雅浦海沟深海沉积物可培养真菌多样性及其反硝化能力研究

自雅浦海沟4个站位的12个沉积物样品中分离得到38株真菌菌株,基于形态观察和ITS序列信息对所得菌株进行分类鉴定,发现它们分别属于青霉属(Penicillium,17株)、曲霉属(Aspergillus,7株)、篮状菌属(Talaromyces,1株)、枝孢属(Cladosporium,6株)、赭霉属(Ochroconis,1株)、Meyerozyma属(1株)、梅拉菌属(Meira,1株)、Cystobasidium属(2株)、Wallemia属(1株)和红酵母属(Rhodotorula,1株)等10个属.其中青霉属、曲霉属和枝孢属菌株为优势菌株,分别占分离菌株总数的44.7%、18.4%和15.8%;而Wallemia属(节担菌纲)菌株是首次从深海环境中分离到.利用PCR技术在分离的深海真菌菌株中检测nir K和P450nor两种反硝化关键酶基因,结果显示nir K基因可在黄曲霉(Aspergillus flavus)、黑曲霉(Aspergillus niger)、杂色曲霉(Aspergillus versicolor)、枝孢菌(Cladosporium sp.)、枝状枝孢(Cladosporium cladosporioides)和产黄青霉(Penicillium chrysogenum)等6种真菌菌株中检测到,而P450nor基因仅在黄曲霉菌株中检测到.同时还通过富集培养方法从上述沉积物样品中分离获得两株反硝化枝孢菌.结果表明,雅浦海沟深海沉积物环境中存在着丰富的真菌资源,它们在深海环境氮循环中起着一定作用,这将增强人们对深海环境可培养真菌多样性及其生态作用的认识.国家海洋局第三海洋研究所基本科研业务费专项资金资助项目(海三科2016039);;国家“973”项目重点基础研究发展计划资助项目(2015CB755903);;国家自然科学基金资助项目(41606145,41776170

南海4种珊瑚附生可培养真菌多样性分析

本研究利用从南海采集的4种珊瑚分离附生真菌,并通过菌株的分类鉴定及系统发育分析获得珊瑚附生可培养真菌的多样性信息.选用酵母麦芽糖琼脂培养基(YM)、马铃薯葡萄糖琼脂培养基(PDA)、马丁氏培养基(Martin)、察氏琼脂培养基(CDA)和孟加拉红培养基(RBM)等5种培养基,采用平板涂布法培养分离附生真菌,基于ITS-rRNA基因序列对真菌菌株进行鉴定和系统发育分析.从4种珊瑚上共分离获得10个属的120株真菌,分别是:青霉属(Penicillum)56株、曲霉属(Aspergillus)25株、枝顶孢属(Acremonium)2株、枝孢属(Cladosporium)5株、毕赤酵母属(Meyerozyma)14株、隔孢伏革属(Peniophora)2株、拟茎点霉属(Phomopsis)1株、裂褶菌属(Schizophyllum)1株、梗孢酵母属(Sterigmatomyces)13株、篮状菌属(Talaromyces)1株.通过对不同珊瑚品种分离获得的附生真菌的比较,发现小叶鹿角珊瑚(Acropora microphthalma)和花鹿角珊瑚(Acropora floride)上分离到的菌株多样性最高,包含6属10种.进一步的分析发现不同珊瑚样品上真菌种类分布有较大差异,隔孢伏革属和裂褶菌属真菌均分离自小叶鹿角珊瑚,梗孢酵母属和篮状菌属均分离自角孔珊瑚(Goniopora sp.),扁脑珊瑚(Platygyra sp.)上则分离到拟茎点霉属.同时还发现不同培养基分离真菌的能力不同,YM培养基分离的菌株最多,CDA培养基分离真菌的多样性最多.以上的研究结果表明,南海珊瑚上存在大量且丰富的真菌资源,可以为后续生物活性物质的开发利用提供良好的菌种资源.国家海洋局第三海洋研究所基本科研业务费专项资金资助项目(海三科2015020,海三科2016039);;国家国际科技合作专项资助项目(2015DFA20500

Breeding high-yield oil-producing strain to use cheap carbon source by UV induced protoplast mutagenesis

【目的】研究并建立利用原生质体紫外诱变技术选育可利用廉价碳源发酵的高产油新菌株的方法。【方法】采用1.5%蜗牛酶和1.0%纤维素酶混合液水解去除细胞壁得到2A00015(近平滑假丝酵母,CAndIdA PArAPSIlOSIS)的原生质体,将其放于紫外灯下诱变及再生壁培养,筛选获得可利用廉价碳源发酵的高产油酵母,并采用气相色谱质谱联用法(gC-MS)测定其脂肪酸组成。【结果】突变效果最好的突变菌株2A00015/25用葡萄糖发酵培养7 d后,其生物量、油脂产率和产油量分别为17.77 g/l、58.12%和10.32 g/l,较原始菌株分别提高了12.45%、23.32%和38.68%;利用废糖蜜发酵培养,其生物量、油脂产率和产油量分别为18.54 g/l、49.44%和9.17 g/l,较原始菌株分别提高了9.09%、21.16%和32.18%。利用废糖蜜培养其产油效率虽低于利用葡萄糖培养,但从环境保护及原材料成本的角度考虑,用废糖蜜作为碳源发酵培养产生油脂更具优势。诱变菌株利用废糖蜜发酵后产生油脂经检测含有8种脂肪酸,其脂肪酸组成与植物油近似,其中不饱和脂肪酸含量占脂肪酸总量的82.4%。【结论】通过利用原生质体紫外诱变技术,成功选育出一株新的可利用廉价碳源的高产油海洋菌株,产油率达到49.4%,提高了21.2%。[Objective] We used UV induced protoplast mutagenesis to study breed a new high-yielding lipid-producing strain which could use cheap carbon source.[Methods] Get the 1.5% glusulase and 1.0% cellulose solution.Hydrolyze to remove the cell wall and obtain the protoplast of 2A00015(Candida parapsilosis).Put it under the ultraviolet lamp for mutagenesis and cultivate regenrated wall.Then screen to get the high oil generated yeast which could be fermented by low-cost carbon source.Determine its fat acid components by gas chromatography with mass spectrometric(GC-MS).[Results] Cultivated the mutant strain with best mutation 2A00015/25 in the glucose.We found that the biomass, oil yield rate and oil production are separately 17.77 g/L, 58.12% and 10.32 g/L, which are separately 12.45%, 23.32% and 38.68% higher than the original strain.We have also cultivated the mutant strain in the waste molasses and found the biomass, oil yield rate and oil production are separately 18.54 g/L, 49.44% and 9.17 g/L, which are separately 9.09%, 21.16% and 32.18% higher than the original strain.The oil yield rate is lower in the waste molasses cultivation than that in glucose cultivation.However, in consideration of environment protection and cost of raw materials, the waste molasses is of much more advantages.It is tested that the fat generated from the waste molasses fermentation consists of eight kinds of fat acid.Its fat acid components are similar to the vegetable oil, in which the content of unsaturated fatty acid comprised 82.4% of the total fatty acid.[Conclusion] Basing on UV induced protoplast mutagenesis, we have successfully bred a new high-yielding oil strain which can make use of the low-cost carbon source, with its oil production rate 49.4% which has increased by 21.2%.深海(微)生物资源勘探与资源潜力评价项目(No.DY125-15-R-01

Study on screening of oleaginous yeast and determination of intracellular lipid content by nile red dyeing

通讯作者：Tel: 86-592-2195679; :mail: [email protected][中文文摘]【目的】建立产油酵母筛选以及胞内油脂含量测定的简便方法。【方法】利用尼罗红与胞内油脂成分结合后在紫外光照射下发出荧光且荧光强弱与油脂含量相关的原理。通过在添加尼罗红的培养基中培养酵母,并观察菌落荧光的方法对385株深海酵母进行产油脂菌株筛选,利用26S rDNA D1/D2区序列分析方法对筛选获得的产油酵母菌株进行鉴定,并以其中的一株高产油脂酵母(2A00015)为试验菌株,建立了一套尼罗红染色快速测定油脂含量的方法。【结果】获得22株产油酵母,其中油脂含量最高可达62.9%,经分子鉴定后显示这22株酵母分别属于(Candida viswanathii)、近平滑假丝酵母(Candidaparapsilosis)、粘质红酵母(Rhodotorula mucilaginosa)、汉逊德巴利酵母(Debaryomyceshansenii)、季也蒙毕赤酵母(Pichia guilliermondii)以及Rhodosporidium paludigenum酵母。尼罗红染色快速测定油脂含量方法的最佳检测条件为:菌悬液OD600小于1.2,尼罗红浓度0.5 mg/L,染色时间5 min,激发波长488 nm,发射波长570 nm。该测定方法得到相对荧光强度与称重法得到油脂含量呈正相关性,R2=0.9637。[英文文摘][Objective] To establish a simple method to screen oleaginous yeast and determine the intracellular lipid content.[Methods] The study is based on the theory that the combina-tion of nile red and intracellular oil will emit fluorescence when induced by UV light and the fluoresence indensity is associated with the lipid content.We cultivate yeast in the culture medium added with nile red,and screen the oleaginous yeast strains from the 385 deep-sea yeasts by measns of obeserving the fluorescence of the yeast colony.We have identified the screened oleaginous yeast strains by the 26S rDNA D1/D2 series analysis method.Designating one of the oleaginous yeasts(2A00015) as the test strain,the lipid content rapid determination method by nile red dyeing was established.[Results] 22 oleaginous yeasts were obtained with the lipid content reaching up to 62.9%.Based on the molecular identification,it showed that the 22 yeasts are separately belong to Candida viswanathii,Candida parapailosis,Rhodotorla mucilaginosa,Debaryomyces hansenii,Pichia guilliermondii and Rhodosporidium paludi-genum.The optimum condition for lipid content determination by nile red dyeing is: bacterium suspension OD600 lower than 1.2,nile red concentration 0.5 mg/L,dyeing time 5 min,excit-ing wavelength 488 nm,emission wavelength 570 nm.The relative fluorescence intensity ob-tained by this method exhibits a positive association with the lipid content obtained by weigh-ing method,which can be explained as R2=0.9637.国家海洋局第三海洋研究所基本科研业务费专项资金项目(No.海三科2010009

Progress of studies on marine filamentous fungi and their bioactive substances

海洋微生物种类繁多 ,是筛选新药的丰富资源宝库 .陆地真菌是仅次于放线菌的多产抗生素的微生物类群 ,而海洋真菌正在成为海洋药源微生物研究的新热点 .海洋真菌产生的生物活性物质已经引起研究者的关注 ,并将成为海洋药物的重要来源 .本文结合国内外文献报道 ,阐述了海洋真菌及其活性物质的研究情况 .The diversity of marine microorganisms provides a luxuriant resource for screening new drugs. Marine filamentous fungi are becoming a hot research field in studies of marine microorganisms for potential pharmaceutical application, whereas terrestrial ones were the second possible producer next to actinomycetales for antibiotics in the land. Some novel bioactive substances have been found derived from marine filamentous fungi, therefore the fungi must be an important resource of marine drugs. Current outcrops of researching marine filamentous fungi and their bioactive secondary metabolites are surveyed in present paper.国家海洋局青年海洋科学基金资助项目 (2 0 0 1313) ;; 国家科技基础性工作项目 (G 99 A 15a

Screening and identification of active microorganisms isolated from the tropical Pacific Ocean

从热带太平洋的生物、海水、沉积物样品中分离到细菌、酵母和霉菌共475株.选择8个指示菌并采用圆形纸片法对分离菌株的发酵液进行抗菌和抗肿瘤活性筛选,获得20个具有抗菌和/或抗肿瘤活性的微生物菌株.细菌、酵母和霉菌活性菌株的筛选得率都比较低,分别为5.4%、2.2%和3.4%,其原因可能与纸片的发酵液的添加量较少和菌株发酵条件的控制有关.同时采用分子生物学方法鉴定了活性菌株,除4株未有结果外,其余菌株分归为9个属,其中芽孢杆菌属7株、占活性菌株的35%,盐单胞菌属2株、占10%,其它菌属各1株、占5%.抑菌谱分析表明,大多数活性菌株对革兰氏阳性细菌具有抑制作用,而来源于鱼体的菌株抑菌谱较广,对细菌、真菌均有拮抗作用,另外还发现一株酵母(Rhodosporidium toruloides)可抑制金黄色葡萄球菌.作者提出“活性指示(activity index)”参数,对活性菌株的抗菌谱和活性强度进行综合评估,也表明源于鱼体的菌株的活性指示值较高.这4株芽孢杆菌尤其是DY-Y-11A1A菌株,具有潜在的后续开发价值.20 bioactive microbial strains,which showed the capacities of anti-microbes and/or anti-tumors,were obtained and screened by means of 8 screening models and a paper-dish technique,from a total of 475 strains consisting of bacteria,yeasts and molds isolated from samples of fishes,sea waters and sediments from the tropical Pacific Ocean.The discovered rate of bioactive strains was quite low.They were 5.4% in the bacterial isolates,2.2% in the yeasts and 3.4% in the molds respectively,probably due to less volume of cultured liquids for each test and of some un-optimal physical and chemical factors for fermentation of the tested microbial isolates.The taxonomy of the 20 bioactive strains,identified by the molecular biology technique,belong to 9 genus,with 7 strains in Bacillus,accounting for 35%,2 in Halomonas for 10% and only 1 in the other each genus for 5% of the total,except for 4 unidentified strains.The spectra of the bioactive strains from 8 models demonstrated that G+ bacteria in the screening models were inhibited by most of the bioactive strains,including Stains Y-2033(Rhodosporidium toruloide)which was interestingly against Staphylococcus aureus;while the strains isolated from fishes showed wide spectra against bacteria,fungi.When the term,"activity index"is suggested by the authors to compare bioactivities among the screened strains in a combination of 2 parameters of spectra and strength of activity,it is found that 4 strains of Bacillus,isolated from fishes,illuminated higher values of the index,which could be the candidates for further developments.中国大洋矿产研究开发协会资助项目(NO.DY105-04-02-06);; 福建省自然科学基金计划资助项目(Z0516050

A STUDY OF MARINE FUNGI AND THE DIVERSITY OF BIOACTIVE SUBSTANCES PRODUCED BY THEM

海洋微生物种类繁多,是筛选新药的丰富资源宝库。近些年来,作为海洋微生物中重要的组成部分的海洋真菌正成为研究的热点,越来越受到国内外学者的关注。本文详细概述了海洋真菌的定义、分布、海洋真菌研究的历史和现状、其所产生的生物活性物质多样性以及存在的问题。The diversity of marine microorganisms provides an abundant resource for screening new drugs.Recent years,marine fungi as an important part of marine microorganisms are becoming a hot research field for the discovery of pharmacologically active substances.More and more attention was paid by domestic and foreign researchers in this field.This review summarizes in detail the definition,research history and status quo,the diversity of bioactive substances produced by marine fungi and unsolved problems.中国大洋矿产研究开发协会资助项目(NO.DY105-2-04-02);; 福建省自然科学基金资助项目(NO.Z0516050)资