Occurrence and Diversity of Candida Genus in Marine Environments

A total of 317 yeast isolates from seawater,sediments,mud of salterns,guts of marine fishes and marine algae wereobtained.The results of routine identification and molecular characterization showed that six isolates among these marine yeastsbelonged to Candida genus as Candida interrnedia for YA01a,Candida parapsilosis for 3eA2,Candida quercitrusa for JHSb,Can-die rugosa for wl8,Candida zeylanoides for TJY13a,and Candida membranifaciens for W14-3.Isolates YA01a (Candida interme-die),wl8 (Candida rugosa),3eA2 (Candida parapsilosis),and JHSb (Candida quercitrusa) were found producing cell-bound lipase,while isolate W14-3 (Candida membranifaciens) producing riboflavin.These marine yeast Candida spp.Seem to have wide potentialapplications in biotechnology.     

Amylase production by the marine yeast Aureobasidium pullulans N13d

The marine yeast strain N13d, producing an extracellular amylase, was isolated from the deep sea sediments of the Pacific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identification methods. The optimal sea water medium for amylase production by this yeast strain was 1.0% peptone and 1.0% soluble starch with pH 4.0. The optimal conditions for amylase production by this yeast strain were with temperature 28 °C, aeration rate 6 Lmin⁻¹ and agitation speed 250 rmin⁻¹. Under these conditions, 58.5 units of amylase activity per mg protein were produced within 56 h of fermentation.     

Intraspecific diversity of Aureobasidium pullulans strains from different marine environments

Totally more than 500 yeast strains were isolated from seawater, sea sediments, mud of sea salterns, marine fish guts and marine algae. The results of routine and molecular biology identification methods show that nine strains among these marine yeasts belong to Aureobasidium pullulans, although the morphologies of their colonies are very different. The marine yeasts isolated from different marine environments indicate that A. pullulans is widely distributed in different environmental conditions. These Aureobasidium pullulans strains include A. pullulans 4#2, A. pullulans N13d, A. pullulans HN3-11, A. pullulans HN2-3, A. pullulans JHSc, A. pullulans HN4.7, A. pullulans HN5.3, A. pullulans HN6.2 and A. pullulans W13a. A. pullulans 4#2 could produce cellulase and single cell protein. A. pullulans N13d could produce protease, lipase, amylase and cellulase. Both A. pullulans HN3-11 and A. pullulans HN2-3 were able to produce protease, lipase and cellulase. A. pullulans JHSc could secrete cellulase and killer toxin. Both A. pullulans HN4.7 and A. pullulans HN5.3 could yield lipase and cellulase. A. pullulans W13a was able to secrete extracellular amylase and cellulase while A. pullulans HN4.7 and A. pullulans N13d could produce siderophores. This means that different A. pullulans strains from different marine environments have different physiological characteristics, which may be applied in many different biotechnological industries.     

Occurrence and diversity of <Emphasis Type="Italic">Pichia</Emphasis> spp. in marine environments

A total of 328 yeast strains from seawater, sediments, mud of salterns, the guts of marine fish and marine algae were obtained. The results of routine identification and molecular methods show that five yeast strains obtained in this study belonged to Pichia spp., including Pichia guilliermondii 1uv-small, Pichia ohmeri YF04d, Pichia fermentans YF12b, Pichia burtonii YF11A and Pichia anomala YF07b. Further studies revealed that Pichia anomala YF07b could produce killer toxin against pathogenic yeasts in crabs while Pichia guilliermondii 1uv-small could produce high activity of extracellular inulinase. It is advisable to test if Pichia ohmeri YF04d obtained in this study is related to central-venous-catheter-associated infection.     

Optimum production and characterization of an acid protease from marine yeast <Emphasis Type="Italic">Metschnikowia reukaufii</Emphasis> W6b

The marine yeast strain W6b isolated from sediment of the South China Sea was found to produce a cell-bound acid protease. The crude acid protease produced by this marine yeast showed the highest activity at pH 3.5 and 40 °C. The optimal pH and temperature for the crude acid protease were in agreement with those for acid protease produced by the terrestrial yeasts. The optimal medium of the acid protease production was seawater containing 1.0% glucose, 1.5% casein, and 0.5% yeast extract, and the optimal cultivation conditions of the acid protease production were pH 4.0, a temperature of 25 °C and a shaking speed of 140 rmin⁻¹. Under the optimal conditions, 72.5 UmL⁻¹ of acid protease activity could be obtained in cell suspension within 48 h of fermentation at shake flask level. The acid protease production was induced by high-molecular-weight nitrogen sources and repressed by low-molecular-weight nitrogen sources. Skimmed-milk-clotting test showed that the crude acid protease from the cell suspension of the yeast W6b had high skimmed milk coagulability. The acid protease produced by M. reukaufii W6b may have highly potential applications in cheese, food and fermentation industries.     

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.     

Preliminary identification of three new isolates in genus Alexandrium (Dinophyceae) from China sea area

The 5.8S ribosomal DNA sequences (5.8S rDNA) and their flanking regions, internal transcribed spacer 1 and spacer 2 (ITS1 and ITS2) of three new isolates in genus Alexandrium (Alexandrium sp. qd1, Alexandrium sp. qd2, Alexandrium sp. gz) from China were amplified, sequenced, and subjected to phylogenetic analysis. Alexandrium sp. gz and Alexandrium sp. qd1 were grouped with high bootstrap values with four strains/species, i.e., A. catenella South Korea strain, A. catenella Japan strain, and two from China, Alexandrium sp. AC03 and Alexandrium sp. AN01 being proposed to be A. catenalla in a previous study. Then Alexandrium sp. gz and Alexandrium sp. qd1 were identified as Alexandrium catenella. As A. catenella was isolated from Qingdao and Guangzhou sea areas, it supposedly distributed at least in these two areas and was genetically different. Alexandrium sp. qd2 differed greatly from species in Alexandrium. It clustered with Symbiodinium californium, Symbiodinium sp. G15 and Gymnodinium sp. Zhao 01 with 100% bootstrap value; so Alexandrium sp. qd2 affiliates to genus Symbiodinium, and is probably a free-living Symbiodinium species.     

Chemical constituents of marine algal-derived endophytic fungus Exophiala oligosperma EN-21

Seven compounds (1–7) were identified from the cultivation of the endophytic fungus Exophiala oligosperma (EN-21) that was isolated from the inner tissue of the marine red alga Laurencia similis. Their structures were identified with spectroscopic and chemical methods as 2-phenoxynaphthalene (1), (2S, 3R, 4E, 8E)-1-O-β-D-glucopyranosyl-3-hydroxy-2-[(R)-2′-hydroxyoctadecanoyl] amino-9-methyl-4, 8-octadeca-diene (2), (22E,24R)-ergosta-7,22-dien-3β,5α,6β-triol (3), (22E, 24R)-3β, 5α, 9α-trihydroxy- ergosta-7, ...     

Exopolysaccharide Production by Four Cyanobacterial Isolates and Preliminary Identification of These Isolates

Four marine cyanobacterial isolates, named 104, 109, 113 and 115, from marine water off China＇s coast can release a large amount of exopolysaccharide （EPS） to medium. The effects of different components in medium on EPS production by the four isolates were investigated. Under the optimal condition, the EPS released by isolates 104,109, 113 and 115 reached 7.48 g L^-1, 8.33 g L^-1, 18.26 g L^-1, and 6.78 g L^-1 within 14 d,respectively. Based on the conventional identification methods for cyanobacteria,these isolates were assigned to genus Cyanothece.     

Iodine content ofSargassum in Southern China

Sodium thiosulphate and neutron activation determination of iodine content in samples of 18Sargassum species collected in Guangdong and Guangxi Province in April 1996 showed large differences (0.47×10⁻³ inSargassum hemiphyllum, 0.56×10⁻³ inSargassum assimile, and 4.5×10⁻³ inSargassum vachellianum). Traditional titration analysis and neutron activation analysis for determination of total iodine yielded similar results showing that certainSargassum species had high capacity to accumulate iodine. Contribution No. 3162 from the Institute of Oceanology, Chinese Academy of Sciences. The project was supported by the National Ninth Five-Year Plan of China (No. 96-916-04-01).     

Comparative studies of inorganic carbon utilization inEmiliania huxleyi and some non-calcifying marine microalgae

Inorganic carbon utilization in the non-calcifying marine microalgae,Nannochloropsis oculata, Phaeodactylum tricornutum andPorphyridium purpureum was compared with high- and low-calcifying strains ofEmiliania huxleyi grown in artificial seawater medium aerated with either air (0.03% V/V CO₂) or CO₂-free air. For high-calcifying strain ofE. oculata andP. tricornutem, similar growth patterns were observed in air-and CO₂-free air-grown cultures.P. purpureum showed a less final cell density in CO₂-free air than in air-grown culture. However, low-calcifying strain ofE. huxleyi was able to grow only in air-grown culture, but not in CO₂-free air-grown culture. Measurements of alkalinity, pH, concentration of dissolved inorganic carbon (DIC) and free CO₂ showed different patterns of DIC utilization. WithN. oculata, P. tricornutum andP. purpureum the pattern of DIC utilization was characterized by an increase of pH and a decrease of DIC but a constant alkalinity in the cultures aerated with air or CO₂-free air, suggesting that bicarbonate utilization was concomitant with an efflux of OH⁻. Both alkalinity and pH were maintained rather constant in air-grown culture of low-calcifying strain ofE. huxleyi, suggesting that diffusive entry of CO₂ could meet the requirement of DIC for its photosynthesis and growth. High-calcifying strain ofE. huxleyi, however, showed a pattern of decrease of alkalinity and DIC but an almost constant pH, indicating that bicarbonate was the major form of inorganic carbon utilised by this organism and bicarbonate uptake is unlikely to be accompanied by an efflux of OH⁻. The final pH values reached byN. oculata, P. tricornutum andP. purpureum in a closed system were 10.75, 10.60 and 9.85 respectively, showing that bicarbonate utilisation is concomitant with an efflux of OH⁻. While the final pH of 8.4 in high-calcifyingE. huxleyi suggests that bicarbonate utilization was not accompanied by an efflux of OH⁻. Contribution No. 3557 from the Institue of Oceanology, Chinese Academy of Sciences. This work was supported by NERC grant GRE3/7853 U. K. and partly supported by Bio-Engineering Center, SSTC 96-C01-05-01.     

Isolation and pharmacological activities of bromophenols from Rhodomela confervoides

Four bromophenols were isolated from the extract of marine red alga,Rhodomela confervoides by column chromatography and HPLC methods. By means of spectroscopic methods inclding IR, MS, 1D, and 2D-NMR techniques, their structures were elucidated as (1) 3-bromo-4,5-dihydroxy benzoic acid methyl ester; (2) bis (2,3-dibromo-4,5-dihydroxybenzyl) ether; (3) 3,4-dibromo-5-(methoxymethyl)-1,2-benzenediol and (4) 3-bromo-4,5-dihydroxy-benzaldehyde. Compound 1 was first isolated from the algae in nature, and 1, 4 were found to have selective cytotoxic activities against KB, Bel 7402 and A549 cells, 2 showed powerful antibacterial activities againstStaphylococcus aurens andPseudomonas aeruginosa. Supported by National “863” Program (No. 2004AA625030, 2001AA620503), Quingdao marine sciences project (No. 04-2-NN-26) and Key Knowledge Innovative Project of the Chinese Academy of Sciences (KZCX3-SW-215).     

Notes on Two Marine Ciliates from the Yellow Sea,China： Placus salinus and Strombidium apolatum（Protozoa, Ciliophora）

The living morphology and infraciliature of two rare marine ciliates, Placus salinus Dietz, 1964 and Strombidium apolatum Wilbert and Song, 2005, collected from the coastal waters near Qingdao, China, were investigated by in vivo observation and protargol impregnation technique. The improved diagnosis for Placus salinus is as follows: medium-sized marine Placus, in vivo (50–60)μm×(30–40)μm; cell elliptical to barrel-shaped; 28–31 somatic kineties; single macronucleus usually ellipsoid and one micronucleus located in the indention of the macronucleus; one contractile vacuole posteriorly positioned. Strombidium apolatum is characterized by: marine strombidium (40–60)μm×(30–45)μmm in vivo, cordiform in shape with somewhat pointed posterior end and conspicuous apical protrusion; extrusomes prominent, about 15μm in length and evenly arranged along the circle kinety; about 16 collar and 5–6 buccal membranelles; one elongate macronucleus and one micronucleus; circle and ventral kineties consisting of about 53 and 45 dikinetids respectively.     

The selection of alkaline protease-producing yeasts fi＇om marine environments and evaluation of their bioactive peptide production

A total of 400 yeast strains from seawater, sediments, saltern mud, marine fish guts, and marine algae were obtained. The protease activity of the yeast cultures was estimated, after which four strains (HN3.11, N11b, YF04C and HN4.9) capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, Issatchenkia orientalis and Cryptococcus cf. aureus. The optimal pH of the protease activity produced by strains HN3.11, YF04C, and HN4.9 was 9.0, while that of the protease produced by strain N11b was 10.0. The optimal temperature for protease activity was 45°C for strains HN3.11, N11b, and YF04C, and 50°C for strain HN4.9. After digestion of shrimp (Penaeus vannamei) protein and spirulina (Arthospira platensis) protein with the four crude alkaline proteases, the filtrate from spirulina (Arthrospira platensis) powder digested by the crude alkaline protease of strain HN3.11 was found to have the highest antioxidant activity (61.4%) and the highest angiotensin I converting enzyme (ACE)-inhibitory activities (68.4%). The other filtrates had much lower antioxidant activity and ACE-inhibitory activities.     

Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.     

BIOCHEMICAL GENETIC STUDIES ON CUTYLEFISH SEPIELLA MAINDRONI （CEPHALOPODA： SEPIIDAE）——ACTIVE LOCI SCREENING OF ISOZYME

Screening of 46 putative enzyme-coding loci and 4 different kinds of tissues of Sepiella maindroni de Rochebrone, 1884 for enzymatic activities using starch gel electrophoretic technique proved that the 21 enzymes such as AAT, AK, ALP, AP, CK, DIA, ES, FBP, G3PDH, GPI, GRS,IDH, LDH, MDH, MEP, MPI, NP, PGDH, PGM, SOD and XO* , were active to Sepiella maindroni after being stained. The tissue exhibiting stable and clear bands was also determined. Among tissues tested, mantle muscle tissue was the best for electrophoretic survey of isozymes. Buccal bulb muscle, eye and liver were fairly good for some special enzymes, such as DIA, ES, MPI, NT, etc.     

Extraction and PTP1B inhibitory activity of bromophenols from the marine red alga Symphyocladia latiuscula

Previously, we had characterized several structurally interesting brominated phenols from the marine red alga Symphyocladia latiuscula collected from various sites. However, Phytochemical investigations on this species collected from the Weihai coastline of Shandong Province remains blank. Therefore, we characterized the chemical constituents of individuals of this species collected from the region. Eight bromophenols were isolated and identified. Using detailed spectroscopic techniques and comparisons with published data, these compounds were identified as 2,3-dibromo-4,5-dihydroxybenzyl methyl ether (1), 3,5-dibromo-4-hydroxybenzoic acid (2), 2,3,6-tribromo-4,5-dihydroxymethylbenzene (3), 2,3,6-tribromo-4,5-dihydroxybenzaldehyde (4), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (5), bis(2,3,6-tribromo-4,5-dihydroxyphenyl)methane (6), 1,2-bis(2,3,6-tribromo-4,5-dihydroxyphenyl)-ethane (7), and 1-(2,3,6-tribromo-4,5-dihydroxybenzyl)-pyrrolidin-2-one (8). Among these compounds, 1 and 2 were isolated for the first time from S. latiuscula. Each compound was evaluated on the ability to inhibit protein tyrosine phosphatase 1B (PTP1B), which is a potential therapeutic target in the treatment of type 2 diabetes. Bromophenols 5, 6, and 7 showed strong activities with IC50 values of 3.9, 4.3, and 3.5 μmol/L, respectively. This study provides further evidence that bromophenols are predominant among the chemical constituents of Symphyocladia, and that some of these compounds may be candidates for the development of anti-diabetes drugs.     

Chemical constituents of marine medicinal mangrove plant <Emphasis Type="Italic">Sonneratia caseolaris</Emphasis>

Twenty-four compounds including eight steroids (1–8), nine triterpenoids (9–16, 24), three flavonoids (20–22), and four benzenecarboxylic derivatives (17–19, 23) were isolated and identified from stems and twigs of medicinal mangrove plant Sonneratia caseolaris. The structures of the isolated compounds were determined by extensive analysis of their spectroscopic data. Among these metabolites, compounds 1, 4–20 and 22–24 were isolated and identified for the first time from S. caseolaris. In the in vitro cytotoxic assay against SMMC-7721 human hepatoma cells, compound 21 (3′,4′,5,7-tetrahydroxyflavone) exhibited significant activity with IC₅₀ 2.8 μg/mL, while oleanolic acid (14), 3,3′-di-O-methyl ether ellagic acid (18), and 3,3′,4-O-tri-O-methyl ether ellagic acid (19) showed weak activity. None of these compounds displayed significant antibacterial activites. Supported by the National Natural Science Foundation of China (No. 30770234); Knowledge Innovation Program of Chinese Academy of Sciences (KZCX2-YW-211-04); Department of Science and Technology of Shandong Province (No.2006GG2205023)     

Impact of redox-stratification on the diversity and distribution of bacterial communities in sandy reef sediments in a microcosm

Relationships between microbial communities and geochemical environments are important in marine microbial ecology and biogeochemistry. Although biogeochemical redox stratification has been well documented in marine sediments, its impact on microbial communities remains largely unknown. In this study, we applied denaturing gradient gel electrophoresis (DGGE) and clone library construction to investigate the diversity and stratification of bacterial communities in redox-stratified sandy reef sediments in a microcosm. A total of 88 Operational Taxonomic Units (OTU) were identified from 16S rRNA clone libraries constructed from sandy reef sediments in a laboratory microcosm. They were members of nine phyla and three candidate divisions, including Proteobacteria (Alpha-, Beta-, Gamma-, Delta-, and Epsilonproteobacteria), Actinobacteria, Acidobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, Verrucomicrobia, Spirochaetes, and the candidate divisions WS3, SO31 and AO19. The vast majority of these phylotypes are related to clone sequences from other marine sediments, but OTUs of Epsilonproteobacteria and WS3 are reported for the first time from permeable marine sediments. Several other OTUs are potential new bacterial phylotypes because of their low similarity with reference sequences. Results from the 16S rRNA, gene clone sequence analyses suggested that bacterial communities exhibit clear stratification across large redox gradients in these sediments, with the highest diversity found in the anoxic layer (15–25 mm) and the least diversity in the suboxic layer (3–5 mm). Analysis of the nosZ, and amoA gene libraries also indicated the stratification of denitrifiers and nitrifiers, with their highest diversity being in the anoxic and oxic sediment layers, respectively. These results indicated that redox-stratification can affect the distribution of bacterial communities in sandy reef sediments.     

Impacts of CO<Subscript>2</Subscript> enrichment on growth and photosynthesis in freshwater and marine diatoms

The physiological responses of Nitzschia palea Kvtzing, a freshwater diatom, to elevated CO2 were investigated and compared with those of a marine diatom, Chaetoceros muelleri Lemmermann previously reported. Elevated CO2 concentration to 700 μl/L increased the dissolved inorganic carbon （D!C） and lowered the pH in the cultures of N. palea, thus enhancing the growth by 4%-20% during the whole growth period. High CO2-grown N. palea cells showed lower levels of dark respiration rates and higher Ik values. Light-saturated photosynthetic rates and photosynthetic efficiencies decreased in N. palea with the doubling CO2 concentration in airflow to the bottom of cultures, although the doubling CO2 concentration in airflow to the surface cultures had few effects on these two photosynthetic parameters. N. palea cells were found to be capable of using HCO5 in addition to gaseous CO2, and the CO2 enrichment decreased their affinity for HCO5 and CO2. Although doubled CO2 level would enhance the biomass of N. patea and C. muelleri to different extents, compared with the marine diatom, it had a significant effect on the specific growth rates of N. palea. In addition, the responses of photosynthetic parameters of IV. palea to doubled CO2 concentration were almost opposite to those of C. muelleri.