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 ℃, while only 213.18 μg g-1 of carotenoid was pro-duced by the wild type under the same conditions.

Optimum Production and Characterization of an Acid Protease from Marine Yeast Metschnikowia reukaufii 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 ℃.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 ℃ 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-molecu-lar-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 Marine Yeasts in Antarctica Environments

A total of 28 yeast strains were obtained from the sea sediment of Antarctica.According to the results of routine identi-fication and molecular characterization,the strains belonged to species of Yarrowia lipolytica,Debaryomyces hansenii,Rhodotorula slooffiae,Rhodotorula mucilaginosa,Sporidiobolus salmonicolor,Aureobasidium pullulans,Mrakia frigida and Guehomyces pullu-lans,respectively.The Antarctica yeasts have wide potential applications in biotechnology,for some of them can produce b-galactosidase and killer toxins.

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 Aureo-basidium 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.

Isolation and identification of a marine killer yeast strain YF07b and cloning of the gene encoding killer toxin from the yeast

It was found that the marine yeast strain YF07b could secrete a large amount of killer toxin against a pathogenic yeast strain WCY which could cause milky disease in Portunus trituberculcttus. The marine yeast strain YF07b was identified to be Pichia anomala according to the results of routine yeast identification and 18S rDNA and ITS sequences. The gene encoding killer toxin in the marine yeast strain YF07b was amplified by PCR technology. After sequencing, the results show that an open reading frame, consisting of 1 281 bp, encoded a presumed protein of 427 amino acids. The sequence of the cloned gene was found to have 99% match with that of the gene encoding killer toxin in Pichia anomalas strain K. A signal peptide including 17 amino acids appeared in the N-terminal domain of the killer toxin. Therefore, the mature protein consisted of 410 amino acids, its molecular mass was estimated to be 47.4 ku and its isoelctronic point was 4.5.

Optimization of medium and cultivation conditions for enhanced exopolysaccharide yield by marine Cyanothece sp. 113

Cyanothece sp. 113 is a unicellular, aerobic, diazotrophic and photosynthetic marine cyanobacterium. The optimal medium for exopolysaccharide yield by the strain was 70.0 g/L of NaCl, and 0.9 g/L of MgSO4 based on the modified F/2 medium for cultivation of marine algae. The optimal cultivation condition for exopolysaccharide yield by this cyanobacterial strain was 29℃, aeration, and continuous illumination at 86.0 μE/M^2/S. Under the optimal conditions, over 18.4 g/L of exopolysaccharide was produced within 12 days. This was so far the highest exopolysaccharide yield produced with strains of Cyanothece sp. obtained.

Screening of protease-producing marine yeasts for production of the bioactive peptides

Over 400 yeast strains from seawater and sediments were obtained, but only five strains named HN2 -3, N13d, N13C, Mb5 and HN3 - 2 among them could form clear zones around their colonies on the double plates with 2.0% casein. Peptides in the hydrolysate produced by the proteases from strains HN2 -3 and N13d had higher angiotensin I-converting-enzyme （ACE）-inhibitory activity. The two marine yeast strains were identified to be Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. After purification of the proteases from the two marine yeast strains, it was found that the optimal pH for them was both 9.0, both of them were serine alkaline protease. However, the optimal temperature for the protease from the strain HN2 -3 was 52℃ while that from strain N13d was 48℃. ACE-inhibitory activity of the peptides in the hydrolysate of shrimp protein produced by the purified protease from the strain HN2 -3 was the highest while antioxidant activity in the hydrolysate of spirulina protein produced by the purified protease from the strain N13d was the highest.

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 pH 6.0. 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 U mg -1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

The selection of alkaline protease-producing yeasts from 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, Nllb, YF04C and HN4.9） capable of secreting extracellular alkaline protease were isolated. The isolated strains were identified as Aureobasidium pullulans, Yarrowia lipolytica, lssatchenkia 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 N1 lb was 10.0. The optimal temperature for protease activity was 45℃for strains HN3.11, N11b, and YF04C, and 50℃ for strain HN4.9. After digestion of shrimp （Penaeus vannamei） protein and spirulina （Arthospira platens＆） protein with the four crude alkaline proteases, the filtrate from spirulina （Arthrospira platensis） powder digested by the crude alkaline protease of strain HNYl 1 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.

Distribution and Diversity of Candida tropicalis Strains in Different Marine Environments

1089 strains of yeasts were obtained from seawater,sea sediments,mud of sea salterns,guts of marine fishes,mangroveplants and marine algae.The results of routine identification and molecular analysis methods show that 44 strains among the marineyeasts obtained in this study belong to Candida tropicalis,which may indicate its wide distribution in different environment,especially in the tropical and subtropical marine environment.The wide distribution of C.tropicalis indicates that it may play an important role in marine environment and the marine environment in turn is a good source for obtaining C.tropicalis.

Selection of Yarrowia lipolytica Strains with High Protein Content from Yeasts Isolated from Different Marine Environments

A total of 78 Yarrowia lipolytica yeast strains from seawater, sediments, mud of salterns, the guts of marine fish, and marine algae were obtained. After the crude protein of the yeasts was estimated by the method of Kjehldahl, we found that seven strains of the marine yeasts grown in soy bean cake hydrolysate with 20 g LZ of glucose for 48h at 28~C contained more than 41.0g protein per 100g of cell dry weight and the cell dry weight was more than 4.4g per L of the culture. Among them, strain SWJ-Ib contained the highest crude protein. The results of Biolog identification and molecular methods further confirmed that they indeed belonged to Y lipolytica.

Occurrence and Diversity of Pichia 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 1 uv-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 luv-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.

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, Can- dia rugosa for wlS, Candida zevlanoides for TJY 13a, and Candida membranifaciens for W 14-3. Isolates YA01 a （Candida intermedia）, wl8 （Candida rugosa）, 3eA2 （Candida parapsilosis）, and JHSb （Candida quercitrusa） were found producing cell-bound lipase, while isolate Wl4-3 （Candida membran（faciens） producing riboflavin. These marine yeast Candida spp. seem to have wide potential applications in bioteehnology.

Marine Yeasts and Their Applications in Mariculture

The terrestrial yeasts have been receiving great attention in science and industry for over one hundred years because they can produce many kinds of bioactive substances. However, little is known about the bioactive substances of marine yeasts. In recent years, it has been found that marine yeasts have wide applications in mariculture and other fields. Therefore,marine yeasts, the bioactive substances from them and the applications of marine yeasts themselves and the bioactive substances they produced are reviewed in this paper.

Strain H_2-419-4 of Haematococcus pluvialis induced by ethyl methanesulphonate and ultraviolet radiation

Two strains H2-410 and H2-419 were obtained from the chemically mutated survivors of wild Haematococcus pluvialis 2 by using ethyl methanesulphonate (EMS). Strains H2-410 and H2-419 showed a fast cell growth with 13% and 20% increase in biomass compared to wild type, respectively. Then H2-419-4, a fast cell growth and high astaxanthin accumulation strain, was obtained by exposing the strain H2-419 to ultraviolet radiation (UV) further. The total biomass, the astaxanthin content per cell, astaxanthin production of H2-419-4 showed 68%, 28%, and 120% increase compared to wild H. pluvialis 2, respectively. HPLC (High Performance Liquid Chromatography) data showed also an obvious proportional variation of different carotenoid compositions in the extracts of H2-419-4 and the wild type, although no peak of carotenoids appeared or disappeared. Therefore, the main compositions in strain H2-419-4, like its wild one, were free of astaxanthin, monoester, and diester of astaxanthin. The asexual reproduction in survivors after exposed to UV was not synchronous, and different from the normal synchronous asexual reproduction as the mother cells were motile instead of non-motile. Interestingly, some survivors from UV irradiation produced many mini-spores (or gamete?), the spores moved away from the mother cell gradually 4 or 5 days later. This is quite similar to sexual reproduction described by Elliot in 1934. However, whether this was sexual reproduction remains questionable, as no mating process has been observed.

Bioflocculant Produced by Klebsiella sp. MYC and Its Appli-cation in the Treatment of Oil-field Produced Water

Seventy-nine strains of bioflocculant-producing bacteria were isolated from 3 activated sludge samples. Among them, strain MYC was found to have the highest and stable flocculating rate for both kaolin clay suspension and oil-field produced water. The bacterial strain was identified as Klebsiella sp. MYC according to its morphological and biochemical characteristics and 16SrDNA sequence. The optimal medium for bioflocculant production by this bacterial strain was composed of cane sugar 20 g L^-1, KH2PO4 2 g L^-1, K2HPO4 5 g L^- 1, （ NH4）2SO4 0.2 g L^-1, urea 0.5 g L^- 1 and yeast extract 0.5 g L^- 1, the initial pH being 5.5. When the suspension of kaolin clay was treated with0.5% of Klebsiella sp. MYC culture broth, the flocculating rate reached more than 90.0% in the presence of 500mg L^-1 CaCI2, while the flocculating rate for oil-field produced water was near 80.0% in a pH range of 7.0 - 9.0 with the separation of oil and suspended particles from the oil-field produced water under similar conditions. The environment-friendly nature of the bioflocculant and high flocculating rate of the strain make the bioflocculant produced by Klebsiella sp. MYC an attractive bioflocculant in oil-field produced water treatment.

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 Pa-cific Ocean. This strain was identified to be Aureobasidium pullulans by 18S rRNA gene sequence analysis and routine yeast identi-fication 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 ℃, 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.

PHYSIOLOGICAL INHIBITORY EFFECT OF OCS IN ARACHIDONIC ACID-RICH PARIETOCHLORIS INCISA (TREBOUXIOPHYCEAE, CHLOROPHYTA)

Parietochloris incisa is an arachidonic acid rich snow green alga. The main physiological profiles, such as ash free dry weight (AFDW), chlorophyll, carotenoid, protein and total fatty acids (TFA), in this alga exposed to old culture supernatant (OCS) at the decline phase or its crude ethyl acetate extracts (CEAE) were investigated by using tubular photobioreactors of different diameters. Results showed that both OCS and CEAE had strong inhibitory effect on the above physiological parameters. The longer the culture was exposed to OCS and the more CEAE were added into the algal culture, the more the above physiological properties were inhibited. Arachidonic acid (AA), the dominant component of fatty acids in this alga, was also seriously inhibited with respect to total TFA, AFDW of cell mass, or culture volume, due to a probable reduction of enzymes activities catalyzing chain elongation from C18:1ω9 to AA. These results incontestably evidenced that some CEAE dissolving substances existing in OCS, like auto inhibitors, inhibited P. incisa growth through feedback. Hence, any efficient removal of auto inhibitors from algal culture to decrease their bioactivity could be good for maximal production of desired products like AA.

Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

Antimicrobial peptide is a polypeptide with antimicrobial activity. Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp （Fenneropenaeus Chinensis） were integrated into Oryza sativa L. subsp, japonica cv. Aichi ashahi by Agrobacterium mediated transformation system. PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in To generation, respectively. RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation, and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants. Four Np3 and Np5 transgenic lines in T1 generation were inoculated with Xanthomonas oryzae pv. oryzae strain CR4, and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4. The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2, Zhe 173 and OS-225. It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

A Carboxymethyl Cellulase from a Marine Yeast(Aureobasidium pullulans 98): Its Purification, Characterization, Gene Cloning and Carboxymethyl Cellulose Digestion

We have reported that A. pullulans 98 produces a high yield of cellulase. In this study, a carboxymethyl cellulase （CMCase） in the supematant of the culture ofA. pullulans 98 was purified to homogeneity, and the maximum production of CMCase was 4.51 U （mg protein）-1. The SDS-PAGE analysis showed that the molecular mass of the purified CMCase was 67.0kDa. The optimal temperature of the purified enzyme with considerable thermosensitivity was 40℃, much lower than that of the CMCases from other ftmgi. The optimal pH of the enzyme was 5.6, and the activity profile was stable in a range of acidity （pH 5,0-6.0）. The enzyme was activated by Na＋, Mg2＋, Ca2＋, K＋, Fe2＋ and Cu2＋, however, it was inhibited by Fe3＋, Ba2＋, Zn2＋, Mn2＋ and Ag＋. Km and Vmax values of the purified enzyme were 4.7mgmL-1 and 0.57 pmol L-1 min-1 （mg protein）-1, respectively. Only oligosaccharides with different sizes were released from carboxymethylcellulose （CMC） after hydrolysis with the purified CMCase. The putative gene encoding CMCase was cloned from A. pullulans 98, which contained an open reading flame of 954bp （EU978473）. The protein deduced contained the conserved domain of cellulase superfamily （glucosyl hydrolase family 5）. The N-terminal amino acid sequence of the purified CMCase was M-A-P-H-A-E-P-Q-S-Q-T-T-E-Q-T-S-S-G-Q-F, which was consistent with that deduced from the cloned gene. This suggested that the purified CMCase was indeed encoded by the cloned CMCase gene in this yeast.