Antifouling activities of marine bacteria associated with sponge (Sigmadocia sp.)

The present study aimed at assessing the antifouling activity of bacteria associated with marine sponges. A total of eight bacterial strains were isolated from the surface of sponge Sigmadocia sp., of them, SS02, SS05 and SS06 showed inhibitory activity against biofilm-forming bacteria. The extracts of these 3 strains considerably affected the extracellular polymeric substance producing ability and adhesion of biofilm-forming bacterial strains. In addition to disc diffusion assay, microalgal settlement assay was carried out with the extracts mixed with polyurethane wood polish and coated onto stainless steel coupons. The extract of strain SS05 showed strong microalgal settlement inhibitory activity. Strain SS05 was identified as Bacillus cereus based on its 16S rRNA gene. Metabolites of the bacterial strains associated with marine invertebrates promise to be developed into environment-friendly antifouling agents.     

Inhibition of norsolorinic acid accumulation to Aspergillus parasiticus by marine actinomycetes

Thirty-six strains of marine actinomycetes were isolated from a sample of marine sediment collected from the Yellow Sea and evaluated in terms of their inhibitory activity on the growth of Aspergillus parasiticus and the production of norsolorinic acid using dual culture plate assay and agar diffusion methods. Among them, three strains showed strong antifungal activity and were subsequently identified as Streptomyces sp. by 16S rRNA gene sequencing analysis. The supernatant from the fermentation of the MA01 strain was extracted sequentially with chloroform and ethyl acetate, and the activities of the extracts were determined by tip culture assay. The assay results show that both extracts inhibited mycelium growth and toxin production, and the inhibitory activities of the extracts increased as their concentrations increased. The results of this study suggest that marine actinomycetes are biologically important for the control of mycotoxins, and that these bacteria could be used as novel biopesticides against mycotoxins.     

SCREENING FOR ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES IN SOME MARINE ALGAE FROM THE FUJIAN COAST OF CHINA WITH THREE DIFFERENT SOLVENTS

Three different solvents viz ethanol, acetone and methanol-toluene (3:1) were used to extract antibiotics from 23 species of marine algae belonging to the Chlomphyta, Phaeophyta and Rhodophyta. Their crude extracts were tested for antibacterial and antifungal activities. Among them, the ethanol extract showed the strongest activity against the bacteria and fungi tested. Four species of the Rhodophyta (Laurenc/a okamurai, Dasya scoparia, Grateloupia filicina and plocamium telfairiae ) showed a wide spectrum of antibacterial activity. Every solvent extract from the four species was active against all the bacteria tested. The test bacterium Pseudomonas solancearum and the fungus Penicilium citrinum were most sensitive to the extracts of marine algae. In general, the extracts of seaweeds inhibited bacteria more strongly than fungi and species of the Rhodophyta showed the greatest activity against the bacteria and fungi tested.     

Screening of marine fungus from Nanji Island and activity of their metabolites against pathogenic Vibrio from Pseudosciaena crocea

Seventy-eight marine fungal strains were isolated from sediment samples collected off the coast of Nanji Island,Wenzhou,Zhejiang Province,China.Antibacterial screening using the agar disc method showed that 19 of the isolated strains could inhibit at least one pathogenic Vibrio from Pseudosciaena crocea.Subsequent screening confirmed that nine strains produced antibacterial metabolites that had activity against one or several types of pathogenic Vibrio.Strain NJ0104 had the widest antimicrobial spectrum and strong activity,particularly against Vibrio parahaemolyticus-MM0810072.A preliminary study of NJ0104 antibacterial metabolites demonstrated that they had thermal stability up to 80°C,ultraviolet stability up to 40 min and pH stability between 4.0-7.0.In addition,the antibacterial metabolites were readily soluble in butanol.To identify the specific strain,the ITS-5.8S rDNA regions of NJ0104 were PCR amplified and sequenced.Based on the combination of phenotypic and genotypic data,the strain was identified as Arthrinium sp.     

Chemical constituents of marine mangrove-derived endophytic fungus Alternaria tenuissima EN-192

A chemical investigation of the ethyl acetate extract of the fermentation broth of Alternaria tenuissima EN-192,an endophytic fungus obtained from the stems of the marine mangrove plant Rhizophora stylosa,resulted in the isolation of nine known secondary metabolites,including four indole-diterpenoids:penijanthine A(1),paspaline(2),paspalinine(3),and penitrem A(4);three tricycloalternarene derivatives:tricycloalternarene 3a(5),tricycloalternarene 1b(6),and tricycloalternarene 2b(7);and two alternariol congeners:djalonensone(8) and alternariol(9).The chemical structures of these metabolites were characterized through a combination of detailed spectroscopic analyses and their comparison with reports from the literature.The inhibitory activities of each isolated compound against four bacteria were evaluated and compounds 5 and 8 displayed moderate activity against the aquaculture pathogenic bacterium Vibrio anguillarum,with inhibition zone diameters of 8 and 9 mm,respectively,at 100 μg/disk.To the best of our knowledge,this is the first report on the secondary metabolites of mangrove-derived Alternaria tenuissima and also the first report of the isolation of indole-diterpenoids from fungal genus Alternaria.     

Antifouling effect of bioactive compounds from selected marine organisms in the Obhur Creek,Red Sea

Three species of sponges and a tunicate were collected from Obhur creek of Jeddah coast for this bioactivity study. In order to assess the antifouling efficacy of selected marine organisms, methanolic extracts of these organisms were tested against different fouling bacterial forms and II-instar stage of the barnacle, Balanus amphitrite. Antibiosis, bioactivity and followed by multivariate analyses were carried out to check the efficacy of antifouling effect of the selected marine organisms. Principal component analysis revealed the exemplary antifouling efficacy of the sponge extracts of Stylissa sp. observed followed by Hyrtios sp. against bacterial forms in the laboratory study. De-trended correspondence analysis confirmed that the contribution of antifouling efficacy of the selected sponge extracts was observed to be more towards Bacillus sp., Vibrio sp. and Alteromonas sp. Moreover, the efficacy of Hyrtios sp. extract(20.430 μg m L~(-1)) followed by Stylissa sp.(30.945 μg m L~(-1)) showed higher against barnacle instar compared with other extracts in the bioactivity assay. Bray-Curtis cluster analysis under paired linkage categorized all the sponge extracts into one major cluster with 75% similarity, and one outlier tunicate. More than 80% similarity observed between Hyrtios sp. and Stylissa sp. Fourier transform infrared spectroscopy(FTIR) showed that the contribution of major peaks found in the marine organisms were towards sulfones, sulfoxides, cyanates and ketones.     

A bacterial pathogen infecting gametophytes of Saccharina japonica (Laminariales, Phaeophyceae)

A newly identified bacterial disease of kelp(Saccharina japonica) gametophytes was found in clone cultures.It is characterized by swollen gametophyte cells in the early period of infection followed by filamentous fading.An alginolytic marine bacterium referred to as A-1 was isolated from the diseased gametophytes.On the basis of 16S rDNA sequencing and morphological,physiological and biochemical characteristics,the bacterium was identified as a strain of the genus Alteromonas.By testing Koch’s postulates,Alteromonas sp.A-1 was further confirmed as the pathogen.The infection process was also investigated using both scanning electron and light microscopy.     

Antialgal and Antilarval Activities of Bioactive Compounds Extracted from the Marine Dinoflagellate Amphidinium carterae

With the global ban on the application of organotin-based marine coatings by the International Maritime Organization, the development of environmentally friendly, low-toxic and nontoxic antifouling compounds for marine industries has become an urgent need. Marine microorganisms have been considered as a potential source of natural antifoulants. In this study, the antifouling potential of marine dinoflagellate Amphidinium carterae, the toxic and red-tide microalgae, was investigated. We performed a series of operations to extract the bioactive substances from Amphidinium carterae and tested their antialgal and antilarval activities. The crude extract of Amphidinium carterae showed significant antialgal activity and the EC50 value against Skeletonema costatum was 55.4 μg m L~(-1). After purification, the isolated bioactive substances(the organic extract C) exhibited much higher antialgal and antilarval activities with EC50 of 12.9 μg m L~(-1) against Skeletonema costatum and LC50 of 15.1 μg m L~(-1) against Amphibalanus amphitrite larvae. Subsequently, IR, Q-TOFMS, and GC-MS were utilized for the structural elucidation of the bioactive compounds, and a series of unsaturated and saturated 16- to 22-carbon fatty acids were detected. The data suggested the bioactive compounds isolated from Amphidinium carterae exhibited a significant inhibiting effect against the diatom Skeletonema costatum and Amphibalanus amphitrite larvae, and could be substitutes for persistent, toxic antifouling compounds.     

Characterization of halophilic C50 carotenoid-producing archaea isolated from solar saltworks in Bohai Bay,China

Halophilic archaea comprise the majority of microorganisms found in hypersaline environments. C50 carotenoids accumulated in archaea cells are considered potential biotechnological products and possess a number of biological functions. Ten red colordes were isolated from brine water in a saltem crystaltizer pond of the Hangu Saltworks, China. 16S rRNA gene sequence analysis showed that the colonies belonged to the extremely halophilic archaea genera Halobacterium and Halorubrum. Two representative strains, Halobacterium strain SP-2 and Halorubrum strain SP-4, were selected for further study on the phenotypic characteristics and effects of salinity and pH on accumulation and composition of pigments in their cells. The archaeal strains were isolated and grown in a culture medium prepared by dissolving yeast extract （10 g/L） and acid-hydrolyzed casein （7.5 g/L） into brine water obtained from a I.ocal salt pond. Their optimum salinity and pH for growth were 250 and 7, respectively, although pigment accumulation （OD490/ mL broth） was highest at pH 8. In addition, at 150-300 salinity, increasing salinity resulted in decreasing pigment accumulation. Analysis of the UV-Vis spectrum, TLC and HLPC chromatograms showed that C50 carotenoid bacterioruberin is the major pigment in both strains.     

Isolation and identification of a bacterium from marine shrimp digestive tract: A new degrader of starch and protein

It is a practical approach to select candidate probiotic bacterial stains on the basis of their special traits. Production of digestive enzyme was used as a trait to select a candidate probiotic bacterial strain in this study. In order to select a bacterium with the ability to degrade both starch and protein, an ideal bacterial strain STE was isolated from marine shrimp (Litopenaeus vannamei) intestines by using multiple selective media.The selected isolate STE was identified on the basis of its morphological, physiological,and biochemical characteristics as well as molecular analyses. Results of degradation experiments confirmed the ability of the selected isolate to degrade both starch and casein. The isolate STE was aerobic, Gram-negative, rod-shaped, motile and non-spore-forming, and had catalase and oxidase activities but no glucose fermentation activity. Among the tested carbon/nitrogen sources, only Tween40, alanyl-glycine, aspartyl-glycine, and glycyl-l-glutamic acid were utilized by the isolate STE. Results of homology comparison analyses of the 16S rDNA sequences showed that the isolate STE had a high similarity to several Pseudoalteromonas species and, in the phylogenetic tree, grouped with P. ruthenica with maximum bootstrap support (100%). In conclusion, the isolate STE was characterized as a novel strain belonging to the genus Pseudoalteromonas.This study provides a further example of a probiotic bacterial strain with specific characteristics isolated from the host gastrointestinal tract.     

Enhanced carotenoid production by a mutant of the marine yeast Rhodotorula sp. hidai

After a serial of UV, EMS and NTG mutagenesis, a mutant named MM of the red marine yeast strain Rhodotorula sp. hidai was obtained. The mutant MM could produce 603.93 μg g⁻¹ of carotenoid within 5 days in the medium containing 4.0 g sucrose, 1.5 g yeast extract, 0.1 g MgSO₄, and 100 mL of sea water, with pH 6.0 and at 30 °C, while only 213.18 μg g⁻¹ of carotenoid was produced by the wild type under the same conditions.     

Optimization of culture conditions and medium composition for the marine algicidal bacterium Alteromonas sp. DH46 by uniform design

Harmful algal blooms (HABs) have led to extensive ecological and environmental issues and huge economic losses. Various HAB control techniques have been developed, and biological methods have been paid more attention. Algicidal bacteria is a general designation for bacteria which inhibit algal growth in a direct or indirect manner, and kill or damage the algal cells. A metabolite which is strongly toxic to the dinoflagellate Alexandrium tamarense was produced by strain DH46 of the alga-lysing bacterium Alteromonas sp. The culture conditions were optimized using a single-factor test method. Factors including carbon source, nitrogen source, temperature, initial pH value, rotational speed and salinity were studied. The results showed that the cultivation of the bacteria at 28°C and 180 r min^?1 with initial pH 7 and 30 salt contcentration favored both the cell growth and the lysing effect of strain DH46. The optimal medium composition for strain DH46 was determined by means of uniform design experimentation, and the most important components influencing the cell density were tryptone, yeast extract, soluble starch, NaNO₃ and MgSO₄. When the following culture medium was used (tryptone 14.0g, yeast extract 1.63g, soluble starch 5.0 g, NaNO₃ 1.6 g, MgSO₄ 2.3 g in 1L), the largest bacterial dry weight (7.36 g L^?1) was obtained, which was an enhancement of 107% compared to the initial medium; and the algal lysis rate was as high as 98.4% which increased nearly 10% after optimization.     

Purification and characterization of 2-haloacid dehalogenase from marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis

2-haloacid dehalogenases constitute a group of dehalogenases which are capable of dehalogenating the halogenated organic compounds. So far, the 2-haloacid dehalogenases have been found in many bacteria, but not in Paracoccus genus. In the present study, one enzyme 2-haloacid dehalogenase(designated as Deh99), induced by DL-2-chloropropionate(DL-2-CPA), was purified from the marine bacterium Paracoccus sp. DEH99, isolated from marine sponge Hymeniacidon perlevis. The enzyme of Deh99 was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography(Q-Sepharose HP), and Superdex 200 gel filtration chromatography. The molecular weight of Deh99 was estimated to be 25.0 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and 50.0 kDa natively by gel filtration chromatography. The enzyme of Deh99 stereospecifically dehalogenated L-2-CPA to produce D-lactate, with an apparent Michaelis-Menten constant(Km) value of 0.21 mmol L-1 for L-2-CPA. The optimal pH and temperature for Deh99 activity were 10.0 and 40℃, respectively. The enzyme of Deh99 acted on short-carbon-chain 2-haloacids, with the highest activity towards monochloroacetate. The activity of Deh99 was slightly affected by DTT and EDTA, but strongly inhibited by Cu2+ and Zn2+. The enzyme of Deh99 shows unique substrate specificity and inhibitor sensitivities compared to previously characterized 2-haloacid dehalogenases and is the reported one about purified 2-haloacid dehalogenase isolated from the bacteria of Paracoccus genus.     

A polysaccharide-degrading marine bacterium Flammeovirga sp. MY04 and its extracellular agarase system

Bacteria of the genus Flammeovirga can digest complex polysaccharides (CPs), but no details have been reported regarding the CP depolymerases of these bacteria. MY04, an agarolytic marine bacterium isolated from coastal sediments, has been identified as a new member of the genus Flammeovirga. The MY04 strain is able to utilize multiple CPs as a sole carbon source and grows well on agarose, mannan, or xylan. This strain produces high concentrations of extracellular proteins (490 mg L-1 ± 18.2 mg L-1 liquid culture) that exhibit efficient and extensive degradation activities on various polysaccharides, especially agarose. These proteins have an activity of 310 U mg-1 ± 9.6 U mg-1 proteins. The extracellular agarase system (EAS) in the crude extracellular enzymes contains at least four agarose depolymerases, which are with molecular masses of approximately 30-70 kDa. The EAS is stable at a wide range of pH values (6.0-11.0), temperatures (0-50℃), and sodium chloride (NaCl) concentrations (0- 0.9 mol L-1). Two major degradation products generated from agarose by the EAS are identified to be neoagarotetraose and neoagarohexaose, suggesting that β-agarases are the major constituents of the MY04 EAS. These results suggest that the Flammeovirga strain MY04 and its polysac-charide-degradation system hold great promise in industrial applications.     

Antifouling potential of bacteria isolated from a marine biofilm

Marine microorganisms are a new source of natural antifouling compounds. In this study, two bacterial strains, Kytococcus sedentarius QDG-B506 and Bacillus cereus QDG-B509, were isolated from a marine biofilm and identified. The bacteria fermentation broth could exert inhibitory effects on the growth of Skeletonema costatum and barnacle larvae. A procedure was employed to extract and identify the antifouling compounds. Firstly, a toxicity test was conducted by graduated pH and liquid-liquid extraction to determine the optimal extraction conditions. The best extraction conditions were found to be pH 2 and 100% petroleum ether. The EC50 value of the crude extract of K. sedentarius against the test microalgae was 236.7 ± 14.08 μg mL-1, and that of B. cereus was 290.6 ± 27.11 μg mL-1. Secondly, HLB SPE columns were used to purify the two crude extracts. After purification, the antifouling activities of the two extracts significantly increased: the EC50 of the K. sedentarius extract against the test microalgae was 86.4 ± 3.71 μg mL-1, and that of B. cereus was 92.6 ± 1.47 μg mL-1. These results suggest that the metabolites produced by the two bacterial strains are with high antifouling activities and they should be fatty acid compounds. Lastly, GC-MS was used for the structural elucidation of the compounds. The results show that the antifouling compounds produced by the two bacterial strains are myristic, palmitic and octadecanoic acids.     

Marine bacterium strain screening and pyrethroid insecticide-degrading efficiency analysis

A pyrethroid insecticide-degrading bacterium, strain HS-24, was isolated from an offshore seawater environment. The strain, which can degrade cypermethrin(CYP) and deltamethrin(DEL), was identified as Methylophaga sp. The optimal culture and degradation conditions for CYP and DEL by strain HS-24 is pH 7 at 28°C. Under optimum culture conditions, strain HS-24 exhibited a broad degradation concentration range of 100, 200, 400, 600, and 800 mg/L for CYP and DEL. The metabolic intermediates were analyzed by NMR, which provided strong evidence that CYP and DEL removal occurred mainly because of a biological process. The toxicity of the degradation products of strain HS-24 was studied simultaneously by measuring the light output of the luminescence bacterium. This demonstrated that the biodegradation ability of strain HS-24 significantly decreased the toxicity of CYP- and DEL-contaminated aquaculture seawater. Finally, the findings of this paper indicate that strain HS-24 is thus revealed as a biological agent for the remediation of marine aquatic environments.     

Enhanced Carotenoid Production by a Mutant of the Marine Yeast Rhodotorula sp. hidai

1 Introduction In recent years, carotenoids have received increasing attention as they have extensive use in medicine, cosmetic, chemistry, food industry and feed industry. In industry, carotenoid and astaxanthin can be used as the additives of food or feeds. Carotenoids can also serve as the precursor of vitamin A in mammals. In recent years, many types of carotenoid have aroused extensive interest because of their many beneficial effects on human health. For in-stance, lycopene and astaxant…