Effect of Halomonas titanicae on fluctuating water-line corrosion of EH40 steel.

The fluctuating water-line corrosion of EH40 steel in sterile and biotic media was investigated with a wire beam electrode. When the coupons were partially immersed in the sterile medium, the position of the low water-line acted as the cathodic zone and the area below the low water-line constantly served as the main anodic zone. The thin electrolyte layers with uneven thickness promoted the galvanic current of the region below the low water-line. Different from the sterile environment, the metabolism of Halomonas titanica with oxygen as the final electron acceptor reduced the dissolved oxygen concentration, which resulted in the position of the low water-line acting as the anodic zone.

Halomonas maris sp. nov., a moderately halophilic bacterium isolated from sediment in the southwest Indian Ocean.

A halophilic, Gram-staining-negative, rod-shaped, flagellated and motile bacterium, strain QX-1 T, was isolated from deep-sea sediment at a depth of 3332 m in the southwestern Indian Ocean. Strain QX-1 T growth was observed at 4-50 °C (optimum 37 °C), pH 5.0-11.0 (optimum pH 7.0), 3-25% NaCl (w/v; optimum 7%), and it did not grow without NaCl. A phylogenetic analysis based on the 16S rRNA gene placed strain QX-1 T in the genus Halomonas and most closely related to Halomonas sulfidaeris (97.9%), Halomonas zhaodongensis (97.8%), Halomonas songnenensis (97.6%), Halomonas hydrothermalis (97.4%), Halomonas subterranea (97.3%), Halomonas salicampi (97.1%), and Halomonas arcis (97.0%). DNA-DNA hybridization (< 26.5%) and average nucleotide identity values (< 83.5%) between strain QX-1 T and the related type strains meet the accepted criteria for a new species. The principal fatty acids (> 10%) of strain QX-1 T are C16:0 (25.5%), C17:0 cyclo (14.0%), C19:0 cyclo ω8c (18.7%), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c, 18.1%). The polar lipids of strain QX-1 T are mainly diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipid, unidentified aminophospholipid, and five unidentified lipids. The main respiratory quinone is Q-9. The G + C content of its chromosomal DNA is 54.4 mol%. Its fatty acid profile, respiratory quinones, and G + C content also support the placement of QX-1 T in the genus Halomonas. These phylogenetic, phenotypic, and chemotaxonomic analyses indicate that QX-1 T is a novel species, for which the name Halomonas maris is proposed. The type strain is QX-1 T (= MCCC 1A17875T = KCTC 82198 T = NBRC 114670 T).

Lipopeptides production by a newly Halomonas venusta strain: Characterization and biotechnological properties.

A halotolerant marine strain PHKT of Halomonas venusta was isolated from contaminated seawater as an efficient biosurfactant producer candidate, on low-value substrate (glycerol). The produced biosurfactants (Bios-PHKT) were characterized as lipopeptides molecules, belonging to surfactin and pumilacidin families, by using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR) and Tandem Mass Spectrometry (MALDI-TOF/MS-MS). Bios-PHKT has a critical micelle concentration (CMC) of 125 mg/L, and showed a high steadiness against a wide spectrum of salinity (0-120 g/L NaCl), temperature (4-121 °C) and pH (2-12), supporting its powerful tensioactive properties under various environmental conditions. Likewise, the cytotoxic test revealed that the biosurfactant Bios-PHKT, at concentrations lower than 125 µg/mL, was not cytotoxic for human HEK-293 cells since the cell survival is over than 80%. Furthermore, Bios-PHKT lipopeptides showed excellent anti-adhesive and anti-biofilm activities, being able to avoid and disrupt the biofilm formation by certain pathogenic microorganisms. In addition, the biosurfactant Bios-PHKT showed a remarkable anti-proliferative activity towards tumor B16 melanoma cell line. Besides, Bios-PHKT exhibited an excellent in vitro and in vivo wound healing process. In light of these promising findings, Bios-PHKT could be successfully used in different biotechnological applications.

Direct binding of benzoate derivatives to two chemoreceptors with Cache sensor domains in Halomonas titanicae KHS3.

Halomonas titanicae KHS3, isolated from a hydrocarbon-contaminated sea harbor in Argentina, is able to grow on aromatic hydrocarbons and displays chemotaxis toward those compounds. This behavior might contribute to the efficiency of its degradation capacity. Using high throughput screening, we identified two chemoreceptors (Htc1 and Htc2) that bind benzoate derivatives and other organic acids. Whereas Htc1 has a high affinity for benzoate (Kd 112 µM) and 2-hydroxybenzoate (Kd 83 µM), Htc2 binds 2-hydroxybenzoate with low affinity (Kd 3.25 mM), and also C3/C4 dicarboxylates. Both chemoreceptors are able to trigger a chemotactic response of E. coli cells to the specific ligands. A H. titanicae htc1 mutant has reduced chemotaxis toward benzoate, and is complemented upon expression of the corresponding receptor. Both chemoreceptors have a Cache-type sensor domain, double (Htc1) or single (Htc2), and their ability to bind aromatic compounds is reported here for the first time.

Polyhydroxyalkanoates (PHA) from Halomonas pacifica ASL10 and Halomonas salifodiane ASL11 isolated from Mariout salt lakes.

Two novel PHA producing bacterial strains were chosen out of 12 strains collected from Mariout salt lakes. Analysis of 16srRNA gene sequence of the two new strains revealed 95.38% and 98.78% similarity to that of Halomonas pacifica and Halomonas salifodiane, respectively. A maximum polymer productivity of 6.9 g/l and 7.1 g/l was recorded by ASL10 and ASL11, respectively. Furthermore, a pH of 7 contributed to the highest polymer production for both strains. Interestingly, both ASL10 and ASL11showed a great ability to tolerate salinity up to 17 g/l NaCL. Moreover, both promising isolates were able to degrade crude oil efficiently by degradation percentages of 69.2% and 67.3% for ASL10 and ASL11, respectively. GCMS, FTIR, NMR, XRD and thermal properties were performed for poly (3 HV-co-3HB) characterization.

A newly isolated strain of Halomonas sp. (HA1) exerts anticancer potential via induction of apoptosis and G2/M arrest in hepatocellular carcinoma (HepG2) cell line.

Marine bacterial strains are of great interest for their ability to produce secondary metabolites with anticancer potentials. Isolation, identification, characterization and anticancer activities of isolated bacteria from El-Hamra Lake, Wadi El-Natrun (Egypt) were the objectives of this study. The isolated bacteria were identified as a moderately halophilic alkaliphilic strain. Ethyl acetate extraction was performed and identified by liquid chromatography-mass spectrophotometry (LC-MS-MS) and nuclear magnetic resonance analysis (NMR). Cytotoxicity of the extract was assessed on the HepG2 cell line and normal human peripheral lymphocytes (HPBL) in vitro. Halomonas sp. HA1 extract analyses revealed anticancer potential. Many compounds have been identified including cyclo-(Leu-Leu), cyclo-(Pro-Phe), C17-sphinganine, hexanedioic acid, bis (2-ethylhexyl) ester, surfactin C14 and C15. The extract exhibited an IC50 of 68 ± 1.8 µg/mL and caused marked morphological changes in treated HepG2 cells. For mechanistic anticancer evaluation, 20 and 40 µg/mL of bacterial extract were examined. The up-regulation of apoptosis-related genes' expression, P53, CASP-3, and BAX/BCL-2 at mRNA and protein levels proved the involvement of P53-dependant mitochondrial apoptotic pathway. The anti-proliferative properties were confirmed by significant G2/M cell cycle arrest and PCNA down-regulation in the treated cells. Low cytotoxicity was observed in HPBL compared to HepG2 cells. In conclusion, results suggest that the apoptotic and anti-proliferative effects of Halomonas sp. HA1 extract on HepG2 cells can provide it as a candidate for future pharmaceutical industries.

Structural characterization and functional properties of novel exopolysaccharide from the extremely halotolerant Halomonas elongata S6.

Production of extracellular polysaccharides by halophilic Archaea and Bacteria has been widely reported and the members of the genus Halomonas have been identified as the most potential producers. In the present work, a novel exopolysaccharide (EPS-S6) produced by the extremely halotolerant newly isolated Halomonas elongata strain S6, was characterized. According to the HPAE-PAD results, EPS-S6 was mainly composed of glucosamine, mannose, rhamnose and glucose (1:0.9:0.7:0.3). EPS-S6 was highly negatively charged and its molecular weight was about 270 kDa. Studies on its functional properties showed that EPS-S6 had several potential features. It has noticeable antioxidant activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH•) inhibition and DNA protection, good ability to inhibit and to disrupt pathogenic biofilms, excellent flocculation of kaolin suspension and interesting emulsifying properties at acidic, neutral and basic pH. Therefore, EPS-S6 could have potential biotechnological concern in several fields such as in food, cosmetic and environmental industries.

Affinity tag effect on the salt rejection potential of Halomonas elongata aquaporin incorporated in thin film nanocomposite membrane.

In this study, effect of affinity tags, Histidine (His) and Glutathione-S-Transferase (GST), on the activity of halophilic aquaporin was analyzed. The gene coding for H. elongata aquaporin was cloned into pET28a vector and expressed in E. coli BL21 successfully. Stopped flow light scattering measurements showed that His-tagged aquaporin is functional. The difference in the filtration parameters caused by affinity tags were determined by using thin film composite nano-filtration (NFC) membranes prepared with the aquaporins. At 100 mM salt concentration, water permeability (L/m2.h) and the % salt rejection of NFC membranes produced with the His-tagged aquaporin was found to be higher than that of the membrane with GST-tagged aquaporin. Salt rejection of His-tagged aquaporin-membrane was found to be 53% with a lower solute permeability value (B). Use of short affinity tag (His tag) for cloning resulted in higher solute rejection ability of TFC membranes prepared with H. elongata aquaporins.

Primary purification of intracellular Halomonas salina ectoine using ionic liquids-based aqueous biphasic system.

Ectoine is a zwitterionic amino acid derivative that can be naturally sourced from halophilic microorganisms. The increasing demands of ectoine in various industries have urged the researches on the cost-effective approaches on production of ectoine. Ionic liquids-based aqueous biphasic system (ILABS) was applied to recover Halomonas salina ectoine from cells hydrolysate. The 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 was used in the ILABS and the recovery efficiency of ILABS to recover ectoine from H. salina cells lysate was evaluated by determining the effects of phase composition; pHs; crude loading and additional neutral salt (NaCl). The hydrophilic ectoine was targeted to partition to the hydrophilic salt-rich phase. A total yield (YB) of 96.32% ± 1.08 of ectoine was obtained with ILABS of phase composition of 20% (w/w) (Bmim)BF4 and 30% (w/w) sulfate salts; system pH of 5.5 when the 20% (w/w) of crude feedstock was applied to the ILABS. There was no significant enhancement on the ectoine recovery efficiency using the ILABS when NaCl was added, therefore the ILABS composition without the additional neutral salt was recommended for the primary purification of ectoine. Partition coefficient (KE) of 30.80 ± 0.42, purity (PE) of 95.82% and enrichment factor (Ef) of 1.92 were recorded with the optimum (Bmim)BF4/sulfate ILABS. These findings have provided an insight on the feasibility of recovery of intracellular biomolecules using the green solvent-based aqueous system in one single-step operation.

Preparation, characterization and functional properties of a novel exopolysaccharide produced by the halophilic strain Halomonas saliphila LCB169T.

A novel exopolysaccharide, designated hsEPS, was successfully prepared from the high-salt-fermented broth of a novel species Halomonas saliphila LCB169T by ethanol precipitation, anion-exchange and gel-filtration chromatography, and its structure was well-characterized by means of chemical and spectral analyses. Results showed that hsEPS was primarily composed of mannose and glucose with a relative weight-average molecular weight of 5.133 × 104 g/mol. It was deduced that the major backbone contained (1→2)-linked α-D-Manp and (1→6)-linked α-D-Manp with branches substituted at C-2 by T-α-D-Manp and at C-6 by the fragment of T-α-D-Manp-(1→2)-α-D-Manp-(1→. A sheet-like structure was observed under high magnification. The water solubility index, water holding capacity, oil holding capacity and foaming capacity of hsEPS were 98.0, 19.3, 1386.7 and 82.2%, respectively. It also exhibited outstanding emulsifying activity against all tested edible oils. Together, the resulted data indicated that hsEPS might serve as an active ingredient in food, cosmetics and detergents.

Highly Efficient Capture of Marine Microbial Strains in Seawater Using Bare Fe3O4 Magnetic Beads.

We develop a method to capture marine bacterial strains at high efficiency to replace the conventional two-step collecting method. Lab-made, Fe3O4 magnetic beads were used to firstly verify the feasibility of capture in artificial seawater, using Bacillus velezensis. Almost 100% of the bacteria could be captured and separated within 10 min. Then, the salinity of capture medium was proved to have the most marked effect on the capture process. After that, the broad application and high efficiency of capture were verified using four different bacterial strains from the Pacific Ocean. Subsequently, through adjusting the salinity, the capture efficiency for Pseudoalteromonas sp. and Halomonas meridiana was increased from 20 to ~ 80% in a seawater system, which was used to simulate the in-situ capture conditions. Finally, mixed strains in seawater were successfully captured, and their genomic DNAs were isolated and analyzed. Bare Fe3O4 magnetic beads were initially applied to capture marine microorganisms and this method is convenient and highly efficient and thus has great potential to replace the conventional two-step method.

Halomonas sambharensis sp. nov., a Moderately Halophilic Bacterium Isolated from the Saltern Crystallizer Ponds of the Sambhar Salt Lake in India.

Two moderately halophilic strains SBS 10T and SSO 06 were isolated from the saltern crystallizer ponds of the hypersaline Sambhar Salt Lake in India. Strains were aerobic, Gram-stain-negative, and rod shaped. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that two strains belong to the genus Halomonas in the Gammaproteobacteria, with highest 16S rRNA gene sequence similarities with Halomonas gudaonensis LMG 23610T (98.2% similarity) and Halomonas campaniensis 5AGT (99.0% similarity). Strains grew optimally at 37 °C, pH 7.5-8.0 in the presence of 5-8% (w/v) NaCl. The major fatty acids of the strain SBS 10T were C18:1ω7c (54.37%), C16:0 (25.69%), C16:1 × 7c/C16:1 × 6c (13.28%), and C12:0 (1.21%). The G+C content was 63.6 mol % (Tm). Phenotypic features, fatty acids profile, and DNA G+C content supported placement of the strain SBS 10T in the genus Halomonas having distinct characteristics with related strains. Analysis of the housekeeping genes: gryB and rpoD and in silico DNA-DNA hybridization between the strain SBS 10T and its type strain Halomonas gudaonensis (LMG 23610T) further revealed the strain SBS 10T to be a distinct species. On the basis of the phenotypic, chemotaxonomic and phylogenetic analysis, the strain SBS 10T is considered to represent a novel species for which the name Halomonas sambharensis is proposed. The type strain is SBS 10T (= MTCC 12313T = LMG 30344T).

Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere.

The synthesis of bioplastic from marine microbes has a great attendance in the realm of biotechnological applications for sustainable eco-management. This study aims to isolate novel strains of poly-ß-hydroxybutyrate (PHB)-producing bacteria from the mangrove rhizosphere, Red Sea, Saudi Arabia, and to characterize the extracted polymer. The efficient marine bacterial isolates were identified by the phylogenetic analysis of the 16S rRNA genes as Tamlana crocina, Bacillus aquimaris, Erythrobacter aquimaris, and Halomonas halophila. The optimization of PHB accumulation by E. aquimaris was achieved at 120 h, pH 8.0, 35 °C, and 2% NaCl, using glucose and peptone as the best carbon and nitrogen sources at a C:N ratio of 9.2:1. The characterization of the extracted biopolymer by Fourier-transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR), and Gas chromatography-mass spectrometry (GC-MS) proves the presence of hydroxyl, methyl, methylene, methine, and ester carbonyl groups, as well as derivative products of butanoic acid, that confirmed the structure of the polymer as PHB. This is the first report on E. aquimaris as a PHB producer, which promoted the hypothesis that marine rhizospheric bacteria were a new area of research for the production of biopolymers of commercial value.

Engineering biosynthesis of polyhydroxyalkanoates (PHA) for diversity and cost reduction.

PHA, a family of natural biopolymers aiming to replace non-degradable plastics for short-term usages, has been developed to include various structures such as short-chain-length (scl) and medium-chain-length (mcl) monomers as well as their copolymers. However, PHA market has been grown slowly since 1980s due to limited variety with good mechanical properties and the high production cost. Here, we review most updated strategies or approaches including metabolic engineering, synthetic biology and morphology engineering on expanding PHA diversity, reducing production cost and enhancing PHA production. The extremophilic Halomonas spp. are taken as examples to show the feasibility and challenges to develop next generation industrial biotechnology (NGIB) for producing PHA more competitively.

Production and characterisation of a marine Halomonas surface-active exopolymer.

During screening for novel emulsifiers and surfactants, a marine gammaproteobacterium, Halomonas sp. MCTG39a, was isolated and selected for its production of an extracellular emulsifying agent, P39a. This polymer was produced by the new isolate during growth in a modified Zobell's 2216 medium amended with 1% glucose, and was extractable by cold ethanol precipitation. Chemical, chromatographic and nuclear magnetic resonance spectroscopic analysis confirmed P39a to be a high-molecular-weight (~ 261,000 g/mol) glycoprotein composed of carbohydrate (17.2%) and protein (36.4%). The polymer exhibited high emulsifying activities against a range of oil substrates that included straight-chain aliphatics, mono- and alkyl- aromatics and cycloparaffins. In general, higher emulsification values were measured under low (0.1 M PBS) compared to high (synthetic seawater) ionic strength conditions, indicating that low ionic strength is more favourable for emulsification by the P39a polymer. However, as observed with other bacterial emulsifying agents, the polymer emulsified some aromatic hydrocarbon species, as well as refined and crude oils, more effectively under high ionic strength conditions, which we posit could be due to steric adsorption to these substrates as may be conferred by the protein fraction of the polymer. Furthermore, the polymer effected a positive influence on the degradation of phenanthrene by other marine bacteria, such as the specialist PAH-degrader Polycyclovorans algicola. Collectively, based on the ability of this Halomonas high-molecular-weight glycoprotein to emulsify a range of pure hydrocarbon species, as well as refined and crude oils, it shows promise for the bioremediation of contaminated sites.

Halomonas borealis sp. nov. and Halomonas niordiana sp. nov., two new species isolated from seawater.

Two Gram-negative strains obtained from tank water in a scallop hatchery in Norway, were phenotypically and genotypically characterized in order to clarify their taxonomic position. On the basis of 16S rRNA gene sequence analysis, these isolates, ATF 5.2T and ATF 5.4T, were included in the genus Halomonas, being their closest relatives H. smyrnensis and H. taeanensis, with similarities of 98.9% and 97.7%, respectively. Sequence analysis of the housekeeping genes atpA, ftsZ, gyrA, gyrB, mreB, rpoB, rpoD, rpoE, rpoH, rpoN and rpoS clearly differentiated the isolates from the currently described Halomonas species, and the phylogenetic analysis using concatenated sequences of these genes located them in two robust and independent branches. DNA-DNA hybridization (eDDH) percentage, together with average nucleotide identity (ANI), were calculated using the complete genome sequences of the strains, and demonstrate that the isolates constitute two new species of Halomonas, for which the names of Halomonas borealis sp. nov. and Halomonas niordiana sp. nov. are proposed, with type strains ATF 5.2T (=CECT 9780T=LMG 31367T) and ATF 5.4T (=CECT 9779T=LMG 31227T), respectively.

Isolation of Gram-positive Firmibacteria as major eicosapentaenoic acid producers from subtropical marine sediments.

In this study, a total of 172 putative omega-3 producers were isolated from 28 sediment samples from the Arabian Gulf employing a selective isolation procedure using marine agar containing 0·1% triphenyl tetrazolium chloride (TTC). Out of these 172 isolates, 19 isolates produced eicosapentaenoic acid (EPA) as confirmed by Gas Chromatography-Mass Spectrometry (GC-MS). The EPA content of the isolated bacterial strain varied from 1·76 to 6·52% of total fatty acids. Among the 19 isolates of EPA producers, while 17 isolates harboured both pfaA gene and Δ6 desaturase gene, only five isolates harboured Δ5 desaturase gene. Two of the EPA positive strains harbour none of the three genes tested. The 16s RNA identification of these isolates revealed that except one, all the EPA producers were Gram-positive marine bacteria belonging to the phylum Firmicutes, family Bacillacea, genera Bacillus and Oceanobacillus. Halomonas pacifica was the only Gram-negative Gamma-Proteobacteria detected to produce EPA from this region. SIGNIFICANCE AND IMPACT OF THE STUDY: Recently, marine bacteria are considered as a promising source of polyunsaturated fatty acid (PUFA) over marine fishes and microalgae. PUFA producers reported from polar and deep-sea sources were restricted to five well-known marine genera under two distinct domains of bacteria such as proteobacteria (Shewanella, Colwellia, and Moritella) and cytophaga group (Flexibacter, Psychroflexus). This study revealed that subtropical marine environment could also be the source of PUFA producing bacteria, and they predominantly belonged to the class of Firmibacteria. This finding opens up new avenue for research to study the inherent mechanism and physiology of such organisms from this unique environment.

Adsorptive removal of bisphenol A from aqueous solutions using phosphonated levan.

In this study, the potential use of phosphonated Halomonas Levan (PhHL) as a natural and cost effective adsorbent for Bisphenol A (BPA), was systematically investigated via the study of the adsorption equilibrium, kinetics, and reuse potential as well as the interpretation of adsorption mechanism. The effects of pH and temperature on the adsorption were also evaluated. The maximum amount of BPA adsorbed on the unit weight of PhHL was determined as 104.8 (∓5.02) mg/g (at 298 K) and the maximum adsorption capacity was calculated as 126.6 mg/g by Sips model. FTIR and XPS studies were conducted to elucidate the adsorption mechanism. Based on the obtained results OH-pi and CH-pi interactions were found to be effective in the adsorption mechanism. The reuse ability was studied with three cycles of adsorption-desorption, and the results showed that the BPA adsorbed per gram of the PhHL decreased 28.6% after the third cycle. This study has shown that PhHL can be used as an effective adsorbent for the removal of BPA from aqueous solutions. The obtained results may be useful in the development of PhHL based adsorption systems for the removal of EDCs with similar chemical properties to BPA.

Highly Efficient Fluorescent Material Based on Rare-Earth-Modified Polyhydroxyalkanoates.

Fluorescent materials play an important role in biomedical fields. However, the main types of fluorescent materials suffer from several disadvantages especially the biotoxicity, which largely restrict its wider applications in biological fields. In this study, a highly efficient rare-earth-modified fluorescent material was successfully designed and fabricated based on polyhydroxyalkanoates, which are known as biodegradable and biocompatible materials. A new Functional-PHA polymer was microbially synthesized by engineered Halomonas bluephagenesis and was used as a basal matrix to generate the rare-earth-modified PHA. N-Acetyl-l-cysteine-grafted PHA (NAL-grafted-PHA) was first produced via a UV-initiated thiol-ene click reaction and the rare earth metal ions (Eu3+ and Tb3+) were subsequently chelated onto the NAL-grafted-PHA through the coordination effect. The composite material exhibited intense photoluminescence properties under UV laser excitation, indicating the excellent features as fluorescent material. The enhanced hydrophilicity and superior biocompatibility of rare-earth-chelated PHA were confirmed, suggesting its great potential application value in biomedical fields.

Structure of the O-specific polysaccharide from a halophilic bacterium Halomonas ventosae RU5S2EL.

Halomonas ventosae RU5S2EL, a halophilic Gram-negative bacterium isolated from salt sediments of lake Elton (Russia), was cultivated and the lipopolysaccharide was extracted by the Westphal procedure. The O-specific polysaccharide (OPS) was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments as well as Smith degradation. The OPS was found to consist of branched pentasaccharide repeating units of the following structure.