低温β-半乳糖苷酶产生菌的筛选、鉴定、生长特性及酶学性质的初步研究

自北冰洋楚科奇海、加拿大海盆、格陵兰海和中国南极中山站近岸的海水、海冰和沉积物样品中分离的322株低温细菌中筛选出73株低温β-半乳糖苷酶产生菌,并对其中1株BCw006进行了研究。通过16S rDNA序列同源性分析表明,该菌株属于交替单胞菌属,定名为Alteromonas sp.BCw006。对该菌株研究表明:该菌最适生长和产酶温度为24℃;在pH6.0—8.0、含0%—6.O% NaCl的培养基条件下菌株能够较好地生长与产酶,其最适生长和产酶NaCl浓度分别为3%和1%,乳糖对β-半乳糖苷酶合成具有明显诱导作用,酵母粉和蛋白胨是菌株生长和产酶较适宜的氮源,酶的最适作用温度为35℃,0℃时能保持35℃下酶活的25%左右,表明其具有较好的适应低温特性。酶对热敏感,60℃时作用30min后酶活力下降了41%。     

南极酵母Rhodotorula sp.NJ298的鉴定及其产虾青素的研究

本研究对产虾青素的南极菌株NJ298进行了鉴定,同时对影响虾青素产量的主要环境因子进行了测试。形态特征、生理生化特征及ITS序列的系统发育分析表明,南极菌株NJ298属于红酵母属(Rhodotorula);虾青素积累主要在NJ298生长的对数期和稳定期;乙酸钠、蛋白胨是合成虾青素的最佳碳、氮源;在0-15℃之间,南极酵母NJ298均可以生长,但是低温更有利于虾青素的积累;pH值在6-8范围内NJ298均可生长,最适pH值为7.5;该菌对盐度要求不严格。     

若尔盖高原泥炭沼泽土嗜冷细菌系统发育分析

结合低温纯培养手段、核糖体DNA扩增片段限制性内切酶分析技术和克隆测序,研究了若尔盖高原泥炭沼泽土中可培养菌的多样性、系统发育和产低温酶特性。从3块采样地的土样中分离到47株表型各不相同的菌株。16S rRNA测序和系统发育分析结果表明,若尔盖高原泥炭沼泽土的可培养低温菌高度相似于分离自极地、高山冻土和冰川等低温环境的嗜冷菌。47株菌分属于4个不同的群,包括高GC含量群、低GC含量群、Beta和Gama变形菌纲等。革兰氏阳性菌以节杆菌属(Arthrobacter)为主,而革兰氏阴性菌以假单胞菌属(Pseudomonas)为主。其他的细菌分属于微小杆菌属(Exiguobacterium)、芽孢八叠球菌属(Sporosarcina)、贪噬菌属(Variovorax)、紫色杆菌属(Janthinobacterium)和寡养单胞菌属(Stenotrophomonas)等不同的类群。产低温酶分析结果表明,在分离到的47株菌中,大多数菌株都能分泌纤维素降解酶,50%以上的菌株具有蛋白酶活性,而以节杆菌的胞外酶产生能力最强。若尔盖高原泥炭沼泽土中存在丰富的低温菌类群,在酶学生物技术上具有潜在应用价值。     

南极菌Pseudoalteromonas sp．S-15-13产胞外多糖的研究及其分子鉴定

本文对影响南极细菌S-15-13生长和胞外多糖产量的主要环境因子进行了研究,同时采用16S rDNA序列分析及系统发育分析方法对其进行了分子鉴定。结果表明：菌株S-15-13最佳产糖条件为：培养时间,56h；培养温度,8℃；碳源,1．0%葡萄糖；NaCl浓度,3．0%；pH,6．0-7．0。16S rDNA序列分析和系统发育分析表明,菌株S-15-13属于假交替单胞菌属(Pseudoaltero- monas)。     

南极阿德雷岛不同基底中真菌的分离培养及初步鉴定

为了探索南极真菌的多样性,进一步揭示其生态学角色和筛选有特定酶活性菌株,本研究对中国第27次南极科学考察采集自阿德雷岛的6份样品进行了真菌的分离培养及其胞外酶活性测定。从6份样品中共分离得到168株真菌,其中大型海藻腐烂物样品中分离到的真菌种类和数量最多,占所分离菌株总数的34.6%;对选取的15株代表菌株进行了真菌ITS1-5.8S-ITS2序列鉴定,结果表明它们分属于4个纲,8个属,其中青霉属5株,假散囊菌属、地丝菌属和枝孢属各2株,生赤壳属、曲霉属、出芽短梗霉属和unclassified Onygenales各 1株,该结果表明南极地区具有丰富的真菌物种多样性,其优势类群青霉菌。胞外酶测定显示产淀粉酶、蛋白酶和纤维素酶产生菌分别占供试菌株的64.6%、45.1%、14.6%,产纤维素酶的菌株可能在南极物质能量循环中扮演着重要角色。     

南极罗斯海沉积物中可培养菌株的分离及胞外水解酶活性检测

研究了南极罗斯海地区沉积物中可培养微生物多样性,并检测了菌株胞外酶活性,为开发利用南极资源奠定基础。采用Zobell2216E培养基、土豆培养基(PDA)及稀释平板划线法分离并获得纯培养的细菌和真菌,分别通过16SrDNA、18S/ITS序列鉴定分析了可培养微生物的多样性和系统发育。从南极罗斯海沉积物样品中获得78株细菌和54株真菌,细菌、真菌的优势菌株分别为嗜冷杆菌属(Psychrobacter)、枝孢属(Cladosporium);78株细菌菌株中有2株与模式菌株的16SrDNA基因序列相似性小于97%,可能是潜在的细菌新物种。细菌API20NE生理生化和真菌产胞外酶活性结果表明,获得的细菌和真菌大多数可检测到胞外水解酶活性。本研究丰富了南极可培养微生物资源,为南极罗斯海地区可培养微生物的多样性研究以及南极微生物资源的应用研究奠定了基础。     

南极阿德雷岛淡水湖沉积物细菌群落研究

应用样品直接稀释涂布平板法,从南极阿德雷岛淡水湖沉积物中分离得到34株菌,并进行了生理生化性质检测。根据其16SrDNA全长序列所进行的系统发育分析表明,分离菌株分属于革兰氏阳性菌:节杆菌(Arthrobacter)、红球菌(Rhodococcus)、葡萄球菌(Staphylococcus);gamma-变形细菌(gamma-proteobacteria):假单胞菌(Pseudomonas)、不动杆菌(Acinetobacter)、寡养单胞菌(Stenotrophomonas);beta-变形细菌(beta-proteobacteria):紫色杆菌(Janthinobacterium);拟杆菌门(Cytophaga-Flexibacteria-Bacteroides):假结核棒杆菌(Cytophaga)、土地杆菌(Pedobacter)等9个属。分离的菌株在4℃条件下能产生多种大分子物质水解酶类,其中32%的菌株能产生脂肪酶,18%的菌能够水解明胶或者产生卵磷脂酶。同时对沉积物垂直方向的可培养细菌生物量进行了分析,表明阿德雷岛企鹅排泄物输入量对微生物分布有较大影响。     

南极菲尔德斯半岛土壤可培养细菌多样性分析

对中国第29次南极科学考察采自南极菲尔德斯半岛区域16个站位的土壤样品进行了细菌的分离培养。经细菌16S r DNA序列测定及系统发育分析,共鉴定得到3个门,13个属的67株细菌。其中优势门为放线菌门(Actinobacteria)和变形菌门(Proteobacteria),优势属为节杆菌属(Arthrobacter)和假单胞菌属(Pseudomonas)。对受到不同外界动物因素影响的4个典型站位的土壤理化性质进行了测定,初步分析了土壤理化因子对可培养细菌群落结构的影响。结果表明在菲尔德斯半岛人活动对土壤中可培养细菌群落的影响显著区别于企鹅和海豹。研究还对产淀粉酶的菌株进行了筛选,发现8株产淀粉酶的菌株。     

青藏高原北部湖泊沉积物中基于不同碳源可培养细菌多样性

前人研究显示微生物群落对有机碳源的利用具有选择性,但是纯菌株对碳源利用的选择性却不甚明了。此外,在盐湖环境中微生物出于能量的考虑可能选择性利用有机碳源以抵抗盐度渗透压。因此,盐度可能间接地影响微生物对有机碳源的利用。然而,目前类似的相关研究较少。采用单碳源纯培养技术、16S rRNA基因序列分析和相关统计方法,研究了青藏高原北部6个湖泊(洱海、青海湖、托素湖、尕海1、尕海2、小柴旦湖)沉积物中基于不同碳源可培养细菌多样性及其与盐度的响应关系。采用7种不同类型的单碳源(甲酸钠、乙酸钠、丙酮酸钠、乳酸钠、葡萄糖、纤维素、混合氨基酸)进行培养筛选。共获得10个细菌分类属的75株纯培养菌株,它们分属于γ-变形菌纲的气单胞菌属(Aeromonas)、盐单胞菌属(Halomonas)、希瓦氏菌属(Shewanella)、假单胞菌属(Pseudomonas)、假交替单胞菌属(Pseudoalteromonas)、弧菌属(Vibrio),β-变形菌纲的脱氯单胞菌属(Dechloromonas)以及芽孢杆菌纲的芽孢杆菌属(Bacillus)、微小杆菌属(Exiguobacterium)和游动球菌属(Planococcus)。其中,盐单胞菌属菌株可利用研究中使用的所有碳源类型,说明这类细菌具有广泛碳代谢途径,可能潜在参与了青藏高原北部湖泊碳循环过程。研究发现所得纯菌株对不同碳链长度碳源的利用具有选择性,即随着盐度增高,某些菌株偏向利用结构更加复杂的碳源。总之,盐度不仅影响着纯培养细菌群落多样性,而且还影响着细菌对碳源类型的选择。     

一株分离自北极海冰的产β-半乳糖苷酶的海单胞菌(Marinomonas sp. BSi20414)的筛选、鉴定和产酶条件研究

从北极海冰中分离筛选出一株产β-半乳糖苷酶的菌株BSi20414,经初步鉴定,该菌株归属于海单胞属(Marinomonas),命名为Marinomonas sp.BS120414.对该菌株的产酶条件实验表明,最适产酶条件为:30℃,180 rpm摇瓶培养96 h,培养基中加1.5%(w/v)乳糖,初始pH 7.0,接种...     

Screening and molecular classification of low-temperature protease from Antarctic microorganism and its characterization

<正> 107 strains producing protease were screened from 260 strains of Ant-arctic psychrophilic bacteria,among which proteolytic activity of five strains wasmore than 45 U ml~(-1).The 16S rRNA gene sequences homology and phylogenet-ic analysis of five Antarctic psychrophilic bacteria showed that NJ276、NJ5-9、NJ16-70、NJ345 belonged to the described genus Pseudoalteromonas and NJ341belonged to the genus Colwellia.The growth and the protease characteristic offour Antarctic psychrophilic bacteria had been studied,and the result showedthat the optimal temperature for growth and protease-producing of four strainswas about 10℃.Their growth and protease-producing were still high during in-cubating 2-5 days.The maximum proteolytic activity occurred at pH 9 for fourAntarctic psychrophilic bacteria.The optimal temperature of protease action ofboth strains NJ276 and NJ5-9 was about 50℃,however,the optimal temperatureof protease action of both strains NJ341 and NJ345 was about 40℃,and theirproteolytic activity under 0℃ exhibited nearly 30% of the maximum activity,but their thermal stabilities were weaker.These results indicated that proteasesfrom NJ341 and NJ345 were low-temperature proteases.     

影响南极海洋石油烃低温降解菌希瓦氏菌NJ49生长和降解率的环境因素研究

以柴油为唯一碳源和能源,从南极海水海冰微生物资源库中筛选到一株石油烃低温降解菌希瓦氏菌NJ49,并对影响其生长和降解率的环境因素(pH、温度、盐度、营养盐和表面活性剂)进行了初步研究。结果表明:希瓦氏菌NJ49可作为低温海域石油烃污染生物修复的菌源,其生长和降解的最适条件为:初始pH7.5,温度15℃,盐度6%,摇瓶装量80ml,最佳氮源硝酸铵,最佳磷源为磷酸二氢钾和磷酸氢二钾的混合物,添加表面活性剂可促进希瓦氏菌NJ49的生长和生物降解率。     

两株南极枝孢霉属真菌Cladosporium sp.NJF4和NJF6的次级代谢产物研究

南极因其独特的自然环境成为潜在、重要的微生物资源库,是产生新型生物活性物质和先导化合物菌株的潜在种源地,南极微生物正在成为创新药物研究新的重要资源。虽然近年来对南极微生物次级代谢产物的研究逐渐增加,但与温带和热带微生物研究相比仍处于初级阶段。对从南极普里兹湾海洋沉积物中获得的两株枝孢霉属真菌Cladosporium sp. NJF4和NJF6进行次级代谢产物分离及结构鉴定,获得20个化合物。化合物结构类型包括甾醇(1)、倍半萜类(7—8)、生物碱类(9—14)、二酮哌嗪(2—5、15—17)、芳香酸(6、18—19)等,其中倍半萜类(7—8)为首次从枝孢霉属真菌中分离得到,以上研究将为丰富南极微生物次级代谢产物库奠定一定的研究基础。     

Phylogenetic analysis of cultivable bacteria isolated from Arctic sea-ice

Phylogenetic analysis based on 16S rDNA of 8 strains of cultivable bacteria isolated from Arctic sea-ice was studied.The results showed that strain BJ1 belonged to genus Planococcus,which was a genus of low mole percent G+C gram-positive bacteria;strain BJ6 belonged to genus Burkholderia of β-proteobacteria and the rest 6 strain all belonged to γ-proteobacteria,of which strain BJ8 was a species of Pseudoalteromonas,strain BJ2-BJ5 and BJ7 were members of genus Psychrobacter.Phylogenetic analysis also indicated that bacteria of genus Psychrobacter of the isolates formed a relatively independent phylogenetic cluster in comparison with other bacteria belonged to genus Psychrobacter.     

Preliminary study on plasma membrane fluidity of Psychrophilic Yeast Rhodotorula sp.NJ298 in low temperature

The ability of cell to modulate the fluidity of plasma membrane was crucial to the survival of microorganism at low temperature.Plasma membrane proteins,fatty acids and carotenoids profiles of Antarctic psychrophilc yeast Rhodotorula sp.NJ298 were investigated at-3 ℃,0 ℃ and 8 ℃.The results showed that plasma membrane protein content was greater at-3 ℃ than that at 8 ℃,and a unique membrane polypeptide composition with an apparent molecular mass of 94.7 kDa was newly synthesized with SDS-PAGE analysis;GC analysis showed that the main changes of fatty acids were the percentage of unsaturated fatty acids(C18:1 and C18:2) and shorter chain saturated fatty acid(C10:0) increased along with the decrease of the culture temperature from 8 ℃ to-3 ℃;HPLC analysis indicated that astaxanthin was the major functional carotenoids of the plasma membrane,percentage of which increased from 54.6±1.5% at 8 ℃ to 81.9±2.1% at-3 ℃.However the fluidity of plasma membrane which was determined by measuring fluorescence anisotropy was similar at-3 ℃,0 ℃ and 8 ℃.Hence these changes in plasma membrane's characteristics were involved in the cellular cold-adaptation by which NJ298 could maintain normal plasma membrane fluidity at near-freezing temperature.     

三种北极微藻对不同温度的适应性研究

为了研究北极微藻对不同环境温度的适应性,对分离自北极王湾地区的三种微藻-玛氏骨条藻(Skeletonema marinoi)、小球藻(Chlorella sp.)和衣藻(Chlamydomonas sp.)在不同温度条件下(0℃、4℃和8℃)的生长曲线和抗氧化酶系统进行了测定.结果表明微藻对温度的适应性因种而异:玛氏骨条藻在4℃具有最快生长速率,最高的超氧化物歧化酶(SOD)和过氧化氢酶(CAT)酶活性,及最低的丙二醛(MDA)含量;小球藻的最快生长速率及酶活均在8℃时,MDA含量最低值出现在0℃;衣藻生长情况相差不大,MDA含量的最低值出现在8℃.抗氧化酶系统变化规律也因藻种及温度而异--在0℃与4℃变化时,玛氏骨条藻抗氧化酶系统的三个指标的差异极显著(P<0.01);在0℃与8℃变化时,玛氏骨条藻与小球藻的SOD活性的差异显著(P<0.05),其余两个指标差异不显著;衣藻在三个温度下的三个指标差异均不显著.总体而言,三种藻对三个温度的适应性均较好:其MDA的含量均较低,三种藻各自具有独特的生理代谢机制来适应不同的温度环境.     

Effect of solar radiation and the subsequent dark periods on two newly isolated and characterized Antarctic marine bacteria

Two strains of psychrotolerant Antarctic marine bacteria were isolated and characterized using biochemical and molecular techniques. Sequencing of 16S rRNA gene showed that UVvi strain belongs to the genus Arthrobacter whereas UVps strain is related to the Flexibacter-Cytophaga-Bacteroides (FCB) group. Response of the strains to solar radiation was studied during the summer of 1999 in Potter Cove, near Jubany station (South Shetland Island, Antarctica). The effect of photosynthetically available radiation (PAR, 400-700 nm), ultraviolet-A (UV-A, 320-400 nm) and ultraviolet-B radiation (UV-B, 280-320 nm) on cell viability was studied using mixed cultures in quartz bottles covered with interferential filters and exposed to solar radiation. In all experiments, four treatments were used: dark (with light screened out), PAR (with UV radiation screened out), PAR+UV-A (UV-B screened out) and PAR+UV-A+UV-B. Under the assayed conditions, PAR+UV-A and PAR+UV-A+UV-B radiation showed similar negative effects on the viability of the studied strains. However, at the end of the exposure time, mortality values in PAR+UV-A+UV-B treatments were higher than those observed under PAR+UV-A treatments. In both PAR+UV-A and PAR+UV-A+UV-B treatments we observed high levels of hydrogen peroxide compared with the dark control. The Arthrobacter UVvi strain showed significant recovery in dark conditions after exposure to the PAR+UV-A but not after the PAR+UV-A+UV-B treatment. This strain proved to be more resistant to UV radiation than the FCB group-related UVps strain. The results showed that UV radiation has a deleterious effect on these Antarctic marine bacteria and also revealed that the analysed components of the Antarctic bacterioplankton may have different responses when they are exposed to the same irradiance conditions.     

极地寡营养细菌AT52(Alteromonas stellipolarissp.nov.)超微结构、生长生理特性及液态富营养发酵培养条件优化

寡营养细菌是一类能利用低浓度有机营养基质的细菌,通过研究1株分离自南极海洋的极地寡营养细菌AT52,发现富营养条件下AT52菌体细胞周身附肢,附肢长短不一,具鞭毛,菌体出芽生殖,由一层原生质膜包围.寡营养饥饿生存的菌体细胞相对较小,近球形,边缘不平滑,无鞭毛附肢等超微结构,细胞体眠未见出芽生殖过程.实验中采用寡营养环境...