as Nocardiopsis sp. by 16S rDNA sequencing studies and thus selected for further optimization studies. Maximum enzyme (1.08 ± 0.09 U/ml) was produced using medium containing Carboxy Methyl Cellulose (Carbon source) and yeast extract (nitrogen source) at 12% NaCl and pH 9.0, incubated at 30 °C for 96 h. Maximum CMCase production at high salt concentration and pH suggests that Nocardiopsis SSL 14 can be used for industrial processes that operate under excessive saline and alkaline conditions.

Extremozymes from halophiles that thrives in extreme hypersaline habitats have been the subject of extensive and expanding research owing to their value in a variety of industrial applications. Esterases and lipases are the most industrially applied enzymes due to their specific hydrolysis of ester bonds. In the present study, esterase and lipase producing extreme and moderate halophilic strains were isolated from brackish water lake Tso Kar of Ladakh in India. All the fifteen isolates were screened for esterase and lipase production. Among the fifteen isolates, two isolates, TKW1 and TKW12, having extracellular and intracellular esterase activity, and one isolate, TKW3, having intracellular lipase activity, were selected for identification using 16S rRNA gene sequence analysis and media optimization to determine the optimum production of esterase/lipase using a "one variable at a time" strategy. The cumulative effect of all the optimized conditions led to maximu m esterase production of about 82.59 U/ml, 136.9 U/ml after 48 hours and 36 hrs by TKW1 and TKW12, respectively, and maximum lipase of about 199.4 U/ml after 60 hrs by TKW3. These strains had marked hal-ophilic esterase and lipase properties, indicating wide potential applications in industrial biotechnology as salt-adapted haloalkaliphilic enzymes. This is the first report of the isolation and molecular identification of an esterase and lipase producing halo-philic bacterium from the Tso Kar brackish water lake. [ABSTRACT FROM AUTHOR]

Quorum Sensing (QS) is a well-studied intercellular communication mechanism in bacteria, regulating collective behaviors such as biofilm formation, virulence, and antibiotic resistance. However, cell–cell signaling in haloarchaea remains largely unexplored. The coexistence of bacteria and archaea in various environments, coupled with the known cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms and the presence of cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms, suggests a possibility for haloarchaea to possess analogous cell–cell signaling or QS systems. Recently, N-acylhomoserine lactone (AHL)-like compounds were identified in haloarchaea; yet, their precise role—for example, persister cell formation—remains ambiguous. This study investigated the capacity of crude supernatant extract from the haloarchaeon Halorubrum saccharovorum CSM52 to stimulate bacterial AHL-dependent QS phenotypes using bioreporter strains. Our findings reveal that these crude extracts induced several AHL-dependent bioreporters and modulated pyocyanin and pyoverdine production in Pseudomonas aeruginosa. Importantly, our study suggests cross-domain communication between archaea and bacterial pathogens, providing evidence for archaea potentially influencing bacterial virulence. Using Thin Layer Chromatography overlay assays, lactonolysis, and colorimetric quantification, the bioactive compound was inferred to be a chemically modified AHL-like compound or a diketopiperazine-like molecule, potentially involved in biofilm formation in H. saccharovorum CSM52. This study offers new insights into putative QS mechanisms in haloarchaea and their potential role in interspecies communication and coordination, thereby enriching our understanding of microbial interactions in diverse environments. [ABSTRACT FROM AUTHOR]

An alkali, salt, and thermo-tolerant strain designated FJAT-45399T was isolated from marine sediment in Fujian Province, China. Strain FJAT-45399T was Gram-stain-positive, rod-shaped, and facultatively aerobic. It shared high 16S rRNA gene sequence similarities with the members of the genus Shouchella. Further, the phylogenetic and phylogenomic analysis also suggested strain FJAT-45399T clustered with the members of the genus Shouchella. Growth of strain FJAT-45399T was observed at 15–55 °C (optimum 45–50 °C), pH 7.0–13.0 (optimum 9.0) and 0–15% (w/v) NaCl (optimum 2%). It contained MK-7 as the menaquinone. The polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG) and an unidentified glycolipid (UGL) and lipid (UL). The major fatty acids (> 10%) were C16:0 (22.8%), iso-C15:0 (21.3%), and anteiso-C15:0 (14.0%). The genomic DNA G + C content was 44.5%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain FJAT-45399T and the most closely related type strain Shouchella clausii DSM 8716T (ANI 94.1% and dDDH 55.4%) were both below the cut-off level for species delineation. Based on the above results, strain FJAT-45399T represents a novel species of the genus Shouchella, for which the name Shouchella tritolerans sp. nov., is proposed. The type strain is FJAT-45399T (= GDMCC 1.3098T = JCM 35613T). [ABSTRACT FROM AUTHOR]

Background: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 μm and a width of 30–250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. Results: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol–maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin–biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. Conclusion: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion. [ABSTRACT FROM AUTHOR]

Xerophilic fungi accumulate a large amount of glycerol in the cytosol to counterbalance the external osmotic pressure. But during heat shock (HS) majority of fungi accumulate a thermoprotective osmolyte trehalose. Since glycerol and trehalose are synthesized in the cell from the same precursor (glucose), we hypothesised that, under heat shock conditions, xerophiles growing in media with high concentrations of glycerol may acquire greater thermotolerance than those grown in media with high concentrations of NaCl. Therefore, the composition of membrane lipids and osmolytes of the fungus Aspergillus penicillioides , growing in 2 different media under HS conditions was studied and the acquired thermotolerance was assessed. It was found that in the salt-containing medium an increase in the proportion of phosphatidic acids against a decrease in the proportion of phosphatidylethanolamines is observed in the composition of membrane lipids, and the level of glycerol in the cytosol decreases 6-fold, while in the medium with glycerol, changes in the composition of membrane lipids are insignificant and the level of glycerol is reduced by no more than 30%. In the mycelium trehalose level have increased in both media, but did not exceed 1% of dry weight. However, after exposure to HS the fungus acquires greater thermotolerance in the medium with glycerol than in the medium with salt. The data obtained indicate the interrelation between changes in the composition of osmolytes and membrane lipids in the adaptive response to HS, as well as the synergistic effect of glycerol and trehalose. [ABSTRACT FROM AUTHOR]

ABSTRACT Concrete hosts a small but diverse microbiome that changes over time. Shotgun metagenomic sequencing would enable assessment of both the diversity and function of the microbial community in concrete, but a number of unique challenges make this difficult for concrete samples. The high concentration of divalent cations in concrete interferes with nucleic acid extraction, and the extremely low biomass in concrete means that DNA from laboratory contamination may be a large fraction of the sequence data. Here, we develop an improved method for DNA extraction from concrete, with higher yield and lower laboratory contamination. To show that this method provides DNA of sufficient quality and quantity to do shotgun metagenomic sequencing, DNA was extracted from a sample of concrete obtained from a road bridge and sequenced with an Illumina MiSeq system. This microbial community was dominated by halophilic Bacteria and Archaea, with enriched functional pathways related to osmotic stress responses. Although this was a pilot-scale effort, we demonstrate that metagenomic sequencing can be used to characterize microbial communities in concrete and that older concrete structures may host different microbes than recently poured concrete. IMPORTANCE Prior work on the microbial communities of concrete focused on the surfaces of concrete structures such as sewage pipes or bridge pilings, where thick biofilms were easy to observe and sample. Because the biomass inside concrete is so low, more recent analyses of the microbial communities inside concrete used amplicon sequencing methods to describe those communities. However, to understand the activity and physiology of microbes in concrete, or to develop living infrastructure, we must develop more direct methods of community analysis. The method developed here for DNA extraction and metagenomic sequencing can be used for analysis of microbial communities inside concrete and can likely be adapted for other cementitious materials.

Solar salterns were placed along the coast and were frequently left unattended after use. While many studies have isolated and identified microorganisms from hypersaline environments, their role and adaptation mechanisms are still unclear. Herein, we elucidated the role of halophiles in salt-polluted areas through the recently reported Halomonas getboli YJPS3-2 from the abandoned saltern. We analyzed the expression levels of genes in the YJPS3-2 strain to identify its adaptation mechanisms to high salinity environments, by representing the process from tidal flats to abandoned salterns with varying salinity gradients. The YJPS3-2 strain primarily overexpresses genes associated with ABC transport to adapt to hypersaline environments. Interestingly, the cheA gene, which recognizes changes in the surrounding, was the most upregulated, and it was also associated with the overexpression of the MS ring and T3SS mechanisms relating to the flagellar activity. The YJPS3-2 recognized the high salt concentration in its surroundings and attempted to accumulate compatible solutes that could withstand high osmotic pressure inside the cell to adapt to the high salinity environment. Furthermore, during this process, the YJPS3-2 strain removed surrounding pollutants and secreted secondary metabolites that could be utilized by neighboring organisms. Our results suggested that this halophilic bacterium has the potential to serve as a pioneering species for thriving the surrounding while adapting to saline environments.

While interest in underground gas storage (UGS) of hydrogen (H2) in salt caverns is increasing in a growing H2 economy, knowledge of the microbial communities inhabiting those hypersaline environments is still scarce. High salt concentrations and limited availability of carbon (C) sources in cavern environments reduce microbial growth rates and metabolic activities. Growth conditions potentially change once H2 is stored in salt caverns. H2 is a universal electron donor that can facilitate autotrophic growth and subsequently growth of heterotrophs. In this study, a mixed culture enriched from hypersaline UGS sites was investigated in microcosm experiments with H2 atmosphere, testing the effect of different salt concentrations and C sources on methanogenesis, sulfate reduction and homoacetogenesis. Sulfate, acetate and lactate concentrations were quantified throughout a time span of 125 days of incubation and correlations with the microbial community structure and function were explored through 16S rRNA gene-based amplicon sequencing at the end of the experiment. Many of the amplicon sequence variants (ASVs) were only assigned to family or order level, reflecting that a large number of ASVs belong to previously undescribed taxa. At 4.4 M NaCl, close to cavern brine salinity, members of the Desulfovibrionales were absent when no C source other than CO2 was offered as the sole C source. This is in line with the finding that no sulfate reduction occurred at these conditions. Acetogenic Halanaerobiia dominated in these high salinity levels. Based on metagenome sequencing of four selected samples, we found that acetogenesis at autotrophic conditions relies on the activity of a halophilic homoacetogen, Acetohalobium sp. and that sulfate reduction can most likely be associated with a so far undescribed member of the Desulfonatronovibrionales. We further discuss the discrepancy between sulfate reduction at heterotrophic conditions, while no sulfate reduction was observed under autotrophic conditions although acetate was produced through the homoacetogenic activity of Acetohalobium. For the application of UGS of H2, this means that the presence and activity of the aforementioned microorganisms must be investigated, as they can eventually lead to the formation of acetate and allow sulfate reduction at relevant concentrations.

Saline wastewater pollution is a critical issue that needs to be addressed. The present study focused on the development of a dual-chambered microbial fuel cell (MFC) treating saline wastewater at the anode. Halophilic exo-electrogenic bacteria enriched from seawater (Arabian Sea, Mumbai, India) were used in the anodic chamber of the MFC. Denitrification using denitrifying bacteria was employed in the cathodic chamber. The maximum power density was significantly increased from 96.77 mW/m2 to 162.09 mW/m2 with a rise in NaCl concentration from 20 to 40 g/L. Nitrate removal in the cathode chamber increased from 80 ± 3% to 89 ± 3.2% with increase in salt concentration from 20 g/L to 40 g/L and concomitantly COD removal in the anode chamber increased from 76 ± 3.8% to 83 ± 4%. Cyclic voltammetry (CV) analysis revealed higher electrochemical activity at 40 g/L salt concentration. Electrochemical impedance spectroscopy (EIS) analysis exhibited that charge transfer and solution resistances were lower when the salinity was increased. Microbial community analysis revealed the presence of Clostridium, Shewanella, and Bacillus as the most abundant genera in the anodic chamber. This study demonstrated the dual applicability of the system targeted for removal of organics from saline wastewater and nitrate removal from contaminated wastewater accompanied by power generation from the MFC. [ABSTRACT FROM AUTHOR]

Extremophiles provide a one-of-a-kind source of enzymes with properties that allow them to endure the rigorous industrial conversion of lignocellulose biomass into fermentable sugars. However, the fact that most of these organisms fail to grow under typical culture conditions limits the accessibility to these enzymes. In this study, we employed a functional metagenomics approach to identify carbohydrate-degrading enzymes from Ethiopian soda lakes, which are extreme environments harboring a high microbial diversity. Out of 21,000 clones screened for the five carbohydrate hydrolyzing enzymes, 408 clones were found positive. Cellulase and amylase, gave high hit ratio of 1:75 and 1:280, respectively. A total of 378 genes involved in the degradation of complex carbohydrates were identified by combining highthroughput sequencing of 22 selected clones and bioinformatics analysis using a customized workflow. Around 41% of the annotated genes belonged to the Glycoside Hydrolases (GH). Multiple GHs were identified, indicating the potential to discover novel CAZymes useful for the enzymatic degradation of lignocellulose biomass from the Ethiopian soda Lakes. More than 73% of the annotated GH genes were linked to bacterial origins, with Halomonas as the most likely source. Biochemical characterization of the three enzymes from the selected clones (amylase, cellulase, and pectinase) showed that they are active in elevated temperatures, high pH, and high salt concentrations. These properties strongly indicate that the evaluated enzymes have the potential to be used for applications in various industrial processes, particularly in biorefinery for lignocellulose biomass conversion. [ABSTRACT FROM AUTHOR]

Since discovery, the halophilic archaeal and bacterial strains were studied for their unique compounds such as pigments, proton pumps, compatible solutes and enzymes. Due to their tolerance to extreme salinities several research on using these organisms in various fields is conducted worldwide. Though sequencing is an efficient way of identifying an organism, the difficulty of selecting an optimal archaea primer for identification of archaea is tedious. Therefore, it is essential to discriminate them as bacteria or archaea before going for sequencing studies. There are several conventional and analytical techniques followed in discriminating and identifying the halophilic archaea and bacteria. The present study is an investigation of various conventional and analytical techniques such as morphological observations, biochemical tests, salinity optima, chloramphenicol susceptibility, growth in sodium taurocholate, lipid profiling by FAME analysis, FTIR and sequencing technique used in identifying halophilic community. This study also explains the disadvantages of each analysis and confirms the tests that can be used in the preliminary confirmation of halophilic archaea or bacteria. In the present study, halophilic archaeal and bacterial community from Solar salterns of Kelambakkam, Chennai, Tamilnadu and Tuticorin, Tamilnadu and Salt evaporation tank of tannery, Vellore, Tamilnadu were isolated and studied by various above-mentioned techniques. From the study, it was identified that FAME analysis out of other analyses can be used as preliminary method to identify and also distinguish the halophilic archaea and bacterial community. [ABSTRACT FROM AUTHOR]

Sebkha is an Arabic word referring to a closed ground depression temporarily occupied by a salt lake. Very few studies on the composition of the microbial communities from these ecosystems in the Algerian High Plateaus have been carried out. To fill this gap, four sebkhas in the eastern High Plateaus of two different Algerian provinces were sampled, in the winter 2020. We employed the 16S rRNA amplicon sequencing to understand the distribution and diversity of prokaryotic communities in these hypersaline soils. Our results indicate that the overall archaeal community in the hypersaline soils was dominated by members of the class Halobacteria followed by members of the yet uncultured phyla Hadarchaeota and Nanohaloarchaeota. Among the bacterial classes, Alphaproteobacteria was by far the most frequently recovered from all samples, whereas the Cyanobacteria phylum dominated in one of the sebkhas. It was evident from data that Halorubrum and Halapricum were the most abundant archaeal genera, whilst Rhodovibrio and Limimonas predominated among Bacteria, and these were present in all samples. Remarkably, the most abundant archaeal OTUs belonged to the families Haloarculaceae (16.6%) and Halobacteriaceae (16.3%). [ABSTRACT FROM AUTHOR]

Salinity has a strong influence on microorganisms distribution patterns and consequently on the relevance of photoheterotrophic metabolism, which since the discovery of proteorhodopsins is considered the main contributor to solar energy capture on the surface of the oceans. Solar salterns constitute an exceptional system for the simultaneous study of several salt concentrations, ranging from seawater, the most abundant environment on Earth, to saturated brine, one of the most extreme, which has been scarcely studied. In this study, pigment composition across the salinity gradient has been analyzed by spectrophotometry and RP-HPLC, and the influence of salinity on microbial diversity of the three domains of life has been evaluated by a metataxonomic study targeting hypervariable regions of 16S and 18S rRNA genes. Furthermore, based on the chlorophyll a and retinal content, we have estimated the relative abundance of rhodopsins and photosynthetic reaction centers, concluding that there is a strong correlation between the retinal/chlorophyll a ratio and salinity. Retinal-based photoheterotrophy is particularly important for prokaryotic survival in hypersaline environments, surpassing the sunlight energy captured by photosynthesis, and being more relevant as salinity increases. This fact has implications for understanding the survival of microorganisms in extreme conditions and the energy dynamics in solar salter ponds. [Display omitted] • The chemotaxonomic value of signature pigments at high salinity is shown. • There is a strong correlation between the retinal/chlorophyll a ratio and salinity. • Retinal-based sunlight capture is essential in hypersaline environments. • Photoheterotrophy gains relevance over photosynthesis as salinity increases. [ABSTRACT FROM AUTHOR]

Hypersaline environments are extreme habitats with a limited prokaryotic diversity, mainly restricted to halophilic or halotolerant archaeal and bacterial taxa adapted to highly saline conditions. This study attempts to analyze the taxonomic and functional diversity of the prokaryotes that inhabit a solar saltern located at the Atlantic Coast, in Isla Cristina (Huelva, Southwest Spain), and the influence of salinity on the diversity and metabolic potential of these prokaryotic communities, as well as the interactions and cooperation among the individuals within that community. Brine samples were obtained from different saltern ponds, with a salinity range between 19.5 % and 39 % (w/v). Total prokaryotic DNA was sequenced using the Illumina shotgun metagenomic strategy and the raw sequence data were analyzed using supercomputing services following the MetaWRAP and SqueezeMeta protocols. The most abundant phyla at moderate salinities (19.5–22 % [w/v]) were Methanobacteriota (formerly "Euryarchaeota"), Pseudomonadota and Bacteroidota , followed by Balneolota and Actinomycetota and Uroviricota in smaller proportions, while at high salinities (36–39 % [w/v]) the most abundant phylum was Methanobacteriota, followed by Bacteroidota. The most abundant genera at intermediate salinities were Halorubrum and the bacterial genus Spiribacter , while the haloarchaeal genera Halorubrum , Halonotius , and Haloquadratum were the main representatives at high salinities. A total of 65 MAGs were reconstructed from the metagenomic datasets and different functions and pathways were identified in them, allowing to find key taxa in the prokaryotic community able to synthesize and supply essential compounds, such as biotin, and precursors of other bioactive molecules, like β-carotene, and bacterioruberin, to other dwellers in this habitat, lacking the required enzymatic machinery to produce them. This work shed light on the ecology of aquatic hypersaline environments, such as the Atlantic Coast salterns, and on the dynamics and factors affecting the microbial populations under such extreme conditions. [Display omitted] • Taxonomic diversity of the prokaryotic community of the saltern decreases with the salinity increase. • Functional metabolic diversity is not significantly correlated with the salt concentration range. • Key taxa produce biomolecules that other community members use for metabolism or to synthesize essential compounds. • Reconstructed bins are found in hypersaline environments worldwide, showing their euryhaline character and wide dispersion. [ABSTRACT FROM AUTHOR]

Soil salinity is one of the most important factors reducing agricultural productivity worldwide. Halophilic plant growth-promoting bacteria (H-PGPB) represent an alternative method of alleviating saline stress in crops of agricultural interest. In this study, the following halophilic bacteria were evaluated: Bacillus sp. SVHM1.1, Halomonas sp. SVCN6, Halomonas sp. SVHM8, and a consortium. They were grown under greenhouse conditions in Solanum lycopersicum at different salinity concentrations in irrigation water (0, 20, 60, and 100 mM NaCl) to determine the effects on germination, fruit quality, yield, and concentration of osmoprotectors in plant tissue. Our results demonstrate the influence of halophilic bacteria with the capacity to promote plant growth on the germination and development of Solanum lycopersicum at higher salinity levels. The germination percentage was improved at the highest concentration by the inoculated treatments (from 37 to 47%), as were the length of the radicle (30% at 20 mM) and plumule of the germinated seed, this bacterium also increased the weight of the plumule (97% at 100 mM). They also improved the yield. The dry weight of the plant, in addition to having an influence on the quality of the fruit and the concentration of osmoprotectors (Bacillus sp. SVHM 1.1) had the greatest effect on fruit yield (1.5 kg/plant at 20 mM), by the otherhand, Halomonas sp. SVHM8 provided the best fruit quality characteristics at 100 mM. According to the above results, the efficiency of halophilic PGPB in the attenuation of salt stress in Solanum lycopersicum has been proven.

The reserves of light conditional oil in reservoirs with low-salinity formation water are decreasing worldwide, necessitating the extraction of heavy oil from petroleum reservoirs with high-salinity formation water. As the first stage of defining the microbial-enhanced oil recovery (MEOR) strategies for depleted petroleum reservoirs, microbial community composition was studied for petroleum reservoirs with high-salinity formation water located in Tatarstan (Russia) using metagenomic and culture-based approaches. Bacteria of the phyla Desulfobacterota, Halanaerobiaeota, Sinergistota, Pseudomonadota, and Bacillota were revealed using 16S rRNA-based high-throughput sequencing in halophilic microbial communities. Sulfidogenic bacteria predominated in the studied oil fields. The 75 metagenome-assembled genomes (MAGs) of prokaryotes reconstructed from water samples were assigned to 16 bacterial phyla, including Desulfobacterota, Bacillota, Pseudomonadota, Thermotogota, Actinobacteriota, Spirochaetota, and Patescibacteria, and to archaea of the phylum Halobacteriota (genus Methanohalophilus). Results of metagenomic analyses were supported by the isolation of 20 pure cultures of the genera Desulfoplanes, Halanaerobium, Geotoga, Sphaerochaeta, Tangfeifania, and Bacillus. The isolated halophilic fermentative bacteria produced oil-displacing metabolites (lower fatty acids, alcohols, and gases) from sugar-containing and proteinaceous substrates, which testify their potential for MEOR. However, organic substrates stimulated the growth of sulfidogenic bacteria, in addition to fermenters. Methods for enhanced oil recovery should therefore be developed, combining the production of oil-displacing compounds with fermentative bacteria and the suppression of sulfidogenesis.

Salinity is one of the strongest environmental drivers of microbial evolution and community composition. Here we aimed to determine the impact of salt concentrations (2.5, 7.5, and 33.2%) on the microbial community structure of reclaimed saltern ponds near San Francisco, California, and to discover prospective enzymes with potential biotechnological applications. Community compositions were determined by 16S rRNA amplicon sequencing revealing both higher richness and evenness in the pond sediments compared to the water columns. Co-occurrence network analysis additionally uncovered the presence of microbial seed bank communities, potentially primed to respond to rapid changes in salinity. In addition, functional annotation of shotgun metagenomic DNA showed different capabilities if the microbial communities at different salinities for methanogenesis, amino acid metabolism, and carbohydrate-active enzymes. There was an overall shift with increasing salinity in the functional potential for starch degradation, and a decrease in degradation of cellulose and other oligosaccharides. Further, many carbohydrate-active enzymes identified have acidic isoelectric points that have potential biotechnological applications, including deconstruction of biofuel feedstocks under high ionic conditions. Metagenome-assembled genomes (MAGs) of individual halotolerant and halophilic microbes were binned revealing a variety of carbohydrate-degrading potential of individual pond inhabitants.

Extremophiles provide a one-of-a-kind source of enzymes with properties that allow them to endure the rigorous industrial conversion of lignocellulose biomass into fermentable sugars. However, the fact that most of these organisms fail to grow under typical culture conditions limits the accessibility to these enzymes. In this study, we employed a functional metagenomics approach to identify carbohydrate-degrading enzymes from Ethiopian soda lakes, which are extreme environments harboring a high microbial diversity. Out of 21,000 clones screened for the five carbohydrate hydrolyzing enzymes, 408 clones were found positive. Cellulase and amylase, gave high hit ratio of 1:75 and 1:280, respectively. A total of 378 genes involved in the degradation of complex carbohydrates were identified by combining high-throughput sequencing of 22 selected clones and bioinformatics analysis using a customized workflow. Around 41% of the annotated genes belonged to the Glycoside Hydrolases (GH). Multiple GHs were identified, indicating the potential to discover novel CAZymes useful for the enzymatic degradation of lignocellulose biomass from the Ethiopian soda Lakes. More than 73% of the annotated GH genes were linked to bacterial origins, with Halomonas as the most likely source. Biochemical characterization of the three enzymes from the selected clones (amylase, cellulase, and pectinase) showed that they are active in elevated temperatures, high pH, and high salt concentrations. These properties strongly indicate that the evaluated enzymes have the potential to be used for applications in various industrial processes, particularly in biorefinery for lignocellulose biomass conversion.

Simple Summary: As one of the two cases of soda lakes in the world, Wadi El-Natrun salt lakes are the intended isolation source in this study. They encompass a characteristic microbial niche of symbionts producing unique bioactive agents, among them carotenoids. Herein, halophilic carotenoid-producing microbes were isolated and purified. Based on the carotenoids content, the potent isolate, namely Virgibacillus halodenitrificans, was molecularly identified, and characterized physiologically, biochemically and morphologically as well. Its carotenoids content was optimized using statistical methods. Under optimized conditions, the maximum productivity was obtained and the extracted carotenoids were identified by UV-Vis analysis, FTIR, Raman spectroscopy, TLC and LC–MS, which confirmed such halophilic carotenoids composed of β-carotene, lutein and β-Apo-8′-carotenal mixture. Meanwhile, the carotenoids were utilized as a powerful antibacterial and antifungal agent. Notably, they exerted higher antibiofilm potentiality at a concentration of 20 μg/mL. Finally, halophilic carotenoids are competing as new biocidal agents, in particular with their biocompatibility and efficiency. Carotenoids, as phytonutrient pigments, are signified by their unique beneficial features that serve human health and the surrounding ecosystem. Haloalkaliphiles from soda lakes produce different natural bioactive molecules; however, their ability to produce carotenoids has been limited. Therefore, this study focused on the screening and isolation of carotenoid-producing haloalkaliphilic microbes. Out of 10 isolates, a powerful carotigenic bacterium was isolated, characterized phenotypically and identified on the molecular level as Virgibacillus halodenitrificans. By employing statistical approaches like Plackett–Burman design and central composite design, the influence of significant nutritional variables on carotenoids production was screened and optimized. Predictive modeling manifested that carotenoid yield was 36.42 mg/mL, a 2.12-fold enhancement compared to the basal conditions through inoculating 1.8% of bacterial biomass on optimized medium containing yeast extract (2 g/mL), peptone (10 g/mL) and NaCl (233.6 g/mL). The carotenoids content was confirmed by UV-Vis spectrum which detected a characteristic unique peak with left and right shoulders at 461 nm, 490 and 522 nm. However, FTIR and Raman spectroscopy showed the presence of several functional groups. Meanwhile, LC–MS confirmed that the examined carotenoids were composed of β-carotene, lutein and β-Apo-8′-carotenal mixture. As a bioactive agent, the carotenoids of V. halodenitrificans DASH showed characteristic antagonistic potency against a wide spectrum of Gram-positive and Gram-negative pathogens. Interestingly, a potent antifungal capacity was observed against Candida albicans, reflecting promising mycocidal efficacy against COVID-19 white fungal post-infections. Furthermore, carotenoids (20 μg/mL) inhibited the biofilm formation of P. aeruginosa and S. aureus by 54.01 ± 3.97% and 80.082 ± 0.895%, respectively. Our results proposed that haloalkaliphiles of Wadi El-Natrun lakes are promising sources of carotenoids that exhibited efficiency as safe, biocompatible and natural bioactive agents for environmental, medical and industrial applications. [ABSTRACT FROM AUTHOR]

Aspergillus penicillioides may be both xerophilic and halophilic. To study whether there is a difference in the mechanisms of adaptation to low water activity and high NaCl concentration the composition of osmolytes and lipids was analysed during growth on two media with 2 M NaCl or 4 M glycerol. Glycerol was the predominant osmolyte of the fungus mycelium grown on both media at all growth stages. On the medium with glycerol, the fungus accumulated twice as much glycerol as on the medium with salt. The proportion of erythritol, arabitol and mannitol was significantly higher on a medium with salt (up to 30% of the total). It was first shown that phosphatidic acids, along with sterols, were the predominant membrane lipids, while phosphatidylcholines and phosphatidylethanolamines, typical for most fungi, were minor. The general pattern of changes in the membrane lipid composition on both media during growth was the increase in the proportions of sterols and unidentified lipid X1 along with the decrease of phosphatidic acids and cardiolipins, but the changes on the salt medium were more pronounced. The degree of phospholipid unsaturation decreased only on the medium with glycerol. The results obtained show main patterns and differences in the mechanisms of adaptation of fungus to low water activity and high salt concentration. [ABSTRACT FROM AUTHOR]

The use of natural polysaccharides as biomaterials is gaining importance in tissue engineering due to their inherent biocompatibility. In this direction, the present study aims to explore the structure and biocompatibility of the EPS produced by Virgibacillus dokdonensis VITP14. This marine bacterium produces 17.3 g/L of EPS at 96 h of fermentation. The EPS was purified using ion exchange and gel permeation chromatographic methods. The porous web-like structure and elemental composition (C, O, Na, Mg, P, S) of the EPS were inferred from SEM and EDX analysis. AFM analysis revealed spike-like lumps with a surface roughness of 84.85 nm. The zeta potential value of −10 mV indicates the anionic nature of the EPS. Initial molecular characterization showed that the EPS is a heteropolysaccharide composed of glucose (25.8%), ribose (18.6%), fructose (31.5%), and xylose (24%), which are the monosaccharide units in the HPLC analysis. The FTIR spectrum indicates the presence of functional groups/bonds typical of EPSs (O-H, C-H, C-O-H, C-O, S=O, and P=O). The polymer has an average molecular weight of 555 kDa. Further, NMR analysis revealed the monomer composition, the existence of two α- and six β-glycosidic linkages, and the branched repeating unit as → 1)[α-D-Xylp-(1 → 2)-α-D-Glcp-(1 → 6)-β-D-Glcp-(1 → 5)]-β-D-Frup-(2 → 2)[β-D-Xylp-(1 → 4)]-β-D-Xylp-(1 → 6)-β-D-Fruf-(2 → 4)-β-D-Ribp-(1 →. The EPS is thermally stable till 251.4 °C. X-ray diffraction analysis confirmed the semicrystalline (54.2%) nature of the EPS. Further, the EPS exhibits significant water solubility (76.5%), water-holding capacity (266.8%), emulsifying index (66.8%), hemocompatibility (erythrocyte protection > 87%), and cytocompatibility (cell viability > 80% on RAW264.7 and keratinocyte HaCaT cells) at higher concentrations and prolongs coagulation time in APTT and PT tests. Our research unveils the significant biocompatibility of VITP14 EPS for synthesizing a variety of biomaterials. [ABSTRACT FROM AUTHOR]

Actinomycetes are Gram-positive filamentous bacteria well known for the production of bioactive compounds. Recently, many halophilic habitats have been explored for isolation of actinomycetes that exhibit biotechnological potentials. In this investigation, a saline habitat of Rajasthan, Sambhar Salt Lake (SSL) was selected to study the actinomycetes population and Carboxy Methyl Cellulase (CMCase) production by native isolates. A total of sixteen actinomycete isolates, halotolerant and moderately halophilic, were obtained using culture-dependent methods and characterized morphologically and biochemically. They were identified as members of Streptomyces, Nocardiopsis, Pseudonocardia, Saccharospolyspora, and Microbispora. Streptomyces was the most dominating genus, followed by Nocardiopsis. Agar plate assay was used for screening the isolates for CMCase production. Thirteen were found to produce the enzyme, apparent by hydrolysis observed on media plates. The highest relative activity of 22.04 was shown by isolate SSL 14 identified as Nocardiopsis sp. by 16S rDNA sequencing studies and thus selected for further optimization studies. Maximum enzyme (1.08 ± 0.09 U/ml) was produced using medium containing Carboxy Methyl Cellulose (Carbon source) and yeast extract (nitrogen source) at 12% NaCl and pH 9.0, incubated at 30 °C for 96 h. Maximum CMCase production at high salt concentration and pH suggests that Nocardiopsis SSL 14 can be used for industrial processes that operate under excessive saline and alkaline conditions. [ABSTRACT FROM AUTHOR]

Brine from Dingyuan Salt Mine (Anhui, China), an athalassohaline hypersaline environment formed in the early tertiary Oligocene, is used to produce table salt for hundreds of millions of people. However, halophiles preserved in this niche during deposition are still unknown. Here, we employed cultivation and high-throughput sequencing strategies to uncover the microbial community and its shift after a long-term storage in the brine collected from Dingyuan Salt Mine. High-throughput sequencing showed (1) in the fresh brine (2021), Cyanobium_stocktickerPCC-6307 spp. (8.46%), Aeromonas spp. (6.91%) and Pseudomonas spp. (4.71%) are the dominant species in bacteria while Natronomonas spp. (18.89%), Halapricum spp. (13.73%), and Halomicrobium spp. (12.35%) in archaea; (2) after a 3-year-storage, Salinibacter spp. (30.01%) and Alcanivorax spp. (14.96%) surpassed Cyanobium_stocktickerPCC-6307 spp. (8.46%) becoming the dominant species in bacteria; Natronomonas spp. are still the dominant species, while Halorientalis spp. (14.80%) outnumbered Halapricum spp. becoming the dominant species in archaea; (3) Alcanivorax spp. and Halorientalis spp. two hydrocarbons degrading microorganisms were enriched in the brine containing hydrocarbons. Cultivation using hypersaline nutrient medium (20% NaCl) combined with high-throughput 16S rRNA gene sequencing showed that (1) the biomass significantly increased while the species diversity sharply declined after a 3-year-storage; (2) Halorubrum spp. scarcely detected from the environment total stocktickerDNA were flourishing after cultivation using AS-168 or NOM medium; (3) twelve possible new species were revealed based on almost full-length 16S rRNA gene sequence similarity search. This study generally uncovered the microbial community and the dominant halophiles in this inland athalassohaline salt mine, and provided a new insight on the shift pattern of dominant halophiles during a longterm storage, which illustrated the shaping of microorganisms in the unique environment, and the adaptation of microbe to the specific environment. [ABSTRACT FROM AUTHOR]

Aim This study aims to determine the biological activity and explore the antimicrobial compounds produced   by a halophilic bacterium, as well as the hemolytic, antioxidant, and antimicrobial properties of extracellular metabolites from Bacillus sp. Methodology The bacterial strain was obtained from the Department of Microbiology at Saveetha Medical College and Hospital, Chennai, India, specifically from the bio-control and microbial product laboratory (BCMPL). The genotype and phenotype of the isolate were characterized while the cultures were maintained in a nutrient broth medium supplemented with 8% sodium chloride (NaCl). The secondary metabolites were extracted using ethyl acetate and concentrated through open evaporation techniques after nine days of growth in the culture medium. Biological compatibility studies were conducted concurrently with the screening of the antimicrobial and antioxidant properties of the secondary metabolites. The chemical composition of the crude metabolites was analyzed using the gas chromatography (GC)-mass spectrometry (MS) technique. Results After phenotypic and genotypic analysis, the obtained potential halophilic bacterium from BCMPL was determined to be Bacillus sp. After the dark brown crude metabolites were extracted, the extracellular metabolites' antimicrobial activity against Staphylococcus aureus ( S. aureus ), Escherichia coli ( E. coli ) , and Candida albicans ( C. albicans ) extracellular metabolites was moderately inhibited. Furthermore, the metabolites exhibited a moderate level of hemolytic and antioxidant activity. The GC-MS method depicted the presence of 12 distinct metabolites, each with a distinct retention time. Conclusion To sum up, the halophilic bacteria that were obtained and identified as  Bacillus species and their crude metabolites demonstrated noteworthy antioxidant and antimicrobial properties. Further investigation may be helpful in identifying possible compounds that Bacillus sp. produces., Competing Interests: Human subjects: All authors have confirmed that this study did not involve human participants or tissue. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work., (Copyright © 2024, Sujitha et al.)

One hundred and sixty-three extreme halophiles were recovered from a single sample collected from an inland solar saltern in Rio Maior. Based on random amplified polymorphic DNA (RAPD) profiles and partial 16S rRNA gene sequencing 125 isolates were identified as members of the Archaea domain within the genus Halorubrum. Two strains, RMP-11 T and RMP-47, showed 99.1 % sequence similarity with the species Halorubrum californiense based on phylogenetic analysis of the 16S rRNA gene sequence. However, phylogenetic analysis based on five housekeeping genes, atpB, EF-2, glnA, ppsA and rpoB', showed Halorubrum coriense as the closest related species with 96.7 % similarity. The average nucleotide identity (ANI) of strains RMP-11 T , RMP-47 and species Hrr. coriense were within the range of 90.0-90.5 %, supporting that strains RMP-11 T and RMP-47 represent a novel species of the genus Halorubrum. These strains formed red-pigmented colonies that were able to grow in a temperature range of 25-50 °C. Polyhydroxyalkanoate (PHA) granules were detected in both strains. The polar lipid profile was identical to the neutrophilic species of the genus Halorubrum. The Rio Maior sample from which both strains were isolated was metagenome sequenced. We identified five metagenome-assembled genomes representing novel Halorubrum species but distinct from the species represented by strains RMP-11 T and RMP-47. Based on phylogenetic, phylogenomic, comparative genomics, physiological and chemotaxonomic parameters, we describe a new species of the genus Halorubrum represented by strains RMP-11 T (=CECT 30760 T  = DSM 115521 T ) and RMP-47 (=CECT 30761 = DSM 115541) for which we propose the name Halorubrum miltondacostae sp. nov., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Salimicrobium sp. PL1-032A was isolated from Pearse Lakes, Western Australia. The sequenced genome consists of a single chromosome (2,705,688  bp) with a GC content of 47.2%. The isolation of Salimicrobium sp. PL1-032A contributes to the collection of culturable extremophiles and offers potential insight into the Pearse Lakes biome., Competing Interests: The authors declare no conflict of interest.