溶藻细菌FS1的溶藻效果与机制初探

近年来,由水体富营养化引发的蓝藻水华频繁暴发,对水体生态系统平衡产生了重大影响,给人类健康也带来严重威胁。生物法除藻具有高效性、环境友好等优点,因此,如果能获得具有较高溶藻效率的溶藻细菌,选择生物法除藻更为理想。从菏泽一富营养化池塘分离得到1株溶藻细菌FS1,经16S rDNA测序分析鉴定为芽胞杆菌属。实验以铜绿微囊藻为研究对象,采用血球计数板法计算反应前后藻细胞的浓度,对不同生长阶段溶藻细菌FS1的溶藻效果进行了探究。停滞期、对数期、稳定期和衰亡期的除藻率分别为7.1%、24.3%、57.0%和45.5%,结果表明,处于稳定期的FS1对铜绿微囊藻的去除效果最佳。细菌溶藻方式的研究结果表明,溶藻细菌是通过分泌溶藻物质间接溶解藻细胞。     

一株溶藻细菌NP23的初步分离鉴别及其溶藻作用研究

从水体中分离得到一株具有溶藻能力的细菌,命名为NP23。经形态特征、生理生化鉴定和16S rDNA序列分析表明,该菌株属于肠杆菌属(Enterobacter)。研究了该菌株对湖泊中优势藻的溶藻效果,初步探讨了其溶藻方式及溶藻物质。结果表明,该菌株对小球藻、惠氏微囊藻、栅藻和蛋白核小球藻具有一定的去除效果,叶绿素a的去除率分别为64.1%、53.1%、87.2%和84.4%,而且在10-108CFU/mL菌浓度范围内,藻的去除率与菌液的浓度成正相关;该菌株对小球藻、栅藻和蛋白核小球藻是间接溶藻,对惠氏微囊藻是直接溶藻;该菌株对栅藻的溶藻物质是蛋白类物质,对蛋白核小球藻的溶藻因子是菌体胞外分泌的具有热稳定性的非蛋白类物质。     

一株中肋骨条藻特异溶藻菌的分离鉴定及溶藻特性

【背景】赤潮频发引起严重的海洋生态学问题,不仅直接影响到海洋生态系统稳定、海洋生物资源可持续利用和水产养殖业等海洋产业的健康发展,而且对人类健康也构成了严重威胁。高效的溶藻细菌是生物法防控赤潮的有效工具之一。【目的】分离得到对中肋骨条藻具有高效溶藻效果的溶藻细菌,并对其进行分子鉴定,研究该菌株的溶藻机理以及溶藻菌所分泌溶藻物质的特性。【方法】采用2216E平板稀释涂布法分离纯化细菌,测定16SrRNA基因序列以鉴定细菌种类,利用显微镜计数溶藻菌处理后的目标藻种计算溶藻率,通过扫描电镜观察溶藻菌对中肋骨条藻的溶藻过程,利用常规生理生化方法研究溶藻菌溶藻物质的特征,并通过透析袋截留法研究溶藻物质分子量大小。【结果】分离得到一株中肋骨条藻高效溶藻菌FDHY-CJ,该菌株属于交替单胞菌属(Alteromonas sp. FDHY-CJ)。该菌株72 h处理赤潮藻结果显示,对中肋骨条藻溶藻率为95.45%,对于其他常见赤潮藻溶藻率低于40%。溶藻菌FDHY-CJ通过胞外分泌物溶藻;溶藻物质的溶藻特性不受反复冻融的影响,但对酸碱性及温度较为敏感;扫描电镜观察结果显示该溶藻菌的溶藻物质直接溶解中肋骨...     

一株溶藻细菌对海洋原甲藻的溶藻效应

从深圳大鹏湾南澳赤潮爆发海域的表层海水中分离得到1株对海洋原甲藻(Prorocentrum micans)具有溶藻活性的海洋细菌,菌株编号为N10。利用液相感染法研究了该溶藻细菌的溶藻效果和溶藻作用方式。结果表明,菌株N10能使藻细胞失去运动活性,并膨胀变形,细胞膜内物质聚集于一端,藻细胞最终破裂死亡。菌悬液接种到藻液中的量越大,初始细菌密度越高,其溶藻效果越强。菌悬液以1∶10的体积比接种到藻液中时,藻细胞在24 h的死亡率为83%,至72 h全部溶解死亡;体积比为1∶20的藻细胞在24 h的死亡率为71%,之后藻细胞密度略有波动,120 h时死亡率达77%;而体积比为1∶100的藻细胞密度在前24 h有所下降,死亡率达39%,之后藻细胞密度又开始明显上升;对照组的藻细胞密度均呈明显上升趋势。菌悬液过滤液和高温加热处理后的菌悬液过滤液对海洋原甲藻均无溶藻活性,表明菌株N10的溶藻方式为直接溶藻。通过16S rRNA序列分析并与GenBank数据进行同源性检索,并结合细菌形态及生理生化特征,菌株N10隶属于黄杆菌科(Flavobacteriaceae)中的Muricauda sp.。     

滇池中溶藻细菌的分离鉴定及其溶藻效应

【背景】藻类水华或赤潮在世界范围内频发,带来各种危害,亟需找到有效途径控制水华或赤潮。溶藻细菌具有杀死藻类控制藻类生物量的能力,可以作为防治水华和赤潮的有效工具。【目的】分离并鉴定滇池中的铜绿微囊藻(Microcystisaeruginosa)及其溶藻细菌,对溶藻菌作用于铜绿微囊藻的溶藻效应进行研究,初步了解其溶藻特性与溶藻机制。【方法】采用LB平板稀释涂布,再经多次划线分离纯化细菌,测定16SrRNA基因序列以鉴定细菌种类;采用毛细管分离的方法分离铜绿微囊藻,并测定其cpcBA基因序列以鉴定蓝藻种类;采用热乙醇法提取叶绿素a,从而计算溶藻效率;基于过氧化氢酶(CAT)、还原型谷胱甘肽(GSH)和丙二醛(MDA)探究藻细胞在溶藻菌处理下的抗氧化系统响应。【结果】共分离获得11株微囊藻和17株针对铜绿微囊藻的高效溶藻菌。选取其中一株生长速度最快的铜绿微囊藻DCM4和一株溶藻效果最好的溶藻菌Sp37 (Bacillus siamensis)进行后续研究。Sp37对DCM4的4 d溶藻率达到92.4%±1.5%,且对微囊藻属的水华微囊藻(M. flos-aquae)和惠氏微囊藻(M.wesenbergii)均有溶藻效果,而对绿藻没有溶藻效果。Sp37的原菌液和无菌滤液对DCM4的4d溶藻率分别为86.8%±4.3%和81.1%±2.2%,两者没有显著差异(P0.05)。Sp37菌体对DCM4的溶藻率为25.4%±7.3%。Sp37无菌滤液经不同温度和pH处理之后的溶藻率与未经处理的无菌滤液的溶藻率无明显差异。Sp37无菌滤液处理藻细胞会使藻细胞的CAT、GSH和MDA含量发生变化。【结论】菌株Sp37对铜绿微囊藻DCM4具有高效的溶藻作用,而且对微囊藻属具有一定的溶藻特异性。Sp37是通过分泌胞外物质间接溶藻,且溶藻物质具有热稳定性和酸碱稳定性。Sp37无菌滤液处理藻细胞会触发藻细胞抗氧化系统,并且会损伤藻细胞膜。Sp37无菌滤液很可能是通过对藻细胞造成氧化胁迫,最终导致藻细胞死亡的。     

稻瘟病拮抗菌株的分离、筛选及鉴定

从水稻病健叶、茎和根组织以及稻田土壤中共分离得到细菌菌株321株。经发酵法初筛,对稻瘟病菌丝生长有抑制作用的有57株,再通过平板对峙法复筛,具有强烈拮抗作用的菌株有5株,其抑菌距离达16mm以上。分别对5个菌株进行形态学观察、生理生化指标进行鉴定,结果有1株(No.156)为Bacillus subtilis,2株(No.171和No.177)为Bacillus pumillus,2株(No.192和No.279)为Bacillus ploymyxa。     

醋酸钙不动杆菌的分离鉴定及溶藻特性

淡水微囊藻水华不仅造成水体动植物缺氧死亡,而且释放藻毒素,影响人类和其它动物的健康。利用液体感染技术,从河南省平顶山市白龟山水库分离一株能够溶解铜绿微囊藻PCC 7806的溶藻菌,命名为溶藻菌5,16S r DNA核苷酸序列测序证实该菌株为醋酸钙不动杆菌。它具有一定的溶藻特异性,只溶解PCC 7806,对FACHB-930和斜生栅藻没有影响,能够促进衣藻和红球藻的生长。最佳溶藻体积比为1∶1。溶藻菌5的菌体和无细胞培养物均具有相同的溶藻效果。显微观察藻细胞被溶解的黄化液显示细菌并未附着在藻细胞周围,也无菌胶膜形成。表明溶藻菌5可能通过释放杀藻物质和与藻竞争营养物质两种机制溶解藻细胞。     

一株赤潮藻溶藻菌的筛选鉴定及溶藻特性分析

为缓解赤潮微藻对海洋生态环境的危害性问题,从潮间带泥样中筛选溶藻菌进行生物学特征分析,通过稀释涂布平板法及平板划线分离法从浙江舟山桃花岛潮间带泥样分离筛选菌株,以中肋骨条藻(Skeletonema costatum)及东海原甲藻(Prorocentrum donghaiense)为受试对象,通过丙酮法提取、测定叶绿素含量变化从中筛选高效抑制赤潮微藻生长的菌株。经形态学观察、生理生化特征检测及16S rDNA序列分析比对初步确定菌株的分类地位。通过不同pH、发酵时间、添加比例等单因素实验,对菌株溶藻特性和溶藻活性物质特性进行研究。从潮间带泥样中共获得43株菌,以菌株2-1-2抑制效果最佳,经鉴定属于芽孢杆菌属,初步命名为Bacillus sp. 2-1-2。溶藻菌Bacillus sp. 2-1-2以胞外分泌溶藻物质的方式进行间接溶藻,最适生长pH为7.0±1.0,菌液最佳发酵时间2—4d、最佳添加比例20%;溶藻活性物质耐热性好,对酸碱耐受性佳,但不耐强酸,在pH=10、80℃处理后溶藻率分别可达(90.57±0.43)%和(89.52±0.96)%。对赤潮藻细胞ROS水平与MDA含量...     

菘蓝内生细菌的分离、筛选和鉴定

筛选和鉴定药用植物菘蓝中对河西走廊常见植物病原菌禾谷镰刀菌、链格孢霉、大斑凸脐蠕孢有拮抗活性的内生细菌。利用常规分离法对菘蓝根、茎、叶、叶柄、花进行内生细菌的分离,采用同步培养法对分离的内生细菌进行活性菌株筛选,并对筛选的活性菌株进行形态观察,生理生化指标测定以及16S r RNA全序列鉴定。结果显示,共分离得到19株内生细菌,其中10株细菌对禾谷镰刀菌有不同程度的抑制作用,占分离菌总数的52.6%,其中菌株G2对禾谷镰刀菌的抑菌效果最强,抑菌率为94.63%,19株细菌均对链格孢霉,大斑凸脐蠕孢有不同程度的抑制作用,占分离菌总数的100%,其中菌株G2、J1、Y5及B2对这两种病原菌的抑菌率最大,均接近100%,经形态学观察,生理生化检测及16S r RNA序列分析,菌株G2为玫瑰色库克菌(Kocuriarosea);菌株J1,Y5为黄杆菌(Microbacterium maritypicum);菌株B2为人参短状杆菌(Brachybacterium ginsengisoli)。从菘蓝中分离筛选到了对河西走廊常见植物病原菌有强抑菌活性的内生细菌,尤其菌株G2、J1、Y5和B2的抑菌性较强,其抑菌活性值得进一步研究。     

南方九孔鲍鲍苗掉板病原菌的药敏测定

为提高鲍鱼培苗的成活率,对分离自广东汕尾一养殖场鲍苗掉板池中(包括水、藻膜和变白鲍苗)的、经回归感染试验证明为致病菌的菌株进行了鉴定和药物敏感性测定。API鉴定表明,这些致病菌株由Vibrio alginolyticus,Vibrio cholerae,Vibrio parahaemolyticus等组成,其中弧菌17株,约占总分离菌株的50％,而溶藻弧菌则为弧菌的优势菌株,有11株,约占弧菌总数的70％。药敏结果显示,绝大多数菌株对链霉素、红霉素和庆大霉素敏感;相反,四环素和新生霉素则对它们没有作用或不敏感。     

Photobiont association and genetic diversity of the optionally lichenized fungus Schizoxylon albescens

The fungus Schizoxylon albescens occurs both as lichen and as saprobe. Lichenized colonies grow on the bark of Populus tremula; saprotrophic morphs grow on dead Populus branches. We wanted to (1) test whether lichenized and saprotrophic S. albescens are genetically distinct, (2) investigate photobiont association and diversity, (3) investigate the interactions between fungi and algae that occur during co-cultivation and (4) test whether Schizoxylon shows algal selectivity during lichenization. Fungal and algal genetic diversity were investigated for three markers. Algae from lichenized thalli were isolated in axenic cultures, and isolate sequence diversity was compared with algae amplified directly from thallus fragments. Co-culture experiments of fungi and algae were performed to study the morphological interaction patterns. Two distinct phylogenetic units are revealed in S. albescens, which are interpreted as phenotypically cryptic species. The algae are related to Coccomyxa and Pseudococcomyxa, and form two distinct sister clades separating samples isolated in cultures from those amplified directly from thallus fragments, indicating that more easily cultured strains of algae are not necessarily major components of the lichens. Schizoxylon albescens interacts with isolated algal strains, similar to fungal-Coccomyxa symbioses in nature. As the system is maintained without difficulty in culture, it can potentially be an easily controlled lichen symbiosis study system under laboratory conditions.     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0.45 microns membrane filters which were then placed upon appropriate media incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37 degrees C in 2.5% CO2 for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia, and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

云南程海湖酵母菌多样性及应用

【目的】针对云南丽江永胜县境内程海湖环境的特殊性,研究高原湖泊环境中酵母菌的多样性,初步探索程海湖环境中酵母菌的利用价值。【方法】对程海湖的湖水和其周边土壤样品中的酵母菌进行分离;应用26S rDNA的D1/D2区域序列分析,并结合形态及生理生化指标对分离获得的酵母菌进行鉴定;采用筛选培养基对已鉴定酵母菌进行产酶定性实验,分析高原湖泊中酵母菌的多样性及可应用性。【结果】分离得到酵母菌64株,对其中63株进行鉴定,归属于9个属22个种(包括4个疑似新种或新变种);地霉属Geotrichum和隐球酵母属Cryptococcus是2种环境中的共有属;在产酶活性筛选中发现有9株产胞外酶活性的菌株,其中YM24373既产蛋白酶又可产淀粉酶。【结论】研究结果显示程海湖中酵母菌组成具有较为丰富的多样性,其应用价值值得进一步研究。     

A membrane filter procedure for assaying cytotoxic activity in heterotrophic bacteria isolated from drinking water

Cytotoxic activity assays of Gram-negative, heterotrophic bacteria are often laborious and time consuming. The objective of this study was to develop in situ procedures for testing potential cytotoxic activities of heterotrophic bacteria isolated from drinking water systems. Water samples were passed through 0·45 μm membrane filters which were then placed upon appropriate media and incubated. After incubation, each membrane filter was transferred to the surface of Y-1 mouse adrenal cells overlaid with 1% agar. The filters were removed after exposure for 15 min. The Y-1 cells were then incubated at 37°C in 2·5% CO₂ for an additional 24 h. The release of putative cytotoxic and cytotonic products from the bacterial colonies was recognized by zones of cellular lysis and injury of Y-1 cells that appeared immediately beneath the membrane. Cytotoxic strains of Aeromonas, Vibrio, Escherichia , and Legionella spp. were readily recognized by this method. About 1% of the bacteria isolated from drinking water also released cytotoxic products. This frequency was dependent upon the primary medium used and the density of bacteria present. The majority of cytotoxic strains isolated from drinking water also expressed protease activity (95%) and haemolytic activity (70%). This in situ membrane filter procedure is a facile method for simultaneously testing many different bacterial colonies.     

The 96-well twin-layer system: a novel approach in the cultivation of microalgae

A novel system for the growth and maintenance of microalgae has been developed that allows the cultivation of a large number of strains with little manual effort. The system is based on a 96-well microtiter plate in which a membrane filter constitutes the bottom of each well. Algal strains are immobilised on the membranes and provided with culture medium through contact with layers of glass fibre located beneath the membranes in a special cultivation chamber. The configuration effectively separates culture medium from algal cells which allows the simultaneous exchange of the culture medium for 96 strains within a few minutes without the need to transfer the algae. If necessary, algal strains can be transferred using multi-channel pipettes. We demonstrate that a large variety of microalgal strains including delicate flagellates can be reliably grown in the system under axenic conditions and without cross-contamination. As an array system, the 96-well twin-layer system using immobilised algae is also amenable to high-throughput and massively parallel applications increasingly sought after in algal bio- and environmental technology.     

新疆两盐湖可培养嗜盐古菌多样性研究

从新疆地区艾比盐湖和艾丁盐湖卤水及泥土样品中分离到86株嗜盐古菌。16S rRNA基因序列分析结果表明,分离自艾比湖的嗜盐古菌分别属于Haloarcula、Halobacterium、Halorubrum、Haloterrigena、Natrinema和Natronorubrum6个属的11个分类单元,而分离自艾丁湖的嗜盐古菌分别属于Haloarcula、Halobiforma、Halorubrum、Haloterrigena、Natrialba、Natrinema6个属的8个分类单元,这一结果表明艾比湖可培养嗜盐古菌生物多样性稍高于艾丁湖。基于16S rRNA基因序列的系统发育分析表明代表菌株ABH15应为Natronorubrum属的中性嗜盐古菌新种,代表菌株ABH07、ABH12、ABH17、ABH19、ABH51和AD30可能是Halobacterium、Halorubrum、Haloterrigena、Haloarcula的新成员。     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

Culturable halophilic archaeal diversity of Ayakekumu salt lake located in Xinjiang, China

Molecular diversity of halophilic archaea from Ayakekumu salt lake was investigated by the polymerase chain reaction (PCR) amplification and culture methods. 19 water samples and 15 soil samples were taken from 19 sites within Ayakekumu salt lake in winter and spring. Under aerobic culture conditions, some halophilic microorganisms were isolated by five different media. The 16S rRNA gene sequences of 62 red strains were amplified by using PCR, determined by the DNA sequencer and analyzed through the BLASTn program subsequently. Results revealed that all sequences belonged to six genera grouped within the Halobacteriaceae. Mostly 16S rRNA gene sequences related to the genera Halorubrum (47%) and Natrinema (24%) were detected. Subsequent analysis by using Shannon index indicated that cultured halophilic archaeal diversities are not significantly different between winter and spring samplings in Ayakekumu salt lake. Similarity values of haloarchaeal 16S rRNA gene sequences to known sequences were less than 97%, suggesting the presence of two novel taxa. In addition, taxonomic characteristics of Natrinema altunense and Halobiforma lacisalsi isolated from Ayakekumu salt lake had been described previously. The discovery of the novel species provides new opportunity to further examine the diversity of these halophilic microorganisms in Ayakekumu salt lake.     

晋阳湖丝状蓝藻的形态学分析与分子鉴定北大核心CSCD

蓝藻是地球上最古老的生物之一,其形态结构较为简单,为产氧型光合作用的原核生物。山西省晋阳湖为华北地区最大的人工湖,该研究以采自晋阳湖水体及岸边附着的蓝藻为材料,采用经典毛细管法分离纯化出5株丝状蓝藻,利用光学显微镜观察其形态结构特征(如细胞形状、藻丝体宽度、是否有鞘)和显微结构,并采用16S rRNA序列分析其系统发育关系,以明确晋阳湖的蓝藻种类,为预防湖泊蓝藻水华的发生、维护水资源环境稳定与生态平衡提供理论数据。结果显示:(1)所分离纯化的5株丝状蓝藻依形态特征归属于3个科,其中2株(JYH005和JYH012)为细鞘丝藻亚科(Leptolyngbyaceae),2株(JYH008和JYH022)为伪鱼腥藻科(Pseudanabaenaceae),1株(JYH010)为沙丝藻科(Desertifilaceae)。(2)基于16S rRNA序列构建的系统发育树显示,5株丝状蓝藻中JYH005为结丝藻属(Nodosilinea)的一种;JYH008可归为Arthronema,该株蓝藻在培养条件下观察到不同的形态特征,可能为新物种;JYH010为沙丝藻属(Desertifilum)的一种;JYH012可归为细鞘丝藻属(Leptolyngbya);JYH022与伪鱼腥藻科聚为一支,由于与该科其他藻相似度低于90%,且不能聚为一支,因此只能归为伪鱼腥藻科。研究表明,基于16S rRNA序列系统发育分析与形态学鉴定结果相一致。该研究结果丰富了山西省晋阳湖丝状蓝藻的多样性,为该湖的资源利用和环境保护提供了一定的科学依据。     

Comparison of various culture methods (Skirrow medium, a blood-free medium and a filtration system enriched in Bolton and Preston broths) for isolation of Campylobacter spp. from raw meat samples

We compared six procedures and investigated the optimal method for isolation of Campylobacter spp. from raw meat samples. Ninety-nine meat samples were enriched in Bolton broth and Preston broth, followed by plating on Skirrow, mCCDA, and blood agar (a membrane filter on its surface) media, respectively. Thirty-nine of 99 samples were positive and 71 Campylobacter were isolated by one or more methods. More than one species of Campylobacter were obtained in 8 (20.5 %) of 39 positive samples and two genotypes were yielded on the same medium (11 samples, 28.2 %) by pulsed-field gel electrophoresis (PFGE) genotyping. Enrichment by Preston broth was significantly better than by Bolton broth (P?<?0.05). Moreover, the latter failed to detect Campylobacter jejuni strains. Skirrow medium was significantly less efficient than mCCDA medium and membrane filtration method (P?<?0.05). Overall, the combination of PC (primary enrichment in Preston broth, followed by selective enrichment on mCCDA agar), PF (primary enrichment in Preston broth, followed by membrane filtration culture onto blood agar), and BF (primary enrichment in Bolton broth, followed by membrane filtration culture onto blood agar) methods provided the optimum isolation rate of Campylobacter spp.