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…     

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.     

Alkaline protease production by a strain of marine yeasts

Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China. The protease had the highest activity at pH 9.0 and 45℃. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min^- 1 and agitation speed 150 r min^-1 . Under the optimal conditions, 623.1 Umg^-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.     

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 were obtained. The results of routine identification and molecular characterization showed that six isolates among these marine yeasts belonged to Candida genus as Candida intermedia for YA01a, Candida parapsilosis for 3eA2, Candida quercitrusa for JHSb, Candia rugosa for wl8, Candida zeylanoides for TJY13a, and Candida membranifaciens for W14-3. Isolates YA01a (Candida intermedia), 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 potential applications in biotechnology.     

Light and electron microscope observations onNephroselmis gaoae sp. nov. (Prasinophyceae)

Nephroselmis gaoae sp. nov. is described on the basis of light and electron microscope observations of cultured material originally collected and isolated from seawater of Jiaozhou Bay, Qingdao, China. The periplasts on the cell body and flagella are covered by five types of scales, two types on the flagella and three on the body. Among these, the morphology and the number of spines of large stellate body scales differ remarkably from those of previously described species ofNephroselmis. Apart from these, the unusual fine structure of the eyespot (stigma) is very characteristic. As in the other species ofNephroselmis, the eyespot lies immediately under the two-membraned chloroplast envelope; unlike the others, however, it is not composed of a number of osmiophilic globules, but consists of about 14 curved rod-shaped osmiophilic bodies arranged loosely and randomly. This feature distinguishes the present new species not only from the other species ofNephroselmis but also from the other motile algal species, the eyespots structure of which had been previously described. Contribution No. 2316 from the Institute of Oceanology, Chinese Academy of Sciences. This paper was presented at the XIVth International Seaweed Symposium held in Brittany, France, 1992.     

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.     

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.     

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.     

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.     

Optimization of culturing condition and medium composition for the production of alginate lyase by a marine <Emphasis Type="Italic">Vibrio</Emphasis> sp. YKW-34

Carbohydrases secreted by marine Vibrio sp. YKW-34 with strong Laminaria cell wall degrading ability were screened, and among them alginate lyase was found to be dominant. The effects of medium composition and culturing condition on the production of alginate lyase by marine Vibrio sp. YKW-34 in flask were investigated in this study. In the culture medium of marine broth, no alginate lyase was produced. The activity of the alginate lyase, after being induced, reached 5 UmL^-1. The best inoculum volume and inoculum age were 10% and 12h, respectively. The optimal temperature for alginate lyase production was 25℃. The fermentation medium was composed of 0.5% of Laminaria powder and 0.2% of KNO3 with an initial acidity of pH 8.0. Alginate could induce alginate lyase production but not as efficiently as Laminaria powder did. The addition of fucoidan, cellulose and glucose had negative effect on the alginate lyase production. Other kinds of nitrogen sources, such as yeast extract, beef extract and peptone, had positive effect on the growth of the microorganism and negative effect on alginate lyase production. In addition, the time course of alginate lyase production under the optimized condition was described. The optimal harvest time was 48 h.     

Genetic transformation of marine cyanobacterium Synechococcus sp. CC9311 (Cyanophyceae) by electroporation

Synechococcus sp.CC9311 is a marine cyanobacterium characterized by type IV chromatic acclimation(CA).A genetic transformation system was developed as a first step to elucidate the molecular mechanism of CA.The results show that Synechococcus sp.CC9311 cells were sensitive to four commonly used antibiotics:ampicillin,kanamycin,spectinomycin,and chloramphenicol.An integrative plasmid to disrupt the putative phycoerythrin lyase gene mpeV,using a kanamycin resistance gene as selectable marker,was constructed by recombinant polymerase chain reaction.The plasmid was then transformed into Synechococcus sp.CC9311 via electroporation.High transformation efficiency was achieved at a field strength of 2 kV/cm.DNA analysis showed that mpeV was fully disrupted following challenge of the transformants with a high concentration of kanamycin.In addition,the transformants that displayed poor growth on agar SN medium could be successfully plated on agarose SN medium.     

Observation of female gamete (Carpogonia) germination of Porphyra yezoensis and P. oligospermatangia

Several papers have reported that part or whole leafy thallus seemingly consisting of zygotospores can give rise to both blades and conchocelis in the same culture ofPorphyra. Study on samples of wild and cultivatedPorphyra yezoensis andP. oligospermatangia were conducted to clarify the origination of the young blades in the culture. It is confirmed that single cells on the blade of both species, which normally intermixed with zygotospores, germinated into young blades. TEM and SEM observation has shown that the single cells ofPorphyra yezoensis had typical features of female gamete (carpogonia) but archeospore. Therefore, the female gametes are responsible in developing leafy thalli. This project was sponsored by the NSFC (No. C-0205-05-39770593)     

Synthesis of three-dimensional agaric-like biomorphic TiO2 by a facile method with Coscinodiscus sp. frustule

The paper aims to expand the application of natural marine algae.Marine diatoms,which have intricate frustule structures,can serve as bio-template for preparing three-dimensional materials.A simple and effective approach to synthesize the corrugated agaric-like biomorphic TiO 2 templated with frustule of Coscinodiscus sp.is reported.In the sol-gel preparation process,the titania-coating on the frustule is prepared through the deposition and condensation with the aid of acetylacetone(acac) as a controlling agent to make the precursor Ti(BuO) 4 hydrolyze slowly.The as-prepared titania-coated frustule and biomorphic TiO 2 is characterized by scanning electron microscopy(SEM) attached with energy dispersive X-ray spectrometer(EMAX) and X-ray diffraction(XRD).The microstructure of the corresponding titania nanoparticles appears to be sphere with the diameters distributed around 10 20 nm.The templating process is repeated for three cycles.Subsequently,the three-dimensional freestanding corrugated agaric-like biomorphic TiO 2 structure is obtained by a selective removal in the NaOH solution.As far as we known,the 3D freestanding corrugated agaric-like biomorphic TiO 2 with greatly increased surface area is obtained for the first time.     

Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council     

A Taxonomic Study of the Genus Tinocladia （Chordariales,Phaeophyta） in China Coasts I. Two new species, T. eudesmoides sp.nov, and T. zhangii sp.nov.

Two new species of the genusTinocladia Kylin (1940)T. eudesmoides Ding et Lu sp. nov. andTinocladia zhangii Ding et Lu sp. nov., are described.T. eudesmoides is mainly characterized by its longitudinally and transversely produced sub-cortical filaments from the medulla, its short uniseriate assimilating filaments, usually composed of 6–10 cells with slightly swollen and narrow ultimate cells, and its unilocular sporangia (60–115)×55–70μm, variable in size.T. zhangii is mainly characterized by its very solid frond, not rip apart by hands, its evident longer primary branches, its short and slender secondary branches, with very evident sub-cortical layer, and filaments divided repeatedly, and its large and small unilocular sporangia, with the latter borne on an evidently swollen pedicel. Project B80981816 supported by the NSFC. Contribution No. 4443 from the Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.     

Sequence comparison of partial cytochrome b genes of two Coilia species

Sequence variation of partial cytochrome b genes between two Coilia species, C. ectenes and C. mystus, was investigated. Of the 402 nucleotides, twenty-seven (6.72%) are polymorphic and all are synonymous substitutions. At the third positions of genetic condon of cytochrome b gene, the two species show an extreme anti-G bias (<4%) and a pronounced bias towards A and C (>68%). There is no amino acid sequence divergence between the partial cytochrome b genes of the two species, indicating a close genetic relationship between them. The k-2p genetic distance of partial cytochrome b segment of the two species is 0.072, suggesting that the species were separated 3.6 Ma ago, in the middle Pliocene. Our result reveals that the cytochrome b gene is an appropriate marker for studies of population genetic structures and phylogeographic patterns of the two species.     

The ultrastructure of separated and cultured cell of Porphyra yezoensis

There are many reports that cells (protoplasts) separated from the thallus ofPorphyra by enzyme can develop to normal leafy thalli in the same way as monospores. But there are few investigations on the subcellular structure of the isolated vegetative cell for comparison with the subcellular structure of monospores. To clarify whether the separated and cultured cells undergo the same or similar ultrastructure changes during culture and germination as monospores undergo in their formation and germination, we observed their ultrastructure, compared them with those of the monospore and found that the ultrastructure of separated and cultured cells did not have the characteristic feature as that of monospore formation, such as production of small and large fibrous vesicles, but was accompanied by vacuolation and starch mobilization like that in monospore germination. The paper also discusses the relations between monospores and separated and cultured cells. Contribution No. 3875 from the Institute of Oceanology, Chinese Academy of Sciences. Project 39770593 supported by NSFC; Project 96-C01-05-01 of the 9th Five-Year Plan supported by Science and Technology Commision of China.     

Cloning and nucleotide sequence of D-hydantoinase gene of marine polyphosphate-accumulating bacterium, Halomonas sp. YSR-3

Hydantoinase is involved in the production of optically pure amino acids from racemic 5-mono-substituted hydantoins. We measured the D-hydantoinase activity in marine Halomonas sp. YSR-3 and amplified the D-hydantoinase gene by PCR. The gene was inserted into vector pGM-T and transformed into E. coli TOP10. The positive transformants with the D-hydantoinase gene were sequenced. The sequenced fragment comprises 1 510 base pairs. The D-hydantoinase gene from YSR-3 is 77% similar to that from Pseudomonas entomophila L4 by searching against the NCBI databse. The protein product of the YSR-3 D-hydantoinase gene is 75%, 73%, and 70% similar to those from Pseudomonas fluorescens Pf-5, Marinomonas sp. MED121, and Burkholderia vietnamiensis G4, respectively. The difference of the D-hydantoinase gene between marine Halomonas sp. YSR-3 and other terrestrial organisms is distinct.     

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.     

ON THE MARINE CILIATE, Pseudocohnilembus hargisi （PROTOZOA, CILIOPHORA, SCUTICOCILIATIDA） WITH COMPARISON OF ITS RELATED SPECIES

The living morphology, nuclear apparatus, infraciliature and silverline system of the poorly-known ciliatePseudocohnilembus hargisi Evans & Thompson, 1964, collected from a shrimp culture pond in Qingdao, China, were examined by observation of the live specimen; and by the pyridinated carbonate method and Chatton-Lwoff technique. The main characteristics: 13–14 (usually 13) longitudinal or slightly spirally arranged somatic kineties; single contractile vacuole with usually 2 (1–2) pores at posterior ends of the fourth and fifth somatic kineties to the right of the buccal cavity. Several related congeners were compared and morphometrically tabulated. Project No. 39970098 supported by NSFC.