Marine Agar Research Articles

First report of seaweed-associated yeast from Indonesia: Species composition and screening of their polysaccharides-degrading enzymes

Abstract. Sibero MT, Frederick EH, Sabdono A, Wijayanti DP, Pringgenies D, Radjasa OK, Zilda DS, Murwani R. 2022. First report of seaweed-associated yeast from Indonesia: Species composition and screening of their polysaccharides-degrading enzymes. Biodiversitas 23: 1408-1419. Yeast has been widely utilized in various industries due to its enzyme properties. Therefore, plenty of studies focus on exploring yeast biodiversity from diverse sources. However, there are limited reports regarding marine yeast biodiversity and its potential from Indonesia. This study aimed to isolate and identify the seaweed-associated yeast from Jepara in Central Java and Sepanjang Beach in Gunung Kidul, Yogyakarta, then examined their potential to produce extracellular polysaccharides-degrading enzymes (EPEs). Marine yeasts were isolated using standard marine agar (STD) and potato dextrose agar (PDA). All isolates were characterized by their salinity tolerance, morphology, and species confirmation using DNA barcoding. Three EPEs consisting of agarase, alginate-lyase, and carrageenase were screened using polysaccharides-enriched agar media. A specific agarase encoding gene was detected with specific primers. In total, 21 seaweed-associated yeast were successfully isolated from 6 seaweeds and noted as facultative marine yeast. The DNA barcoding study discovered that these yeasts belonged to 5 genera, consisted of Aureobasidium, Candida, Debaryomyces, Hortaea, and Rhodotorula. Moreover, Candida was noted as the most abundant genus (71.42%). It was noted that Hortaea werneckii MTJ.11 and MTJ.13 were positive for all enzymes; Aureobasidium melanogenum MTGK.31 gave a positive result for agarase and carrageenase; while [Candida] zeylanoides MTGK.23 only exhibited alginate-lyase activity. Unfortunately, none of the primers used to detect the presence of genes encoding polysaccharide-degrading enzymes were successfully amplified.

Pacificimonas pallium sp. nov., an Isolated Bacterium from the Mantle of Pacific Oyster Crassostrea gigas in Germany, and Prediction of One-Carbon Metabolism

A yellow bacterium from marine agar, strain WHA3T, was isolated from the mantel of the Pacific oysters Crassostrea gigas in the Wilhelmshaven Sea in northern Germany. Based on the 16S rRNA gene sequence, strain WHA3T had a high similarity to Pacificimonas flava JLT2015T (95.80%) and 94.79% to Pacificimonas aurantium JLT2012T. Furthermore, the dDDH and ANI value analysis between WHA3T and other closest type strains were lower than 70% and 95%, respectively. The percentage of conserved proteins (POCP) and the average amino acid identity (AAI) value against Pacificimonas flava JLT2015T and Pacificimonas aurantium JLT2012T represented in the ranges of higher than 50% and 60%, respectively. Strain WHA3T contained ubiquinone-10 (Q-10) as the predominant quinone, and the major fatty acids were C16:1 ω7c and C18:1 ω7c. Granules of polyhydroxyalkanoates (PHAs) were absent. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, several sphingoglycolipids, an unknown phospholipid, an unknown glycolipid, and an unknown polar lipid. The polyamines contained spermidine and spermine. The DNA G + C content of strain WHA3T was 61.69%. An analysis of the whole-genome sequence in the frame of genome mining strain WHA3T predicted the presence of genomes for one-carbon metabolism, TonB-dependent transporters, vitamin B12 transporter, iron siderophore receptor protein, and other genes, some of which play important roles against restricted nutrient sources. The extract of strain WHA3T moderately inhibited the growth of Candida albicans DSM 1665. The polyphasic taxonomic analysis results suggested that strain WHA3T could be separated from its closest type strains. Strain WHA3T represents a novel species in the genus Pacificimonas, for which we propose the name Pacificimonaspallium sp. nov., with the type strain WHA3T (= DSM 111825T = NCCB 100832T).

Bioprospecting of bacterial symbionts of sponge Spongia officinalis from Savu Sea, Indonesia for antibacterial potential against multidrug-resistant bacteria

Abstract. Prastiyanto ME, Kartika AI, Darmawati S, Radjasa OK. 2022. Bioprospecting of bacterial symbionts of sponge Spongia officinalis from Savu Sea, East Nusa Tenggara, Indonesia for antibacterial potential against multidrug-resistant bacteria. Biodiversitas 23: 1118-1124. Marine sponge Spongia sp. has been reported to have potential as an antibacterial agent. However, less information on the potential of bacterial symbionts of Spongia sp. as an antibacterial agent has been documented. The present investigation involves isolating bacterial symbionts of sponge Spongia officinalis and the characterization of antibacterial potential against multidrug-resistant bacteria isolated from clinical specimens. Spongia officinalis was collected from Savu Sea, East Nusa Tenggara, Indonesia and its symbionts were isolated with Zobell marine agar media. The overlay method was used to screen the antibacterial activity against selected six MDR bacteria. Antibacterial activity was determined by measuring the diameter of the inhibition zone. Identification of active bacterial symbionts was carried out based on the 16S rRNA gene sequencing. The results revealed that four out of 10 symbionts showed antibacterial activity against MDR bacteria with an inhibition index ranging from 4.8 to 12.6 mm. Prastiyanto-1A and Prastiyanto-1E isolates demonstrated antibacterial activity against ESBL- Escherichia coli and ESBL + CRE- Klebsiella pneumoniae subsp. pneumoniae, Pratiyanto-2A isolate showed antibacterial activity against MRSA, while Prastiyanto-4A isolates showed antibacterial activity against CRPA. The selected four isolates were identified as Bacillus subtilis, Bacillus mojavensis and Bacillus simplex using 16S rRNA gene sequencing and BLASTn analysis. These results provide information about the potential of bacterial symbionts of S. officinalis as natural antibacterial sources against MDR bacteria.

Screening, Characterization, and Isolation of Pigments from Bacteria in Mesophotic Depths of the Benham Bank Seamount, Philippine Rise Region

The exploration for pigment-producing bacteria and structurally novel pigment continues to increase, and the marine environment has recently become an attractive research site for these investigations because of its rich yet untapped biodiversity. In this study, 16S rRNA gene sequencing and HPLC (high-performance liquid chromatography) profiling of the microbial pigments produced by marine heterotrophic bacteria were described. Seven phenotypically distinct isolates of marine pigmented heterotrophic bacteria (MPHB) were isolated from near-bottom waters and coral reef sediments at mesophotic depths of the Benham Bank Seamount – namely, isolates BR14, BR61, BR63, BR100, BR101, BR144, and BR146. Four promising isolates were subjected to 16S rRNA gene sequencing and revealed that strains BR61 and BR100 were related to members of the genus Cytobacillus, whereas isolates BR144 and BR146 clustered with Meridianimaribacter flavus and Pseudoalteromonas rubra, respectively. Growth of the seven isolates on three media – natural sea water (NSW) Reasoner’s 2A (R2A), marine agar (MA), and tryptic soy agar – revealed variable pigment production and growth yield. MA appeared to be a superior substrate, resulting in darker pigmentation and higher biomass yield. Through a liquid-liquid partitioning approach, isolates BR14 and BR100 produced pigments that were acetone-soluble, whereas isolates BR101, BR144, and BR146 are hexane soluble. Moreover, the BR146 red pigment was proposed to be a mixture of putative prodiginine analogs. The putative prodiginines produced by isolate BR146 can stain fabrics, supporting a proof-of-concept that marine bacteria can be utilized as fabric colorants. The data presented here provided new insights into the utilization of local Philippine marine microbial resources for natural marine pigments with industrial applications.

Histidinibacterium aquaticum sp. nov., Isolated from Salt-Field Sea Water.

A Gram stain-negative, aerobic, motile, and rod-shaped bacterium designated 176SS1-4T was isolated from the salt-field sea water. Strain 176SS1-4T grew well at 25°C on marine agar. The taxonomic affiliation of the novel isolate was identified using the polyphasic approach. By comparative 16S rRNA gene sequence, it could be shown that strain 176SS1-4T branches within the family Rhodobacteraceae and class Alphaproteobacteria and is related to Histidinibacterium lentulum B17T (96.0% sequence similarity). The digital DNA-DNA hybridization and average nucleotide identity between strain 176SS1-4T and H. lentulum B17T was 19.2% and 77%. The genome comprises of 3,905,029bp with a G + C content of 67.7mol. Ubiquinone 10 (Q-10) was the major respiratory quinone. The major fatty acids were summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), C16:0, C19:0 cyclo ω8c, C18:0, and C18:1 ω6c 11-methyl and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylcholine. On the basis of the genotypic and phenotypic characteristics, strain 176SS1-4T can be placed as a novel species within the genus Histidinibacterium; the name Histidinibacterium aquaticum sp. nov. has been proposed, with type strain 176SS1-4T (= KACC 19891T = LMG 31032T).

Mongoliitalea daihaiensis sp. nov., isolated from Daihai Lake in Inner Mongolia.

A novel Gram-negative strain, designated X100-76T, was isolated from Daihai Lake in Inner Mongolia, Republic of China. The strain was non-motile, non-spore-forming, long-rod-shaped, oxidase positive and catalase positive. Colonies incubated at 33°C on 2216 marine agar medium for 3days were circular, smooth, transparent, convex with clear edges, orange-red in colour and approximately 1.0mm in diameter. Growth occurred at pH 6.5-12 (optimum pH 7.5), in 0-5.0% (w/v) NaCl (optimum 1.5%, w/v) and at 4-40°C (optimum 28-33°C). Phylogenetic analysis based on 16S rRNA gene sequences showed that X100-76T belongs to the genus Mongoliitalea and is most closely related to Mongoliitalea lutea MIM18T with 98.3% sequence similarity. The total genome size of X100-76T was 4,816,617bp with a G + C content of 39.6%. The digital DNA-DNA hybridization and average nucleotide identity values between strain X100-76T and M. lutea MIM18T were both below the recommended cut-off values. Chemotaxonomic analysis revealed iso-C15:0, Sum Feature 4 (anteiso-C17:1 B and/or iso-C17:1 I), C15:1ω6c, Sum Feature 9 (C16:0 10-methyl and/or iso-C17:1ω9c), C16:0 and iso-C15:1 G as the major fatty acids, menaquinone MK-7 as the major isoprenoid quinone, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol as the major cellular polar lipids. The results of polyphasic analysis demonstrated that X100-76T represents a novel species within the genus Mongoliitalea, for which the name Mongoliitalea daihaiensis sp. nov. is proposed. The type strain is X100-76T (= CGMCC 1.18762T = KCTC 82458T).

Marine bacterium Seonamhaeicola algicola strain CC1 as a potential source for the antioxidant carotenoid, zeaxanthin

Currently, there are only six species in the genus Seonamhaeicola, i.e., Seonamhaeicola aphaedonensis, S. algicola, S. marinus, S. acroporae, S. maritimus, and S. sediminis. These bacteria have typical yellow or orange color. Among the identified strains, only S. marinus that had been reported to have a yellow polyene flexirubin pigment. However, the presence of carotenoid pigments has not been reported in this genus. Recently, we successfully isolated a new strain, S. algicola strain CC1, bacterium that was found in association with a red seaweed, Halymenia sp., collected from the coast of South Malang, Indonesia. The strain was grown well in the Zobell marine agar 2216E producing yellowish pigments. According to the 16S rRNA sequencing analysis and BLAST search, the strain is closely related to S. algicola strain Gy8, with 99.78% identity. The pigment composition was separated and analyzed by a high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and the strain was found to produce zeaxanthin as the major component, which appeared at a retention time (tR) of 28.89 min, showing a typical mass spectrum with a molecular ion at m/z 568.5 [M]+ and four product ions at m/z 261.4 [M−307]+, 476.6 [M−92]+, 429.3 [M−139]+, and 536.5 [M− 32]+. Other carotenoids, including zeaxanthin cis isomers, β-cryptoxanthin, β-carotene cis isomer, and β-carotene, are as minor components. The novel and noteworthy finding of this report is the identification of a Seonamhaeicola species that produces carotenoids and can be used as a source of zeaxanthin.

Abundance of Culturable Marine Heterotrophic Bacteria in Ulva lactuca Associated with Farmed Seaweeds Kappaphycus spp. and Eucheuma denticulatum

Ulva lactuca is one of the macro-epiphytes of farmed seaweeds Kappaphycus spp. and Eucheuma denticulatum in Tawi-Tawi, southern Philippines, especially during the summer season, which affects the growth and health of farmed seaweeds. In this study, the abundance of culturable heterotrophic marine bacteria from U. lactuca associated with farmed seaweeds Kappaphycus spp. and E. denticulatum was investigated in the seaweed farms of Tongsibalo, Sibutu, Tawi-Tawi, southern Philippines, using serial dilution procedure. Results revealed that the average bacterial counts obtained from U. lactuca associated with Kappaphycus alvarezii, K. striatus, and E. denticulatum were 2.48 x 1010 CFU g-1, 1.14 x 1012 CFU g-1, and 1.32 x 1011 CFU g-1, respectively. In addition, agar-digesting bacteria were observed from U. lactuca samples associated with K. alvarezii and K. striatus manifested by the depression and liquefaction of the marine agar after 2-3 days which were suspected as pathogenic bacteria causing ice-ice disease. Therefore, U. lactuca may serve as a vector for these potential pathogens to farmed seaweeds.

Shallow Hydrothermal Vent Bacteria and Their Secondary Metabolites with a Particular Focus on Bacillus.

Extreme environments are hostile for most organisms, but such habitats represent suitable settings to be inhabited by specialized microorganisms. A marine shallow-water hydrothermal vent field is located offshore in northeast Taiwan, near the shallow shore of the southeast of Kueishantao Island (121°55′ E, 24°50′ N). Research on extremophilic microorganisms makes use of the biotechnological potential associated with such microorganisms and their cellular products. With the notion that extremophiles are capable of surviving in extreme environments, it is assumed that their metabolites are adapted to function optimally under such conditions. As extremophiles, they need specific culture conditions, and only a fraction of species from the original samples are recovered in culture. We used different non-selective and selective media to isolate bacterial species associated with the hydrothermal vent crab Xenograpsus testudinatus and the sediments of its habitat. The highest number of colonies was obtained from Zobell marine agar plates with an overall number of 29 genetically distinct isolates. 16S RNA gene sequencing using the Sanger sequencing method revealed that most of the bacterial species belonged to the phylum Firmicutes and the class Bacilli. The present study indicates that hydrothermal vent bacteria and their secondary metabolites may play an important role for the reconstruction of the evolutionary history of the phylum Procaryota.

Characterization of active lead molecules from Lissocarinus orbicularis with potential antimicrobial resistance inhibition properties

BackgroundMarine organisms are the potential contributors of novel bioactive molecules. Nevertheless, their biodiversity and the versatility of bioactive metabolites have not been fully explored. Hence, the aim of the present study was to investigate the potentials of gut associated bacteria from a marine crab for the production of novel antibacterial compound. MethodsAerobic gut autochthonous bacteria isolated from marine crab (Lissocarinus orbicularis) collected from Pazhayar coastal area in Nagapattinam district of Tamil Nadu, India were screened for antibacterial activity. Optimization for bacterial growth and antimicrobial compound production, extraction, purification and characterization were studied. ResultsIn the present study, eight morphologically distinct colonies of L. orbicularis gut associated aerobic bacterial isolates (Iso1-Iso8) on Zobell marine agar plate were selected. Isolates were screened for antimicrobial activity against human bacterial pathogens such as Salmonella paratyphi, Vibrio cholera, Vibrio parahaemolyticus, Aeromonas hydrophila and Listeria monocytogenes. On the basis of screening results, isolate 5 (Iso5) was selected as the most potential strain and identified as Paenibacillus polymyxa using biochemical and 16S rRNA sequencing methods. The sequence data was submitted to NCBI (Gene bank Accession No: MK583465). Optimization of P. polymyxa for growth and antimicrobial compound production revealed incubation period (36 h), agitation (150 rpm), pH 8.0, 35 °C, 2.5% salinity, 2% glucose and 1% yeast extract as carbon and nitrogen sources respectively were the ideal conditions and mass culture was done with these parameters. Antimicrobial compound from the cell free supernatant of mass culture medium was extracted using ethanol. The lowest minimum inhibitory concentration (MIC) of 16 μg/ml was observed against of both V. parahaemolyticus and V. cholerae. GC–MS analysis of the active ethanol fraction showed the presence of different components such as dodecane (96.72%), Tridecane (1.69%), Undecane, 2,6-dimethyl- (1.69%), Tetradecane (1.12%) and Dodecane, 2,6,11-trimethyl- (1.12%). ConclusionThe present study showed that the gut associated autochthonous bacteria of marine crabs are one of the potential sources of antibacterial compound. However, further studies are needed for the identification of the antimicrobial compound.

Characterization and Modeling of Thermostable GH50 Agarases from Microbulbifer elongatus PORT2

Viewing the considerable potential of marine agar as a source for the sustainable production of energy as well as nature-derived pharmaceutics, this work investigated the catalytic activity of three novel GH50 agarases from the mesophilic marine bacterium Microbulbifer elongatus PORT2 isolated from Indonesian coastal seawaters. The GH50 agarases AgaA50, AgaB50, and AgaC50 were identified through genome analysis; the corresponding genes were cloned and expressed in Escherichia coli BL21 (DE3). All recombinant agarases hydrolyzed β-p-nitrophenyl galactopyranoside, indicating β-glycosidase characteristics. AgaA50 and AgaB50 were able to cleave diverse natural agar species derived from Indonesian agarophytes, indicating a promising tolerance of these enzymes for substrate modifications. All three GH50 agarases degraded agarose, albeit with remarkable diversity in their catalytic activity and mode of action. AgaA50 and AgaC50 exerted exolytic activity releasing differently sized neoagarobioses, while AgaB50 showed additional endolytic activity in dependence on the substrate size. Surprisingly, AgaA50 and AgaB50 revealed considerable thermostability, retaining over 75% activity after 1-h incubation at 50 °C. Considering the thermal properties of agar, this makes these enzymes promising candidates for industrial processing.

Gephyromycinifex aptenodytis gen. nov., sp. nov., isolated from gut of Antarctic emperor penguin Aptenodytes forsteri.

A novel actinobacterium NJES-13T was isolated from the gut of Antarctic emperor penguin Aptenodytes forsteri. The new isolate produces bioactive gephyromycin metabolites and exopolysaccharides (EPS). Cells were Gram-negative, motile with the peritrichous flagella, and with a faint layer of extracellular slime. Colonies were yellow when grown on marine agar, ISP1, 2, 4 and TSAmedia. The strain developed clusters of coccoid, anddivided by binary fission in the early phase of growth. The cell clusters were gradually disrupted during the stationary phase and formed short rod-shape cells which were interconnected by viscous EPS showing a three-dimensional net-like morphology, and contained polyhydroxyalkanoates (PHA)granules inside the cells. Growth of strain NJES-13T was observed at 15-45°C, at pH 6.0-9.0 with 0.5-9.0% (w/v) NaCl. The complete genomic size of strainNJES-13T was 3.45Mb with a DNA G + C content of 67.0mol%. The combined polyphasic taxonomic characterizations presented in this study unequivocally separated strain NJES-13T from all known genera in the family Dermatophilaceae. Thus, strain NJES-13T represents a novel species of a new genus, for which the name Gephyromycinifex aptenodytis gen. nov., and sp. nov. is proposed. The type strain is NJES-13T (= CCTCC 2019007T = KCTC 49281T). Genetic prediction of secondary metabolite biosynthesis revealed a 44.5kb-long biosynthetic gene cluster (BGC) of type III polyketide synthase (PKS)as well as four other BGCs, indicating its great potential to produce novel bioactive metabolites derived from the gut microbiota of animals living in the extreme habitats in the Antarctica.

Marinomonas vulgaris sp. nov., a marine bacterium isolated from seawater in a coastal intertidal zone of Zhoushan island.

A Marinomonas-like, Gram-stain-negative, strictly aerobic and rod to ovoid-shaped bacterium, designated as strain A79T, was isolated from the seawater mixtures of oyster shells and brown algae in a coastal intertidal zone of Zhoushan, China. The strain was positive for oxidase and catalase. Colonies grown on marine agar for 48h were round, milky white, smooth and moist with the diameter of 2-3mm. Growth was observed at 15-30°C (optimum, 25℃), pH 5.5-9.5 (optimum, pH 8.5) and with 0.5-8% (w/v) NaCl (optimum, 2-2.5%). The G + C content based on the genome sequence was 46.0%. The only respiratory quinone was Q-8. The main polar lipids contained phosphatidylglycerol, phosphatidylethanolamine, unidentified glycolipids, unidentified phospholipid and three unidentified lipids. The major fatty acids (> 10%) were C16:0, Summed feature 3 (comprising C16:1 ω6c and/or C16:1 ω7c) and summed feature 8 (comprising C18:1 ω6c and/or C18:1 ω7c). The 16S rRNA gene sequence similarity between strain A79T and Marinomonas pollencensis IVIA-Po-185T was 97.4%, the similarities with other type strains of the genus Marinomonas were 93.8-96.7%. Based on the results, Marinomonas vulgaris sp. nov. was proposed as a novel species. The type strain is A79T (= MCCC 1K05799T = KCTC 82519T = JCM 34473T).

Halomonas azerica sp. nov., Isolated from Urmia Lake in Iran.

A novel moderately halophilic, Gram-staining negative and facultative aerobic bacterium, designated as TBZ9T, was isolated from water of Urmia Lake in Azerbaijan region of Iran. The cells were found to be rod-shaped and motile, growing in the form of creamy, convex and shiny colonies. The strain could grow in the presence of NaCl at concentrations 1-17% (w/v) (optimum, 3%), temperatures 10-40°C (optimum, 30°C) and pH 6.0-11.0 (optimum, pH 7.0) on marine agar. Strain TBZ9T 16S rRNA gene sequence was related to the genus Halomonas showing highest similarities to Halomonas arcis AJ282T (98.4%), Halomonas songnenensis NEAU-ST10-39T (98.0%) and Halomonas lutescens Q1UT (97.8%). In the phylogenetic trees, strain TBZ9T formed a distinct branch closely related to a subclade inside the Halomonas genus. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strain TBZ9T and H. arcis AJ282T (20.0%, 74.0%) and H. songnenensis NEAU-ST10-39T (19.8%, 75.2%) indicated that TBZ9T represents a distinct species. Evaluation of fatty acid contents determined C10:0, C16:0, C12:0 3-OH, summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) as major cellular fatty acids. The major quinone of strain TBZ9T was Q-9. Polar lipid patterns consisted of phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unidentified phospholipids (PL) and four unidentified polar lipids (L). The average DNA G + C content of strain TBZ9T is 55.4mol%. Results from phenotypic, chemotaxonomic and molecular analysis suggest that the strain TBZ9T represents a novel species within the genus Halomonas for which the name Halomonas azerica sp. nov. is proposed. The type strain is TBZ9T (= KACC 21783T = LMG 25775T).

Combining Culture-Dependent and Culture-Independent Methods: New Methodology Insight on the Vibrio Community of Ruditapes philippinarum.

Vibrios represent a natural contaminant of seafood products. V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus are the most hazardous species to human health. Given the worldwide consumption of mollusc products, reliable detection of Vibrio species is recommended to prevent human vibriosis. In this study, culture-dependent and -independent methods were compared and integrated to implement knowledge of the Manila clam Vibrio community composition. Here, 16S and recA-pyrH metabarcoding were applied to compare the microbial communities of homogenate clam samples (culture-independent method) and their culture-derived samples plated on three different media (culture-dependent method). In addition, a subset of plated clam samples was investigated using shotgun metagenomics. Homogenate metabarcoding characterized the most abundant taxa (16S) and Vibrio species (recA-pyrH). Culture-dependent metabarcoding detected the cultivable taxa, including rare species. Moreover, marine agar medium was found to be a useful substrate for the recovery of several Vibrio species, including the main human pathogenic ones. The culture-dependent shotgun metagenomics detected all the main human pathogenic Vibrio species and a higher number of vibrios with respect to the recA-pyrH metabarcoding. The study revealed that integration of culture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity.

The Characterization of Stenotrophomonas maltophilia Isolated from Marine Sponge Producing Salt Tolerant Proteases

Protease is an important industrial enzyme and salt tolerant protease in which has desirable properties that could enhance its uses not only in industries, but also in agriculture and environmental. Marine organisms usually harbour halophilic microorganisms which produce salt tolerant protease. In this study, salt tolerant protease producing bacteria from marine sponge were isolated and screened on skim milk marine agar supplemented with different NaCl concentrations (1.5 % w/v). Out of 11 isolates, 8 isolates (S1-1, S1-2, S1-3, S2-1, S2-3, S2-4, S2-5 and S2-6) showed clearing zones with ability to digest casein on the skim milk agar. Morphologically, these strains are gram negative bacilli which grow in yellow colonies and were found to be catalase-positive but oxidase-negative. They are also non-lactose fermenter that produce gelatinase but not α-amylase. The ribosomal 16S rRNA sequencing was used to identify each isolate (Acc. number of S1-1 for MT645770, S1-2 for MT645 771, S1-3 for MT645 772, S2-1 MT645 773, S2-3 MT645 774, S2-4 MT645 775, S2-5 MT645 776 and S2-6 MT645 7767). The 16S rRNA sequences showed that these isolates were highly similar to Stenotrophomonas maltophilia (S2-6, 99.87%) and to a related strain Pseudomonas hibiscicola (S1-1, S1-2, S1-3, S2-1, S2-3, S2-4, S2-5 (97.16-99.9 %). Further proteolytic studies were carried out using skim milk agar with 1.0 %, 2.0 %, 3.0 % and 4.o % (w/v) of NaCl concentrations. All isolates were able to hydrolyze casein which produced clear zones surrounding each colony at 1.0 % and 2.0 % (w/v) salt. However, only isolate S1-5 and S2-6 showed proteolytic activities at 3.0 % (w/v) salt but none of them at 4.0 % (w/v). The ability of these isolates to produce protease which active at higher salt may indicate their potential to be the sources for enzyme with useful properties.

Aestuariibaculum sediminum sp. nov., a marine bacterium isolated from a tidal flat in Zhoushan.

A Gram-staining-negative, non-motile, strictly aerobic bacterium, designated as strain TT11T, was isolated from a sediment sample of a tidal flat connected in Zhoushan, China. Cells of strain TT11T are spherical, halotolerant, catalase- and oxidase-positive, and produce carotenoid-like pigments. Colonies were 0.5-1.0mm diameter, smooth, round, convex and orange-yellow after growth on marine agar at 30°C for 24h. Growth of the strain TT11T was observed at 10-40°C (optimum, 35°C), at pH 6.0-9.5 (optimum, pH 6.5), and in the presence of 0-8.0% (w/v) NaCl (optimum, 0.5-1.0%). The results of 16S rRNA gene sequence analysis revealed that strain TT11T represents a member of the genus Aestuariibaculum and was closely related to Aestuariibaculum suncheonense SC17T (97.2%) and Aestuariibaculum marinum IP7T (96.8%). The G + C content of the genome was 34.6%. The only respiratory quinone was MK-6. The major fatty acids (> 10%) were iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH. The major polar lipids contained phosphatidylethanolamine, phosphoglycolipid, four unidentified aminolipids, four unidentified lipids and two unidentified glycolipids. On the basis of these genomic, chemotaxonomic and phenotypic characteristics, we propose a novel species Aestuariibaculum sediminum sp. nov. with the type strain TT11T (= KCTC 82195T = MCCC 1K04734T).

Environment variables, composition and metabolic characteristics of culturable sediment bacteria isolated around Gökçeada Island, Aegean Sea, Turkey

Determination of bacteriological diversity in marine ecosystems is of great importance in terms of understanding the functioning of the marine ecosystem, as well as being an indicator of alarm for pollution and sudden changes in marine environments. In this study, we aimed to investigate the composition and metabolic characteristics, relative to variable environmental parameters in the sediment sampling points, of cultivable heterotrophic bacteria taken seasonally from the coastal areas of Gökçeada Island, Turkey between February and November of 2015. The counts of the total cultivable heterotrophic aerobic bacteria (HAB) were tested by using the spread plate technique with Marine agar. The living and dead bacteria counts (TB) were tested with an epifluorescent microscope by means of the DAPI–CTCstaining method. The enzyme activation and metabolic responses of the bacterial isolates were screened against certain substrates by using the VITEK 2 Compact 30 automated micro-identification system. Variable environmental parameters were recorded in-situ in the seawater samples surrounding the sediment samples with multiparameter instrument. The spatial variability of trophic parameter values was tested by spectrophotometric methods. The mean HAB and TB counts of sediment samples were recorded as 35x109±0.5 and 79x1010±0.9 CFU g−1, respectively. During the study period, sixteen bacteria species belonging to six classes. Bacilli (6.0%), Gammaproteobacteria (71.1%), Betaproteobacteria (3.6%), Actinobacteria (1.2%), Cocci (1.2%), and Alphaproteobacteria (16.9 %) were recorded. Arylamidase was determined to be the highest enzyme activity. These results showed that nitrogen mineralization occurs at a high rate in the sediment samples of Gökçeada Island. Temporal and spatial variations in total and heterotrophic bacteria levels between seasons were significant (p<0.05) in the study area. There were no significant station variations between TB and HAB levels (p>0.05). It was determined that trophic parameters related to primary environmental conditions affect bacterial composition, frequency, and metabolites in coastal areas.

Marine actinomycetes from Malaysia marine environments with antagonistic potential

Marine actinomycetes have garnered the attention of researchers worldwide due to their ability to produce unique and novel bioactive compounds. This study aimed at assessing the diversity and antagonistic potential of marine actinomycetes isolated from Langkawi Island, Kedah and Tioman Island, Johor. In a total of 215 isolates were successfully recovered of which 52% were obtained from Starch–yeast Extract Agar (SYE), 31.4% from Marine Agar (MA) and 16.3% from Actinomycetes Isolation Agar. Fourteen isolates were found to be positive of Polyketide Synthase Type I (PKS-I) and/or Non-ribosomal Peptide Synthetases (NRPS) genes and these isolates were then subjected to antimicrobial evaluation test against 3 Gram-positive bacteria – Staphylococcus aureus, Methicillinresistant S. aureus, Bacillus subtilis; 4 Gram-negative bacteria – Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens; and 2 fungi – Candida albicans, Candida parapsilosis. Most isolates were able to inhibit at least one test microorganism. There is, however, no direct correlation between the presence of the biosynthetic genes and antagonistic potential as demonstrated by the lack of antimicrobial activity though PKS-I and/or NRPS genes were present. Molecular identification using the 16S rRNA gene revealed 10 isolates belonging to the genus Streptomyces and each representative of the genera Actinomadura, Rhodococcus, Gordonia and Salinispora. Among the isolates, Streptomyces sp. T55 is a potent antibacterial actinomycete while Streptomyces sp. T109 is a good antifungal agent. Further characterization of marine actinomycetes was conducted using scanning electron microscopy, culture characteristics on several media (SYE, MA, Potato Dextrose Agar, ISP2, ISP3, ISP4, Czapek Agar), NaCl tolerance and carbon sources utilization profile using BIOLOG. Findings from this study demonstrate the potential of Malaysia’s marine environment as a new resource of actinomycetes with biosynthetic capabilities which can be further explored for the development of the natural product.

&lt;p&gt;&lt;strong&gt;Isolation, screening and identification of amylase and catalase producing bacterial strains from marine sediments&lt;/strong&gt;&lt;/p&gt;

Marine sediments are valuable source of industrially useful enzymes. Here, we attempted isolation, screening and identification of bacterial strains from marine sediments which produce industrially important enzymes amylase and catalase. Marine sediment samples were collected and cultured on zobell marine agar medium. After incubation, the isolates that showed amylase and catalase activity were selected for the assay. The strains AM01 and Ca07 showed the highest amylase and catalase activity, respectively. The selected strains were further sequenced for identification. Morphological studies indicated that the isolates were Gram -ve, rod shaped and non-motile organism. The phenotypic characterization and 16S rRNA of the strains AM01 and Ca07 revealed them to be Klebsiella pneumoniae and K. quasipneumoniae, respectively