Halophilic Conditions Research Articles

Draft genome sequence of Halomonas sp. SSL-5, a halophilic Mn(II)-oxidizing, perchlorate-tolerant bacterium.

Halomonas sp. SSL-5 is a Mn(II)-oxidizing, perchlorate-tolerant halophilic bacterium isolated from an Australian hypersaline lake. The genome sequence contains 27 contigs, and the genome is 3.4 Mb with a GC content of 67.2%. The sequence provides information for future studies of Mn(II) oxidation and perchlorate resistance under halophilic conditions.

High Salt Electrolyte Solutions Challenge the Electrochemical CO2Reduction Reaction to Formate at Indium and Tin Cathodes

AbstractFormate is a promising product of the electrochemical CO2reduction reaction (eCO2RR) that can serve as feedstock for biological syntheses. Indium (In) has been shown as a selective electrocatalyst of eCO2RR with high coulombic efficiency (CE) for formate production at small scale at biocompatible non‐halophilic that is low salt conditions. Ohmic losses and challenges on potential/current distribution arise for scaling‐up, where higher salt loads are advantageous for minimizing these. Higher salt concentration within the solution or halophilic conditions also enable the use of halophilic biocatalysts. We optimized eCO2RR with halophilic media by introducing tin (Sn) as a more sustainable alternative to In. At 3 % NaCl providing a catholyte conductivity (of 70 mS cm−1, the maximum specific formate production rates (rformate) of 0.143±0.030 mmol cm−2 h−1and 0.167±0.027 mmol cm−2 h−1were achieved at In and Sn electrocatalysts, respectively. Decrease inrformateand CE, in addition to higher variation between replicates was observed with further increase in NaCl concentration above 3 % (>70 mS cm−1) up to 10 % (=127 mS cm−1). This study sets the foundation for integrated microbial synthesis by halophiles.

Gillisia lutea sp. nov., isolated from marine aluminium residues from the Mediterranean sea.

A novel Gram-reaction-negative, facultatively anaerobic, rod-shaped, non-motile, non-spore forming, orange-pigmented bacterium identified as M10.2AT, was isolated from marine residues submerged on the Malva-rosa beach (València, Spain), on the western coast of the Mediterranean Sea. This strain was catalase-positive and oxidase-negative and grew under mesophilic, neutrophilic and halophilic conditions. With respect to the 16S rRNA gene sequences, M10.2AT showed similarities with Gillisia mitskevichiae DSM 19839T and Gillisia hiemivida IC154T (97.57 and 97.50 % gene sequence similarity, respectively). The genome of M10.2AT was sequenced and has been deposited in the DDBJ/ENA/GenBank databases under the accession code JAKGTH000000000. The genomic DNA G+C content was 36.13 %. Its adscription to a novel species of the genus Gillisia was confirmed by the genomic indexes average nucleotide identity by blast (ANIb) and digital DNA-DNA hybridisation (dDDH). The major fatty acids were iso-C15 : 0, iso-C15 : 1G, iso-C16 : 0 3-OH, iso-C17 : 0 3-OH and summed feature 3 (C16 : 1ω7c/C16 : 1ω6c). According to the results of this polyphasic study, strain M10.2AT represents a novel species of the genus Gillisia, for which name Gillisia lutea sp. nov. (type strain M10.2AT = CECT 30308T = DSM 112385T) is proposed.

Optimization of 4-aminobutyric acid feeding strategy and clustered regularly interspaced short palindromic repeats activation for enhanced value-added chemicals in halophilic Chlorella sorokiniana

Chlorella sorokiniana (CS) is a prominent microalga with vast potential as a biocarrier for carbon mitigation toward a green process. However, challenges remain in achieving high biomass levels and production rates. Therefore, a systematic feeding strategy using 4-aminobutyric acid (GABA) and CRISPR technology was applied to improve microalgal productivity. At first, GABA increased protein content by 1.4-fold, while intermittent supplementation during cultivation resulted in a 1.58-fold and 2.13-fold increase in biomass and pigment content, respectively. Under halophilic conditions, the optimal approach involved repeated feeding of 5 mM GABA at the initial and mid-log phases of growth, resulting in biomass, protein, and pigment levels of 6.74 g/L, 3.24 g/L, and 49.87 mg/L. CRISPRa mediated glutamate synthase and using monosodium glutamate (MSG) as a cheap precursor for GABA has effectively enhanced the biomass, protein, and lutein content, thus offers a cost-effective approach to commercialize high-valued chemical using algae towards a low-carbon paradigm.

Bacillus thuringiensis EM-A1: A novel bacterium for high concentration of ammonium elimination with low nitrite accumulation

The biological elimination of high concentration of ammonium from wastewater has attracted increasing attention in recent years. However, few studies on the efficient elimination of high concentration of ammonium by a single bacterium have been reported. Here, the efficient elimination of NH4+-N (>99%) and total nitrogen (TN) (>77%) were attained by Bacillus thuringiensis EM-A1 under 150 rpm at pH 7.2 with sodium succinate and a carbon/nitrogen ratio of 15 at 30 °C with an inoculum size (as measured by absorbance at 600 nm) of 0.2. Strain EM-A1 effectively eliminated 100 mg/L of inorganic nitrogen with maximal NH4+-N, NO3−-N, and NO2−-N elimination rates of 4.88, 2.57, and 3.06 mg/L/h, respectively. The elimination efficiencies of NH4+-N were 99.87% and 97.13% at initial concentrations of 500 and 1000 mg/L, respectively. Only 0.91 mg/L of NO2−-N was accumulated with the elimination of 1000 mg/L NH4+-N. A concentration of 5 mg/L exogenous hydroxylamine was toxic and further inhibited heterotrophic nitrification and aerobic denitrification (HN-AD). The NH4+-N and NO2−-N elimination capacities of strain EM-A1 were specifically inhibited by 2-Octyne (OCT) over 4 μmol/L and diethyldithiocarbamate (DDC) over 0.5 mmol/L, respectively. Above 25 mg/L procyanidin (PCY) inhibited the bioconversion of NO3−-N and NO2−-N. The results demonstrated that strain EM-A1 had HN-AD capacity under halophilic conditions, and has great potential for use in the treatment of nitrogen pollution wastewater; this study also provides new insights into this strain's nitrogen elimination mechanism, helping advance environmental biotechnology.

Arcobacter roscoffensis sp. nov., a marine bacterium isolated from coastal seawater.

A novel Gram-negative, aerobic, motile, rod-shaped, beige-pigmented bacterium, strain ARW1-2F2T, was isolated from a seawater sample collected from Roscoff, France. Strain ARW1-2F2T was catalase-negative and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA sequences revealed that strain ARW1-2F2T was closely related to Arcobacter lekithochrous LFT 1.7T and Arcobacter caeni RW17-10T(95.8 and 95.5 % gene sequence similarity, respectively). The genome of strain ARW1-2F2T was sequenced and had a G+C content of 28.7%. Two different measures of genome similarity, average nucleotide identity based on blast and digital DNA-DNA hybridization, indicated that strain ARW1-2F2T represents a new Arcobacter species. The predominant fatty acids were C16 : 1 ω7c/C16 : 1 ω6c and C18 : 1 ω7c/C18 : 1 ω6c. The results of a polyphasic analysis supported the description of strain ARW1-2F2T as representing a novel species of the genus Arcobacter, for which the name Arcobacter roscoffensis sp. nov. is proposed with the type strain ARW1-2F2T (DSM 29169T=KCTC 52423T).

Lipase B from Candida antarctica in Highly Saline AOT-Water-Isooctane Reverse Micelle Systems for Enhanced Esterification Reaction

Butyl oleate synthesis by the lipase B from Candida antarctica (CalB) under extreme halophilic conditions was investigated in the present research through the AOT/Water/Isooctane reverse micellar system. The impact of aqueous content (Wo=H2OSurfactant) and NaCl variation on the enzymatic activity of CalB in the butyl oleate reaction in reverse micelles was explored. The results indicated that, based on the increase of NaCl, it is remarkable to achieve higher enzymatic activity up to 444.85 μmolmin at 5 M NaCl and Wo = 10, as the best esterification conditions at pH 7.2 and 30 °C. However, it was clear that butyl oleate synthesis by lipase CalB increased based on the reduction in the average reverse micelle size, where reverse micelle sizes were determined by dynamic light scattering (DLS). This increase in butyl oleate synthesis demonstrated the potential of reverse micelles as systems that enhance mass transport phenomena in heterogeneous biocatalysis. Furthermore, reverse micelles are promising systems for extreme halophilic lipases research.

Maritalea mediterranea sp. nov., isolated from marine plastic residues.

A novel Gram-reaction-negative, aerobic, motile, rod-shaped, grey bacterium, strain P4.10XT, was isolated from plastic debris sampled from shallow waters in the Mediterranean Sea (Valencia, Spain). P4.10XT was catalase- and oxidase-positive, and grew under mesophilic, neutrophilic and halophilic conditions. The 16S rRNA gene sequences revealed that P4.10XT was closely related to Maritalea myrionectae DSM 19524T and Maritalea mobilis E6T (98.25 and 98.03 % sequence similarity, respectively). The DNA G+C content of the genome sequence of P4.10XT was 53.66 %. The genomic indexes average nucleotide identity by blast (ANIb) and digital DNA-DNA hybridization (dDDH) confirmed its classification as representing a novel species of the genus Maritalea. The predominant fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c) and C18 : 1 ω7c 11-methyl. The results of this polyphasic study confirm that P4.10XT represents a novel species of the genus Maritalea, for which the name Maritalea mediterranea sp. nov. is proposed (type strain P4.10XT=CECT 30306T = DSM 112386T).

Biochemical characterization of a psychrophilic and halotolerant α-carbonic anhydrase from a deep-sea bacterium, Photobacterium profundum.

Prokaryotic α-carbonic anhydrases (α-CA) are metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and proton. We had reported the first crystal structure of a pyschrohalophilic α-CA from a deep-sea bacterium, Photobacterium profundum SS9. In this manuscript, we report the first biochemical characterization of P. profundum α-CA (PprCA) which revealed several catalytic properties that are atypical for this class of CA's. Purified PprCA exhibited maximal catalytic activity at psychrophilic temperatures with substantial decrease in activity at mesophilic and thermophilic range. Similar to other α-CA's, Ppr9A showed peak activity at alkaline pH (pH 11), although, PprCA retained 88% of its activity even at acidic pH (pH 5). Exposing PprCA to varying concentrations of oxidizing and reducing agents revealed that N-terminal cysteine residues in PprCA may play a role in the structural stability of the enzyme. Although inefficient in CO2 hydration activity under mesophilic and thermophilic temperatures, PprCA exhibited salt-dependent thermotolerance and catalytic activity under extreme halophilic conditions. Similar to other well-characterized α-CA's, PprCA is also inhibited by monovalent anions even at low concentrations. Finally, we demonstrate that PprCA accelerates CO2 biomineralization to calcium carbonate under alkaline conditions.

Forced Biomineralization: A Review.

Biologically induced and controlled mineralization of metals promotes the development of protective structures to shield cells from thermal, chemical, and ultraviolet stresses. Metal biomineralization is widely considered to have been relevant for the survival of life in the environmental conditions of ancient terrestrial oceans. Similar behavior is seen among extremophilic biomineralizers today, which have evolved to inhabit a variety of industrial aqueous environments with elevated metal concentrations. As an example of extreme biomineralization, we introduce the category of “forced biomineralization”, which we use to refer to the biologically mediated sequestration of dissolved metals and metalloids into minerals. We discuss forced mineralization as it is known to be carried out by a variety of organisms, including polyextremophiles in a range of psychrophilic, thermophilic, anaerobic, alkaliphilic, acidophilic, and halophilic conditions, as well as in environments with very high or toxic metal ion concentrations. While much additional work lies ahead to characterize the various pathways by which these biominerals form, forced biomineralization has been shown to provide insights for the progression of extreme biomimetics, allowing for promising new forays into creating the next generation of composites using organic-templating approaches under biologically extreme laboratory conditions relevant to a wide range of industrial conditions.

Draft Genome Sequence of Halomonas sp. Strain KAO, a Halophilic Mn(II)-Oxidizing Bacterium.

Halomonas sp. strain KAO is an aerobic, Mn(II)-oxidizing, halophilic bacterium. The draft genome of the isolate contains 47 contigs encompassing 3.7 Mb and a GC content of 64.22%. This sequence will provide essential information for future studies of Mn(II) oxidation, particularly under halophilic conditions.

Exploring the potential of halotolerant bacteria for biodegradation of polycyclic aromatic hydrocarbon

The present study aimed to determine the degradation and transformation of three-ring PAHs phenanthrene and anthracene by Cryptococcus sp. MR22 and Halomonas sp. BR04 under halophilic conditions. The growth progress of Cryptococcus sp. MR22 and Halomonas sp. BR04 on anthracene and phenanthrene was monitored by colony-forming unit (CFU) technique. The growth of the bacteria was maintained at a maximum concentration of 200mg/L of all tested hydrocarbon, indicating that Cryptococcus sp. MR22 and Halomonas sp. BR04 significantly perform in the removal of the PAH-contaminated medium at low concentrations. The fit model to represent the biodegradation kinetics of both PAHs was first-order rate equation The extract prepared from cells supplemented with three different substrates exhibited some enzymes such as hydroxylase, dioxygenase, laccase and peroxidase. The results suggest that both strains had an impressive ability in the degradation of aromatic and aliphatic hydrocarbon but also could tolerate in the extreme salinity condition.

Pectinase Producing Bacteria Isolation from Halophilic Soil, Water Samples and Partial Purification of the Enzyme

Pectinases are protease enzymes capable of degrading pectin, which is one of the most important polysaccharide found in nature. Pectinase enzymes find uses in paper-pulp industry, textile industry, food industry and other industries where lignocellulosic material is utilized. Commercially pectinase enzymes are obtained from bacterial and fungal culture grown on decaying fruits and vegetables. In the present study, an attempt was made to isolate pectinase producing bacteria growing in halophilic conditions. Soil (sand) and water samples containing high concentrations of salts from beaches of Taiwan and Karwar, Karnataka were collected and screened for pectinase activity using modified M9 media. Among the obtained 16 bacterial strains, strain 15 showed the highest pectinase activity during screening and was identified to be a Bacillus sp. from morphological features and biochemical tests. This strain was selected and its cultural conditions were standardized for maximum production of pectinase enzyme. Maximum enzyme activity was obtained at pH 11, temperature 40°C, and incubation period of 24 hours. To enhance the productivity modification were tried out in culture media related to nitrogen source and carbon source in which the nitrogen source did not have any significance in activity of enzyme but glucose seemed to be the best carbon source for the growth of bacteria. Further the obtained enzyme was partially purified using the salting out method, dialysis followed by desalting, gel filtration chromatography and ion-exchange chromatography.

High Production of Chitinolytic Activity in Halophilic Conditions by a New Marine Strain of Clonostachys rosea.

Twenty-eight fungal strains have been isolated from different natural marine substrates and plate screened for their production of chitinolytic activity. The two apparent best producers, Trichoderma lixii IG127 and Clonostachys rosea IG119, were screened in shaken cultures in media containing 1% colloidal chitin, 1% yeast nitrogen base and 38‰ NaCl, for their ability to produce chitinolytic enzymes under halophilic conditions. In addition, they were tested for optimal growth conditions with respect to pH, salinity and temperature. The Trichoderma strain appeared to be a slight halotolerant fungus, while C. rosea IG119 clearly showed to be a halophilic marine fungus, its optimal growth conditions being very coherent for life in the marine environment (i.e., pH 8.0, salinity 38‰). Due to its high and relatively fast activity (258 U/L after 192 h of growth) accompanied by its halophilic behaviour (growth from 0 to 160‰ of salinity), C. rosea was selected for further studies. In view of possible industrial applications, its medium for chitinolytic enzyme production was optimized by Response Surface Methodology using 1% colloidal chitin and different concentrations of corn step liquor and yeast nitrogen base (0–0.5%). Time course of growth under optimized condition showed that maximum activity (394 U/L) was recorded after 120 h on medium containing Corn Steep Liquor 0.47% and Yeast Nitrogen Base 0.37%. Maximum of productivity (3.3 U/Lh) was recorded at the same incubation time. This was the first study that demonstrated high chitinolytic activity in a marine strain of C. rosea.

Biodegradation of long chain alkanes in halophilic conditions by Alcanivorax sp. strain Est-02 isolated from saline soil

In this study, through a multistep enrichment and isolation procedure, a halophilic bacterial strain was isolated from unpolluted saline soil, which was able to effectively and preferentially degrade long chain alkanes (especially tetracosane and octacosane). The strain was identified by 16S rRNA gene sequence as an Alcanivorax sp. The growth of strain Est-02 was optimized at the presence of tetracosane in different NaCl concentrations, temperatures, and pH. The consumption of different heavy alkanes was also investigated. Optimal culture conditions of the strain were determined to be as follows: 10% NaCl, temperature 25-35°C and pH 7. Alcanivorax sp. strain Est-02 was able to use a wide range of aliphatic substrates ranging from C14 to C28 with clear tendency to utilize heavy chain hydrocarbons of C24 and C28. During growth on a mixture of alkanes (C14-C28), the strain consumed 60% and 65% of tetracosane and octacosane, respectively, while only about 40% of the lower chain alkanes were degraded. This unique ability of the strain Est-02 in efficient and selective biodegradation of long chain hydrocarbons could be further exploited for remediation of wax and heavy oil contaminated soils or upgrading of heavy crude oils. Comparison of the sequence of alkane hydroxylase gene (alkB) of strain Est-02 with previously reported sequences for Alcanivorax spp. and other hydrocarbon degraders, showed a remarkable phylogenetic distance between the sequence alkB of Est-02 and other alkane-degrading bacteria.

Ecotoxic potential of a presumably non-toxic azo dye

Microbes have potential to convert non-toxic azo dyes into hazardous products in the environment. However, the role of microbes in biotransforming such presumably non-toxic dyes has not been given proper attention, thereby, questions the environmental safety of such compounds. The present study assessed salinity driven microbial degradation of an unregulated azo dye, Acid orange 7 (AO7), under moderately halophilic conditions of textile effluent. The halophilic microbial consortium from effluent decolorized ~97% AO7 (50–500mgL−1). The consortium efficiently decolorized the dye at different pH (5–8) and salinity (5–18% NaCl). The 16S rRNA sequence analyses confirmed the presence of Halomonas and Escherichia in the consortium. The FTIR and GC-MS analyses suggested microbial consortium degrade AO7 following symmetric and asymmetric cleavage and yield carcinogenic/mutagenic aromatic byproducts viz. aniline, 1-amino-2-naphthol, naphthalene, and phenyldiazene. In contrast to AO7, the biodegraded products caused molecular, cellular and organism level toxicity. The degraded products significantly reduced: radicle length in root elongation assay; shoot length/biomass in plant growth assays; and caused chromosomal abnormalities and reduced mitotic index in Allium cepa bioassay. We demonstrated that under saline conditions of textile effluent, halophilic microbes convert a presumably non-toxic azo dye into hazardous products. The study calls to review the current toxicity classification of azo dyes and develop environmentally sound regulatory policies by incorporating the role of environmental factors in governing dye toxicity, for environmental safety.

Exploring the potential of halophilic bacteria from oil terminal environments for biosurfactant production and hydrocarbon degradation under high-salinity conditions

The wastewater from oil production can be exceptionally saline and contain a complex mixture of hydrocarbons, many of which are highly toxic. This study aimed to identify and characterize 141 halophilic bacteria isolated from production water and activated sludge from Marine Terminal Almirante Barroso (Brazil) and evaluate their potential for biosurfactant production and biodegradation of distinct petroleum hydrocarbons. Sequencing and phylogenetic analysis of the 16S rRNA gene revealed that the halophilic bacteria retrieved are distributed among 20 genera and four phyla (Proteobacteria, Firmicutes, Actinobacteria and Flavobacteria). RAPD fingerprinting was used to differentiate isolates at the infra-specific level, revealing 79 different genetic profiles. GC-MS analysis carried out with eight strains confirmed their ability to efficiently degrade alkanes and aromatic compounds under halophilic conditions, with preference for aromatic degradation. Eleven strains showed significant ability for reduction of the surface tension (from 72 to 40 mN/m) and for emulsification (up to 71%) of four different types of oils (mineral, soybean, diesel and kerosene). Results gathered in this study demonstrate a high taxonomic and genetic diversity of the halophilic bacterial strains isolated from the oil terminal samples and an outstanding potential for further use in biotechnological processes such as biosurfactant production or bioremediation.

The methylaspartate cycle in haloarchaea and its possible role in carbon metabolism.

Haloarchaea (class Halobacteria) live in extremely halophilic conditions and evolved many unique metabolic features, which help them to adapt to their environment. The methylaspartate cycle, an anaplerotic acetate assimilation pathway recently proposed for Haloarcula marismortui, is one of these special adaptations. In this cycle, acetyl-CoA is oxidized to glyoxylate via methylaspartate as a characteristic intermediate. The following glyoxylate condensation with another molecule of acetyl-CoA yields malate, a starting substrate for anabolism. The proposal of the functioning of the cycle was based mainly on in vitro data, leaving several open questions concerning the enzymology involved and the occurrence of the cycle in halophilic archaea. Using gene deletion mutants of H. hispanica, enzyme assays and metabolite analysis, we now close these gaps by unambiguous identification of the genes encoding all characteristic enzymes of the cycle. Based on these results, we were able to perform a solid study of the distribution of the methylaspartate cycle and the alternative acetate assimilation strategy, the glyoxylate cycle, among haloarchaea. We found that both of these cycles are evenly distributed in haloarchaea. Interestingly, 83% of the species using the methylaspartate cycle possess also the genes for polyhydroxyalkanoate biosynthesis, whereas only 34% of the species with the glyoxylate cycle are capable to synthesize this storage compound. This finding suggests that the methylaspartate cycle is shaped for polyhydroxyalkanoate utilization during carbon starvation, whereas the glyoxylate cycle is probably adapted for growth on substrates metabolized via acetyl-CoA.

Fermentative Production of Pyranone Derivate I from Marine Vibrio sp. SKMARSP9: Isolation, Characterization and Bioactivity Evaluation.

Pyranone derivative I was isolated from fermented broth of isolated marine bacterial strain Vibrio sp. SKMARSP9. The compound I was characterized, and evaluated for its antimicrobial properties. The isolated strain was identified based on 16S rRNA based phylogenetic analysis. The molecular analysis data suggested that this strain is closely related to Vibrio ruber, Vibrio sp. MSSRF10 and Vibrio rhizosphaerae. The best fermentative growth of this isolate was achieved under halophilic conditions and grew efficiently at 30°C in the presence of 12% NaCl. The compound I production by this strain is associated with growth. The unpurified extract is hydrophobic in nature, and released only during late growth phase. The extract was purified and characterized by spectral data using NMR, DEPT, and ESI-MS. The purity of I was 97% which was confirmed by HPLC. The pyranone derivative I exhibited >50% antioxidant activity and broad spectrum antimicrobial properties against gram negative and gram positive strains. Molecular docking analysis revealed that this pyranone derivative I may be a potential candidate at pharmaceutical sector.

A single aromatic core mutation converts a designed "primitive" protein from halophile to mesophile folding.

The halophile environment has a number of compelling aspects with regard to the origin of structured polypeptides (i.e., proteogenesis) and, instead of a curious niche that living systems adapted into, the halophile environment is emerging as a candidate "cradle" for proteogenesis. In this viewpoint, a subsequent halophile-to-mesophile transition was a key step in early evolution. Several lines of evidence indicate that aromatic amino acids were a late addition to the codon table and not part of the original "prebiotic" set comprising the earliest polypeptides. We test the hypothesis that the availability of aromatic amino acids could facilitate a halophile-to-mesophile transition by hydrophobic core-packing enhancement. The effects of aromatic amino acid substitutions were evaluated in the core of a "primitive" designed protein enriched for the 10 prebiotic amino acids (A,D,E,G,I,L,P,S,T,V)-having an exclusively prebiotic core and requiring halophilic conditions for folding. The results indicate that a single aromatic amino acid substitution is capable of eliminating the requirement of halophile conditions for folding of a "primitive" polypeptide. Thus, the availability of aromatic amino acids could have facilitated a critical halophile-to-mesophile protein folding adaptation-identifying a selective advantage for the incorporation of aromatic amino acids into the codon table.