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.     

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.     

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.     

Antimicrobial activities of novel cultivable bacteria isolated from marine sponge Tedania anhelans

Marine sponge Tedania anhelans distributes throughout the intertidal zone of Fujian, southeastern China, and is a potential source of natural bioactive products. The sponge harbors a large number of bacterial groups that have been identified using various techniques, including fluorescent in situ hybridization (FISH). Fractionation of dissociated sponge allowed isolation of 25 bacterial species. Based on 16S rRNA gene sequencing, phylogenetic analysis attributed most of these eubacteria to α-Proteobacteria, γ-Proteobacteria, Cytophaga/Flavobacterium/Bacteroidetes (CFB group), and the family Bacillaceae of Gram-positive bacteria. In sequence similarity, five putatively novel species were identified with less than 98% similarity to other strains in the NCBI database. Tests for antimicrobial activities were performed against Gram-positive bacteria, Gram-negative bacteria, fungi, antitumor indicators Escherichia coli 343/591 (with DNA repair deficiency), regular E. coli 343/636 (with different DNA repair capacity), and 10 bacterial isolates exhibited inhibitory bioactivities. Among these strains, three isolates were detected involving function gene NRPS-A domains, which were most closely related to the amino acid sequences of linear gramicidin synthetase and pyoverdine synthetase. These results contribute to our knowledge of the microbes associated with marine sponges and further reveal novel bacterial resources for the screening of bioactive marine natural products.     

Antibacterial activity of extracts from <Emphasis Type="Italic">Zostera marina</Emphasis> against pathogens of <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> skin ulceration disease

The purpose of this study was to investigate the antibacterial activity of extracts from Zostera marina against the pathogens of Apostichopus japonicus skin ulceration disease. When 95% ethanol (v/v) solvent was used to extract Zostera marina at 50°C, aqueous extract (ZA) showed obvious bacteriostatic effects on the tested bacterial strains (inhibition halo diameters between 8.23 mm and 13.62 mm), whereas the ethyl acetate extract (ZE) was almost inactive. The minimal inhibitory concentration (MIC) of ZA against four pathogens were homogeneous at 12.8 g L⁻¹. ZA components were analyzed by thin layer chromatography (TLC) assay and six fractions were obtained. In another study, the six fractions showed inhibitory effects against the tested bacteria while their functions seemed to counteract the ZA activity.     

Marine antifouling behavior of the surfaces modified by dopamine and antibacterial peptide

Marine biofouling causes serious harms to surfaces of marine devices in transportation, aquaculture, and offshore construction. Traditional antifouling methods pollute the environment. A novel and green antifouling strategy was developed to prevent effectively the adhesion of bacteria and microalgae. An antifouling surface was fabricated via coating Turgencin B_Mox2 (TB) onto dopamine-modified 304 stainless steel (304 SS). The surface physical and chemical properties before and after modification were characterized by Fourier transform infrared spectrometer (FTIR), X-ray photoelectron spectrometer (XPS), contact angle measurement (CA), 3D optical profilometer, ellipsometer, and atomic force microscope (AFM). Antimicrobial peptide was coated onto the surface of 304 SS successfully, and the surface morphology and wettability of the modified sample were modified. Moreover, cytocompatibility of the peptide was evaluated by co-culture of peptide and cells, indicating promising cell biocompatibility at the modified sample surface. At last, antifouling performance and electrochemical corrosion were tested. Results show that the adhesion rates of Vibrio natriegens and Phaeodactylum tricornutum on the antifouling surface were reduced by 99.85% and 67.93%, respectively from those of untreated samples. Therefore, the modified samples retained superior corrosion resistance. The study provide a simply and green way against biofouling on ship hulls and marine equipment.

     

A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations

Axenic microalgal strains are highly valued in diverse microalgal studies and applications. Antibiotics, alone or in combination, are often used to avoid bacterial contamination during microalgal isolation and culture. In our preliminary trials, we found that many microalgae ceased growing in antibiotics at extremely high concentrations but could resume growth quickly when returned to an antibiotics-free liquid medium and formed colonies when spread on a solid medium. We developed a simple and highly efficient method of obtaining axenic microalgal cultures based on this observation. First, microalgal strains of different species or strains were treated with a mixture of ampicillin, gentamycin sulfate, kanamycin, neomycin and streptomycin (each at a concentration of 600 mg/L) for 3 days; they were then transferred to antibiotics-free medium for 5 days; and finally they were spread on solid f/2 media to allow algal colonies to form. With this method, five strains of Nannochloropsis sp. (Eustigmatophyceae), two strains of Cylindrotheca sp. (Bacillariophyceae), two strains of Tetraselmis sp. (Chlorodendrophyceae) and one strain of Amphikrikos sp. (Trebouxiophyceae) were purified successfully. The method shows promise for batch-purifying microalgal cultures.     

Chemical Defense of Soft Coral Sinularia polydactyla from the Red Sea Against Marine Biofilm-Forming Bacteria

Soft corals are an important group of organisms that live in reef ecosystems. In this study, the chemical defense of soft coral Sinularia polydactyla against biofilm-forming bacteria was assessed. The soft coral S. polydactyla was collected from the Obhur creek of the Red Sea and the bioactive compounds were extracted under different conditions using methanol and hexane as solvents. Results revealed that the bioactive compounds produced by the soft coral S. polydactyla were active against the growth, hydrophobicity and extracellular polymeric substances production of the biofilm-forming bacteria. However, the damage or injury in soft coral tissue caused a decrease in the activity against biofilm-forming bacteria. GC-MS analysis revealed that sesquiterpenes were abundant in normal coral sample extract while cembranoids were abundant in damaged coral sample extracts. In general, the results indicated that the soft corals either under stress or with damage may not have a strong chemical defense against the colonizing marine organisms and competitors.     

Inhibitory activity of an extract from a marine bacterium Halomonas sp. HSB07 against the red-tide microalga Gymnodinium sp. （Pyrrophyta）

In recent years, red tides occurred frequently in coastal areas worldwide. Various methods based on the use of clay, copper sulfate, and bacteria have been successful in controlling red tides to some extent. As a new defensive agent, marine microorganisms are important sources of compounds with potent inhibitory bioactivities against red-tide microalgae, such as Gymnodinium sp. （Pyrrophyta）. In this study, we isolated a marine bacterium, HSB07, from seawater collected from Hongsha Bay, Sanya, South China Sea. Based on its 16S rRNA gene sequence and biochemical characteristics, the isolated strain HSB07 was identified as a member of the genus Halomonas. A crude ethyl acetate extract of strain HSB07 showed moderate inhibition activity against Gymnodinium sp. in a bioactive prescreening experiment. The extract was further separated into fractions A, B, and C by silica gel column chromatography. Fractions B and C showed strong inhibition activities against Gymnodinium. This is the first report of inhibitory activity of secondary metabolites of a Halomonas bacterium against a red-tide-causing microalga.     

Contribution of trimethylamine N-oxide on the growth and pressure tolerance of deep-sea bacteria

Trimethylamine N-oxide (TMAO) is widely dispersed in marine environments and plays an important role in the biogeochemical cycle of nitrogen. Diverse marine bacteria utilize TMAO as carbon and nitrogen sources or as electron acceptor in anaerobic respiration. Alteration of respiratory component according to the pressure is a common trait of deep-sea bacteria. Deep-sea bacteria from different genera harbor high hydrostatic pressure (HHP) inducible TMAO reductases that are assumed to be constitutively expressed in the deep-sea piezosphere and facilitating quick reaction to TMAO released from fish which is a potential nutrient for bacterial growth. However, whether deep-sea bacteria universally employ this strategy remains unknown. In this study, 237 bacterial strains affiliated to 23 genera of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria were isolated from seawater, sediment or amphipods collected at different depths. The pressure tolerance and the utilization of TMAO were examined in 74 strains. The results demonstrated no apparent correlation between the depth where the bacteria inhabit and their pressure tolerance, regarding to our samples. Several deep-sea strains from the genera of Alteromonas, Halomonas, Marinobacter, Photobacterium, and Vibrio showed capacity of TMAO utilization, but none of the isolated Acinebacter, Bacillus, Brevundimonas, Muricauda, Novosphingobium, Rheinheimera, Sphingobium and Stenotrophomonas did, indicating the utilization of TMAO is a species-specific feature. Furthermore, we noticed that the ability of TMAO utilization varied among strains of the same species. TMAO has greater impact on the growth of deep-sea isolates of Vibrio neocaledonicus than shallow-water isolates. Taken together, the results describe for the first time the TMAO utilization in deep-sea bacterial strains, and expand our understanding of the physiological characteristic of marine bacteria.

     

Inhibition of Aspergillus parasiticus and cancer cells by marine actinomycete strains

Ten actinomycete strains isolated from the Yellow Sea off China's coasts were identified as belonging to two genera by 16 S r DNA phylogenetic analysis: Streptomyces and Nocardiopsis. Six Streptomyces strains(MA10, 2SHXF01-3, MA35, MA05-2, MA05-2-1 and MA08-1) and one Nocardiopsis strain(MA03) were predicted to have the potential to produce aromatic polyketides based on the analysis of the KSα(ketoacyl-synthase) gene in the type II PKS(polyketides synthase) gene cluster. Four strains(MA03, MA01, MA10 and MA05-2) exhibited significant inhibitory effects on mycelia growth(inhibition rate 50%) and subsequent aflatoxin production(inhibition rate 75%) of the mutant aflatoxigenic Aspergillus parasiticus NFRI-95. The ethyl acetate extracts of the broth of these four strains displayed significant inhibitory effects on mycelia growth, and the IC50 values were calculated(MA03: 0.275 mg m L-1, MA01: 0.106 mg m L-1, MA10: 1.345 mg m L-1 and MA05-2: 1.362 mg m L-1). Five strains(2SHXF01-3, MA03, MA05-2, MA01 and MA08-1) were selected based on their high cytotoxic activities. The ethyl acetate extract of the Nocardiopsis strain MA03 was particularly noted for its high antitumor activity against human carcinomas of the cervix(He La), lung(A549), kidney(Caki-1) and liver(Hep G2)(IC50: 2.890, 1.981, 3.032 and 2.603 μg m L-1, respectively). The extract also remarkably inhibited colony formation of He La cells at an extremely low concentration(0.5 μg m L-1). This study highlights that marine-derived actinomycetes are a huge resource of compounds for the biological control of aflatoxin contamination and the development of novel drugs for human carcinomas.     

Antialgal and antilarval activities of bioactive compounds extracted from the marine dinoflagellate <Emphasis Type="Italic">Amphidinium carterae</Emphasis>

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 mL^?1. After purification, the isolated bioactive substances (the organic extract C) exhibited much higher antialgal and antilarval activities with EC₅₀ of 12.9 μg mL^?1 against Skeletonema costatum and LC₅₀ of 15.1 μg mL^?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.     

Heterotrophic bacterial floras in industrial area and unpolluted marine environments around Qingdao

From Oct. 1999 to Oct. 2000, the heterotrophic bacterial floras in the industrial marine environment around the Qingdao Power Plant (QPP) and in the unpolluted marine environments were investigated. The results showed that the numbers of the heterotrophic bacteria around QPP were much higher than those in unpolluted environments, and the average numbers in QPP Seawater, QPP Sediment, Unpolluted Seawater and Unpolluted Sediment were 5.4×10⁴cfu(mL)⁻¹, 5.0×10⁵cfug⁻¹, 3.0×10²cfu(mL)⁻¹ and 1.3×10⁵cfug⁻¹ respectively. Totally, 118 strains were isolated from QPP and 99 of them were Gram-negative. One hundred and twenty one strains were isolated from the unpolluted environments and 104 of them were Gram-negative. All the Gram-negative bacteria belonged to 13 genera. The distribution of the bacteria was varied in different marine environments. The results showed that the unpolluted marine environments contained much more Vibrio than seawater and sediment around QPP.     

海洋土曲霉C23-3与不同类型海洋微生物共培养对其次生代谢产物的影响

【目的】研究海洋土曲霉C23-3与不同类型海洋微生物共培养对其次生代谢产物的影响,挖掘土曲霉菌株C23-3的次生代谢潜力。【方法】将副溶血弧菌、枯草芽孢杆菌和胶红酵母的活菌或灭活菌体分别以不同时间与土曲霉C23-3共培养;采用DPPH自由基清除法和Ellman法分别评价各提取物的抗氧化和乙酰胆碱酯酶抑制活性,采用滤纸片法比较各提取物的抗菌活性,采用HPLC指纹图谱分析各提取物化学成分的差异。【结果与结论】发现不同类型的微生物与土曲霉共培养均可激活一些原本沉默的活性次级代谢产物;菌株及共培养物的化学拮抗能力、HPLC指纹图谱中的特征代谢产物变化揭示在活菌培养体系中土曲霉占竞争优势;而且灭活菌体中可能存在某些信号物质,在12 d时对土曲霉群体自感应信号分子丁内酯-I的产生具有比活菌更强的刺激作用。研究结果为深入挖掘海洋真菌代谢潜力及抗老年痴呆等活性先导化合物的发现提供基础。     

Response of marine microalgae,heterotrophic bacteria and their relationship to enhanced UV-B radiation

Ozone depletion in the stratosphere has enhanced solar UV-B radiation reaching the Earth surface and has brought about significant effects to marine ecosystems. The effects of enhanced UV-B radiation on marine microalgae, heterotrophic bacteria and the interaction between them are discussed. The effects on marine microalgae have been proved to occur at molecular, cellular and population levels. Enhanced UV-B radiation increases microalgal flavonoid content but decreases their chlorophyll content and pho-tosynthesis rate; this rachation induces genetic change and results in DNA damage and change of protein content. There have been fewer studies on the effects of UV-B radiation on marine heterotrophic bacteria. Establishment of a nucroalgal ecological dynamic model at population and community levels under UV-B radiation has gradually become a hotspot. The effects of enhanced UV-B radiation on microalgae communities, heterotrophic bacterial populations and interaction between them will become a focus in the near future. This paper will make an overview on the studies concerning the effects of enhanced UV-B radiation on marine microal-gae and heterotrophic bacteria and the interaction between them.     

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.     

Rapid, sensitive detection of Vibrio anguillarum using loop-mediated isothermal amplification

Vibrio anguillarum is an important bacterial pathogen of aquatic organisms and a significant problem in aquatic farming. The rapid detection and identification of V. anguillarum, and other pathogens that infect marine organisms, is crucial to effective disease management. In this study, we developed a loop-mediated amplification (LAMP) assay to detect V. anguillarum in an hour in a single tube without the need for thermal cycling. Conserved regions of the metalloproteinase (empA) gene of V. anguillarum served as the targets for primer design. A fragment of the empA gene was amplified at 65°C in the presence of the primer mixture and Bst DNA polymerase. In the optimized LAMP assay, 6.7 pg of V. anguillarum DNA could be detected. Six strains of V. anguillarum and 17 strains of non-V. anguillarum bacteria were used in this study to evaluate the species specificity of the primers. The six V. anguillarum strains gave a positive result in the LAMP assay. This method was also validated in V. anguillarum-infected fish. This LAMP method is more sensitive than PCR in the detection of V. anguillarum and shows good species specificity. The LAMP assay is therefore an effective method for the quick detection of V. anguillarum both in the laboratory and in the field.     

16S rRNA gene phylogenesis of culturable predominant bacteria from diseased Apostichopus japonicus (Holothuroidea, Echinodermata)

Cultured Apostichopus japonicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopus japonicus individuals.     

Responses in growth, lipid accumulation, and fatty acid composition of four oleaginous microalgae to different nitrogen sources and concentrations

Nitrogen deficiency is an effective strategy for enhancing lipid production in microalgae. Close relationships exist among lipid production, microalgal species, and nitrogen sources. We report growth, lipid accumulation, and fatty acid composition in four microalgae （Chloroeoccum ellipsoideum UTEX972, Chlorococcum nivale LB2225, Chlorococcum tatrense UTEX2227, and Scenedesmus deserticola JNU19） under nitrate- and urea-nitrogen deficiencies. We found three patterns of response to nitrogen deficiency： Type-A （decrease in biomass and increase in lipid content）, Type-B （reduction in both biomass and lipid content）, and Type-C （enhancement of both biomass and lipid content）. Type-C microalgae are potential candidates for large-scale oil production. Chlorococcum ellipsoideum, for example, exhibited a neutral lipid production of up to 239.6 mg/（L＇d） under urea-nitrogen deficiency. In addition, nitrogen deficiency showed only a slight influence on lipid fractions and fatty acid composition. Our study provides useful information for further screening hyper-lipid microalgal strains for biofuel production.     

Identification of the toxic compounds produced by Sargassum thunbergii to red tide microalgae

The inhibitory effects of methanol extracts from the tissues of three macroalgal species on the growths of three marine red tide microalgae were assessed under laboratory conditions.Extracts of Sargassum thunbergii(Mertens ex Roth) Kuntz tissue had stronger inhibitory effects than those of either Sargassum pallidum(Turner) C.Agardh or Sargassum kjellmanianum Yendo on the growths of Heterosigma akashiwo(Hada) Hada,Skeletonema costatum(Grev.) Grev,and Prorocentrum micans Ehrenberg.Methanol extracts of S.thunbergii were further divided into petroleum ether,ethyl acetate,butanol,and distilled water phases by liquid-liquid fractionation.The petroleum ether and ethyl acetate fractions had strong algicidal effects on the microalgae.Gas chromatography-mass spectrometry analyses of these two phases identified nine fatty acids,most of which were unsaturated fatty acids.In addition,pure compounds of four of the nine unsaturated fatty acids had effective concentrations below 5 mg/L.Therefore,unsaturated fatty acids are a component of the allelochemicals in S.thunbergii tissue.