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.     

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.     

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.     

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.     

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.     

Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.     

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.     

近江牡蛎肠道细菌及其产酶能力

从近江牡蛎肠道中分离出21株菌,其中, 9株为革兰阳性菌, 12株革兰阴性菌。为寻找能分泌多种酶的有益微生物,研究了它们蛋白酶、脂肪酶、淀粉酶、纤维素酶的产酶能力。结果表明,有61 .9%的菌株能分泌蛋白酶或淀粉酶(各有13株菌);有52. 4%的菌株能分泌脂肪酶(11株菌);有33. 3%的菌株能产纤维素酶(7株菌)。在这21株菌中,产4种酶的有5株菌,产3种酶的3株菌,产两种酶的5株菌,产1种酶的3株菌,不产酶的仅有5株菌。不产酶的菌株仅占细菌总数的23. 8%,而各种产酶菌则高达76. 2%,由此表明细菌在近江牡蛎对食饵消化过程的重要性。     

Studies on culture condition and extracellular hydrolase of psychrophilic bacteria from Arctic sea ice

Arctic sea ice in the polar region provides a cold habitat for microbial community. Arctic sea ice microorganisms are revealed to be of considerable importance in basic research and potential in biotechnological application. This paper investigated the culture condition and extraceIlular hydrolase of 14 strains of different Arctic sea ice bacteria. The results showed that optimal growth temperature of strains is 15 ℃ or 20 ℃. The optimal pH is about 8.0. They hardly grow at acid condition. 3 % NaCl is necessary for better growth. These strains have different abilities in producing amylase, protease, eellulase and lipase. Pseudoalteronomas sp. Bsi429 and Pseudoalteronomas sp. Bsi539 produced both cellulose, protease and lipase. These results provide a basis for further developing and exploiting the cold adapted marine enzyme resources.     

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes (protease, amylase, and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus, which can produce protease, amylase, and cellulase. Virgibacillus, however, can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.     

Amylase Production by the Marine Yeast Aureobasidium pullulans N13d

1 Introduction Amylases are enzymes which hydrolyze starch molecules to give diverse products including dextrin and pro-gressively smaller polymers composed of glucose units(Windish et al., 1965; Pandey et al., 2000; Chi et al.2001). Amylase is a kind of very important enzyme andconstitutes a class of industrial enzymes sharing approximately 25% of the enzyme market (Sindhu et al.1997; Rao et al., 1998). Amylases are universally distributed throughout the animal, plant and microbial kingdoms…     

Characterization and identification of enzyme-producing microflora isolated from the gut of sea cucumber Apostichopus japonicus

Gut microorganisms play an important role in the digestion of their host animals. The purpose of this research was to isolate and assess the enzyme-producing microbes from the Apostichopus japonicus gut. Thirty-nine strains that can produce at least one of the three digestive enzymes(protease,amylase,and cellulase) were qualitatively screened based on their extracellular enzyme-producing abilities. The enzyme-producing strains clustered into eight groups at the genetic similarity level of 100% by analyzing the restriction patterns of 16 S rDNA amplified with Mbo I. Phylogenetic analysis revealed that 37 strains belonged to the genus Bacillus and two were members of the genus Virgibacillus. Enzyme-producing capability results indicate that the main enzyme-producing microflora in the A. japonicus gut was Bacillus,which can produce protease,amylase,and cellulase. Virgibacillus,however,can only produce protease. The high enzyme-producing capability of the isolates suggests that the gut microbiota play an important role in the sea cucumber digestive process.     

Occurrence and Diversity of Pichia spp. in Marine Environments

A total of 328 yeast strains from seawater, sediments, mud of saltems, 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 luv-small, Pichia ohmeri YF04d, Pichiafermentans 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.     

九孔鲍养殖水体中产酶菌群的研究

为了解九孔鲍养殖环境中细菌生理菌群的生态作用,对分离自养殖水体的33株菌株的产酶能力和种类进行了研究。结果表明,具有分泌胞外蛋白酶、脂肪酶和纤维素酶能力的菌株分别为39.4%、57.6%和30.3%;在这33株菌中,产至少一种胞外酶的菌株比例高达63.6%。API鉴定结果表明,在有能力分泌胞外蛋白酶、脂肪酶和纤维素酶的菌株中,弧菌菌群所占的比例分别为46%、42%和40%,而其中河流弧菌又占各弧菌菌群比例的83.3%、62.5%和100%。充分阐明弧菌,尤其是河流弧菌为优势菌,在鲍鱼养殖微生物态中起的重要作用。     

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.     

Distribution and diversity of <Emphasis Type="Italic">Candida tropicalis</Emphasis> strains in different marine environments

1089 strains of yeasts were obtained from seawater,sea sediments,mud of sea saltems,guts of marine fishes,mangrove plants and marine algae.The results of routine identification and molecular analysis methods show that 44 strains among the marine yeasts 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 importam role in marine environment and the marine environment in turn is a good source for obtaining C.tropicalis.     

具有多种胞外酶的对虾肠道黏附菌的分离和鉴定

用对虾饲料培养基从健康凡纳滨对虾肠道分离出500株黏附细菌,以产淀粉酶、脂肪酶和蛋白酶能力为指标,筛选出产该3种消化酶的细菌90株,占总菌株的18%.对其中生长较快的69株进行16S rDNA 基因测序,确定其分类地位.结果显示,69株菌分别属于不动杆菌属(Acinetobacter)、芽孢杆菌属(Bacillus)、葡萄球菌属(Staphylococcus)、假交替单胞菌属(Pseudoalteromonas)、气单胞菌属(Aeromonas)、嗜盐单胞菌属(Halomonas)、利斯顿氏菌属(Listonella)、莫拉氏菌属(Moraxella)等,其中数量最多是芽胞杆菌属,占鉴定细菌总数的53.62%,数量最少是气单胞菌属和嗜盐单胞菌属,均占鉴定细菌总数的2.90%.表明对虾肠道黏附菌群中具有较多能分泌多种消化酶的细菌,可进一步开发为促进对虾消化功能的益生菌     

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.     

Bacteriological analysis of the digestive tube of the mud snail (Bullacta exarata Philippi) and its rearing shoal

The bacterial flora in the digestive tube of Bullacta exarata Philippi and its rearing shoal were investigated. A total of 157 strains of heterotrophic bacteria, isolated from crop, stomach intestine and other parts of the digestive tube, mainly belong to the genera Photobacterium, Bacillus, Pseudomonas Vibrio and some genera of the family Enterobacteriaceae. There are significantly more varieties of bacteria in crop than in stomach and intestine. A total of 173 strains of bacteria were isolated from the rearing shoal, belonging to 13 genera. The 5 predominant genera, such as Bacillus and Photobacterium, are the same as those in the digestive tube, but greatly differ in percentages. The number of heterotrophic bacteria and Vibrio in rearing shoal change in line with the alteration of the temperature, and are significantly affected by the use of pesticides.     

Molecular identification of Prorocentrum (Dinophyceae) species

A fragment of a large sub-unit ribosomal DNA (LrDNA) of 12 strains ofProrocentrum species was amplified by polymerase chain reaction (PCR). The PCR products were digested by 3 restriction endonucleases (Cfo I, Hae III, and RSA I) and then resolved in agarose gels. Results show that different species had different RFLP patterns, except forP. arcuatum (ME 131), which had the same pattern toP. micans (ME160 and 04). The same fragment of 19 strains of the genus was also amplified and subjected to denaturing gradient gel electrophoresis (DGGE). 11 different patterns were resolved. Different cultures of a same species had the same pattern. The results of RFLP and DGGE analyses showed that eight newly isolated epibenthicProrocentrum species were different from each other, and also from other cultured ones examined in this study.P arcuatum (ME132) could not be differentiated fromP. micans (ME160 and 04), it was probably mis-identified, since they are quite different morphologically.P. redfieldii (ME138) could also not be distinguished formP. triestinium (ME132), it should be regarded as a synonym ofP. triestinium. Unexpectedly, a restriction site was found inP. micans, compared with previous sequence data. Project supported by National Basic Research Priorities Program (2001CB409701, 2001CB409710) and supported by NSFC (40376040, 40025614)