Halorubrum salinarum sp. nov., an extremely halophilic archaeon isolated from a saturated brine pond of a saltern.

A novel extremely halophilic archaeon, strain RHB-CT, was isolated from a saturated brine pond of a solar saltern in Bolinao, Pangasinan, Philippines. Colonies were orange-red-pigmented, smooth, convex and round on a solid modified growth medium containing 25 % (w/v) of total salts. Cells of strain RHB-CT on the solid modified growth medium were ovoid-shaped (0.89-2.66 µm long), while the cells in a liquid modified growth medium were rod-shaped (1.53-5.65 µm long and 0.45-1.03 µm wide). The strain was Gram-stain-negative, motile and strictly aerobic. Strain RHB-CT grew with NaCl concentrations ranging from 10 to 30 % (w/v; optimum, 20-25 %), at pH 6.5-8.5 (optimum, pH 7.0-7.5) and at 20-55 °C (optimum, 40-45 °C). Furthermore, the strain grew even in the absence of Mg2+; however, when supplemented with Mg2+, growth was observed optimally at 0.2-0.4 M Mg2+. The 16S rRNA gene phylogeny inferred that the strain is a member of the genus Halorubrum and was related to Halorubrum xinjiangense CGMCC 1.3527T (99.0 %), Halorubrum sodomense DSM 3755T (98.8 %), Halorubrum coriense Ch2T (98.8 %), Halorubrum trapanicum NRC 34021T (98.4 %) and Halorubrum distributum JCM 9100T (98.1 %). The rpoB' gene sequences also showed that strain RHB-CT is related to Hrr. xinjiangense JCM 12388T (97.1 %), Hrr. distributum JCM 9100T (97.1 %), Hrr. coriense JCM 9275T (96.5 %), Hrr. californiense JCM 14715T (96.5 %), Hrr. trapanicum JCM 10477T (96.3%), Hrr. sodomense JCM 8880T (96.2%) and Hrr. tebenquichense DSM 14210T (95.6 %). The DNA G+C content of strain RHB-CT was 68.7 mol% (genome). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain RHB-CT and the closely related species of Halorubrum were below 40 and 90 %, respectively, which are far below the thresholds to delineate a new species. The polar lipids of strain RHB-CT were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulphate and sulfated mannosyl glycosyl diether. Based on dDDH and ANI values, and the significant morphological and physiological differences from known taxa, it is hereby suggested that strain RHB-CT represents a novel species of the genus Halorubrum, for which the name Halorubrum salinarum sp. nov. is proposed. The type strain is RHB-CT (=KCTC 4274T=CMS 2103T).

Marine Oomycetes (Halophytophthora and Salispina): A Potential Source of Fatty Acids with Cytotoxic Activity Against Breast Adenocarcinoma Cells (MCF7).

Marine oomycetes are ubiquitous, fungus-like eukaryotes known to produce fatty acids with potential anticancer activity. The long chain omega-3 and omega-6 fatty acids are currently popular and considered as safe when used as nutraceuticals in cancer treatment. In this study, crude fatty acids from three marine oomycetes, Halophytophthora spp. (T12GP1 and T12YBP2) and Salispina hoi (USTCMS 1611), were explored for their cytotoxic and apoptotic potentials against human breast adenocarcinoma cells (MCF7) and normal human dermal fibroblasts (HDFn). Extracts from mycelia mats consisted of diverse saturated, monounsaturated, and polyunsaturated fatty acids such as linoleic, α-linolenic, γ-linolenic, eicosatrienoic and eicosapentaenoic acids. The crude fatty acids from all three oomycetes in in vitro assays for cytotoxicity showed no toxicity (30% toxicity values) on HDFn cells. On MCF7 cells, however, IC50 values of 23.44, 15.63, and 26.15 µg/mL were obtained with extracts from Halophytophthora T12GP1 and T12YBP2 and S. hoi, respectively. Treated MCF7 cells exhibited deformed cell membrane in MTT assay and also aggregation of DNA and disruption of nuclear membrane aggregation in nuclear staining; further, green signals indicative of apoptosis was recorded in caspase 3/7 assay.

Degradation of Polycyclic Aromatic Hydrocarbons by Moderately Halophilic Bacteria from Luzon Salt Beds.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants which are highly toxic due to their carcinogenic and mutagenic effects. They are released into the environment by incomplete combustion of solid and liquid fuels, accidental spillage of oils and seepage from industrial activities. One of the promising processes mitigating PAHs is through biodegradation. However, conventional microbiological treatment processes do not function well at high salt concentrations. Hence, utilization of halophilic bacteria should be considered. OBJECTIVES: This study aimed to assess the ability of halophilic bacteria isolated from local salt beds in Pangasinan and Cavite, the Philippines, to degrade PAHs pyrene, fluorene and fluoranthene. METHODS: Polycyclic aromatic hydrocarbon-tolerant halophilic bacteria collected from two sampling sites were phenotypically characterized, molecularly identified and tested to determine their potential to degrade the PAHs pyrene, fluorene and fluoranthene at a hypersaline condition. Best PAH degraders were then assayed to identify the optimal degradation using such parameters as pH, temperature and PAH concentration. Testing for enzyme degradation was also done to determine their baseline information. Extraction and analysis of degraded PAHs were performed using centrifugation and UV-vis spectrophotometry. RESULTS: Twelve isolates from both collection sites tolerated and grew in culture with selected PAHs. These were identified into four genera (Halobacillus, Halomonas, Chromohalobacter, and Pontibacillus). Selected best isolates in a series of biodegradation assays with the above-mentioned parameters were Halobacillus B (Collection of Microbial Strains (CMS) 1802) (=trueperi) (Gram-positive) for pyrene and fluoranthene, and Halomonas A (CMS 1901) (Gram-negative) for fluorene. Degrader biomass and PAH degradation were invariably negatively correlated. Qualitative tests with and without peptone as a nitrogen source implied enzymatic degradation. DISCUSSION: Polycyclic aromatic hydrocarbons utilized by these halophilic bacteria served as a sole source of carbon and energy. Implications of biodegradation of the two best isolates show that high molecular weight (HMW) (4-ring) pyrene tends to be degraded better by Gram-positive bacteria and low molecular weight (3-ring) fluorene by Gram-negative degraders. CONCLUSIONS: Halophilic bacteria constitute an untapped natural resource for biotechnology in the Philippines. The present study demonstrated their potential use in bioremediation of recalcitrant hydrocarbons in the environment. COMPETING INTERESTS: The authors declare no competing financial interests.

TLC Screening Profile of Secondary Metabolites and Biological Activities of Salisapilia tartarea S1YP1 Isolated from Philippine Mangroves.

The Salisapilia species are estuarine oomycetes of the mangrove and saltmarsh ecosystem. To date, reports on the secondary metabolites and biological activities of these microorganisms are wanting. In this study, secondary metabolites in broth ethyl acetate extracts (BEAE) and mycelial ethyl acetate extracts (MEAE) of Salisapilia tartarea S1YP1 isolated from yellow senescent mangrove leaves were screened by Thin Layer Chromatography (TLC). Extracts were assayed for antioxidant, antibacterial, α- glucosidase inhibition, and cytotoxic activity. The TLC detected anthraquinones, anthrones, flavonoids, phenols, and triterpenes in both BEAE and MEAE. Coumarins were detected in BEAE but not in MEAE. Quantifying the total phenolics and total flavonoids content of the extracts in terms of gallic acid and quercetin equivalents, respectively shows that BEAE has higher total phenolic and flavonoid contents than MEAE. BEAE exhibited significant antioxidant activities through measurements of free radical scavenging activity against DPPH, hydroxyl, nitric oxide, and superoxide anion radicals as well as the ability to chelate Fe2+ metal ion. BEAE significantly inhibited in a dose-dependent manner α-glucosidase activity and selectively inhibited HepG2 cell proliferation. Antioxidant, α- glucosidase inhibitory, and cytotoxic activities have not been observed for MEAE. Both BEAE and MEAE do not have antibacterial activity.