溶藻细菌BS03分离、鉴定及其对塔玛亚历山大藻生长的影响

从福建漳江口红树林区筛选出一株对产贝毒赤潮原因藻——塔玛亚历山大藻(Alexandrium tamatense)具有强溶藻能力的细菌,命名为BS03,通过生理生化及16S rDNA序列分析对该菌株进行鉴定,并探讨了菌株BS03对塔玛亚历山大藻的溶藻特性和溶藻代谢产物的初步性质.结果发现,菌株BS03属于微泡菌属(Microbulbifer sp.)相似性达99％;对塔玛亚历山大藻的杀藻效果具有一定浓度效应,在一定浓度范围内处理浓度越高,溶藻效果越好;菌株BS03对处于不同生长时期的塔玛亚历山大藻都表现出较好的杀藻效果,其中对处于延滞期的塔玛亚历山大藻表现出最佳的杀藻效果,处理96 h后,抑藻率达98.17％;不同生长期菌株对塔玛亚历山大藻溶藻作用无明显差异;菌株BS03通过间接作用方式溶藻,所分泌的胞外活性物质的分子量小于1 kDa,耐酸碱、具热稳定性,推测为非蛋白质、非核酸和非多糖类物质.     

一株溶藻菌株的分离鉴定及溶藻特性

从某湖泊中分离出一株具有溶藻作用的菌株W4,并且研究了该菌株溶解铜绿微囊藻的作用效果、作用方式以及铜绿微囊藻与溶藻菌株所处生长期对溶藻效果的影响,并对溶藻菌株进行了生理生化鉴定.结果表明,该菌株对铜绿微囊藻具有很好的去除效果,加入特定量的菌株培养液,8d后铜绿微囊藻的去除率可达99.5%.该菌株通过间接作用方式溶解铜绿微囊藻,并且该菌株分泌的抑藻活性物质为非蛋白质类物质.菌株W4对铜绿微囊藻的溶解作用受藻细胞所处生长期的影响,但不受该菌株所处生长期的影响.根据形态特征及生理生化试验初步鉴定W4菌株为链霉菌属.     

寒区水体中溶藻铜绿假单胞菌的分离和性质研究

我国北方,尤其是东北地区的一些水体由于富营养化导致的水华现象呈增加趋势,成为急需解决的水环境问题.溶藻菌可从生态调控的角度来控制水华的爆发,因此,在富营养化水体治理方面逐渐得到重视.本文从寒区水华爆发水体中分离获得了一株溶藻细菌JM1,系统发育分析表明,菌株JM1与铜绿假单胞菌的同源性最高.VITEK系统鉴定表明,菌株JM1与铜绿假单胞菌的相似度为98%.菌株JM1在不同的碳源和氮源培养基中可产生不同颜色和p H值的代谢产物,而以葡萄糖和蛋白胨分别为碳源和氮源时发酵液的溶藻效果明显好于其它培养基.菌株JM1的溶藻效果随藻细胞初始密度的降低而增强,随发酵液添加量的增加而增加;当菌体细胞的密度达到1011CFU·m L-1时,可单一抑制藻细胞的生长;当菌体细胞的密度大于105CFU·m L-1时,菌体细胞可促进发酵液的溶藻作用.发酵液作用于藻细胞6 h时可明显影响其恢复能力,当暴露时间达18 h时,藻细胞彻底丧失了恢复能力.菌株JM1发酵液在中性和偏碱性的藻液中的溶藻效果最强,并且经过酸化、碱化和高温处理后,仍然保持较高的溶藻活性.本研究可为基于微生物作用过程的水华控制提供优势的土著菌种及其应用上的技术支持.     

营养物质对铜绿微囊藻生长和藻际细菌的影响

菌-藻"体系在水生态平衡中发挥重要作用,为探明蓝藻水华时期的菌藻关系,研究了原位营养刺激后藻际微生物对铜绿微囊藻生长的影响.结果表明,LB培养基可抑制铜绿微囊藻生长,抑藻率为86.49%;而乙酸钠、葡萄糖和柠檬酸钠可促进藻细胞生长,增长率均在50%以上.对LB培养基和蛋白胨刺激后的藻际细菌进行溶藻菌的筛选,共分离出6株具有强效抑藻作用的菌株,其中Bacillus sp.A1抑藻率高达97.55%.16S rRNA高通量测序表明,向铜绿微囊藻中投加营养物质均可改变藻际细菌群落结构并增加物种丰富度,添加LB培养基和蛋白胨可促使藻际细菌群落数量剧增.生理生化响应表明,藻细胞受LB培养基和蛋白胨胁迫后,活性氧（ROS）水平和丙二醛（MDA）含量激增,细胞氧化损伤严重;超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性先急剧上升后下降,抗氧化酶系统受损.扫描电镜显示,LB培养基可使藻细胞逐渐萎缩,最终破碎;而藻际细菌显著增多.因此,在蓝藻水华暴发的水体进行适当的外部营养刺激可在一定程度上实现溶藻菌原位抑藻.     

Isolation and algae-lysing characteristics of the algicidal bacterium B5

Water blooms have become a worldwide environmental problem.Recently,algicidal bacteria have attracted wide attention as possible agents for inhibiting algal water blooms.In this study,one strain of algicidal bacterium B5 was isolated from activated sludge.On the basis of analysis of its physiological characteristics and 16S rDNA gene sequence,it was identified as Bacillusfusiformis.Its algae- lysing characteristics on Microcystis aeruginosa,Chlorella and Scenedesmus were tested.The results showed that:(1)the algicidal bacterium B5 is a Gram-negative bacterium.The 16S rDNA nucleotide sequence homology of strain B5 with 2 strains of B.fusiformis reached 99.86%,so B5 was identified as B.fusiformis;(2)the algal-lysing effects of the algicidal bacterium B5 on M.aeruginosa, Chlorella and Scenedesmus were pronounced.The initial bacterial and algal cell densities strongly influence the removal rates of chlorophyll-α.The greater the initial bacterial cell density,the faster the degradation of chlorophyll-α.The greater the initial algal cell density,the slower the degradation of chlorophyll-α.When the bacterial cell density was 3.6 x l07 cells/ml,nearly 90% of chlorophyll-αwas removed.When the chlorophyll-αconcentration was less than 550μg/L,about 70% was removed;(3)the strain B5 lysed algae by secreting metabolites and these metabolites could bear heat treatment.     

Spatial heterogeneity of cyanobacterial communities and genetic variation of Microcystis populations within large, shallow eutrophic lakes (Lake Taihu and Lake Chaohu, China)

Cyanobacteria, specifically Microcystis, usually form massive blooms in eutrophic freshwater lakes. Cyanobacterial samples were collected from eight sites of both Lake Taihu and Lake Chaohu in late summer to determine the diversity and distribution pattern of cyanobacteria and Microcystis in large, shallow, entropic lakes with significant spatial heterogeneity and long-term Microcystis bloom. Molecular methods based on denaturing gradient gel electrophoresis and clone library analysis were used. A similar heterogeneous distribution pattern of cyanobacteria in both lakes was observed. Most parts of these two lakes with high trophic level were dominated by Microcystis. However, in the regions with low trophic levels as well as low concentrations of chlorophyll a, Synechococcus occupied a considerable percentage. Different morphospecies and genotypes dominated the bloom-forming Microcystis populations in these two lakes. Microcystis viridis and Microcystis novacekii were dominant in Lake Chaohu, whereas Microcystis flos-aquae was dominant in Lake Taihu. Only 2 of the13 Microcystis operational taxonomic units were shared between these two lakes. Analysis of molecular variance based on 16S to 23S internal transcribed spacer sequences indicated the significant genetic differentiation of Microcystis between these two lakes (F_st = 0.19, p < 0.001). However, only 19.46% of the genetic variability was explained by the population variation between lakes, whereas most (80.54%) of the genetic variability occurred within the lakes. Phylogenetic analysis revealed no phylogeographic structure of Microcystis population in these two lakes, as illustrated by their cosmopolitan nature. Our results revealed that spatial heterogeneity within lakes has more impact on the cyanobacterial diversity than geographical isolation in a local scale.     

杀藻细菌色素Deinoxanthin的理化性质及其制剂研发

塔玛亚历山大藻(Alexandrium tamarense)是一种有毒甲藻,常引发赤潮,对海洋环境及人类健康造成极大的威胁.前期研究发现,耐辐射球菌属的一株细菌Y35能够通过分泌杀藻化合物Deinoxanthin达到控制塔玛亚历山大藻生长的效果.为了加强Deinoxanthin的应用效果,对其在不同条件下的稳定性进行了研究.结果表明,杀藻化合物Deinoxanthin有较高的热稳定性,且在中性和碱性条件下稳定,在光照和紫外线辐射下易分解.而通过壳聚糖和海藻酸钠对Deinoxanthin进行包埋固定化,从而构建缓释微球提高了Deinoxanthin的稳定性.杀藻制剂能够高效地表现出杀藻效果,杀藻制剂的研发为杀藻细菌色素应用于藻华治理奠定了基础.     

Off-flavor compounds from decaying cyanobacterial blooms of Lake Taihu

The effect of cyanobacterial bloom decay on water quality and the complete degradation of cyanobacterial blooms in a short period were examined by an enclosure experiment in Gonghu Bay of Lake Taihu,China.Water quality parameters as well as taste and odor compounds during the breakdown of cyanobacterial blooms were measured.Results showed that the decay of cyanobacterial blooms caused anoxic water conditions,decreased pH,and increased nutrient loading to the lake water.The highest concentrations of dimethyl sulfide (DMS),dimethyl trisulfide (DMTS),and β-cyclocitral were observed in the anoxic water,at 62331.8,12413.3,and 1374.9 ng/L,respectively.2-Methylisoborneol was dominant during the live growth phase of cyanobacterial blooms,whereas DMS and DMTS were dominant during the decomposition phase.Dissolved oxygen,pH,and chlorophyll a were negatively correlated with DMS,DMTS,and β-cyclocitral,whereas total phosphorus,total nitrogen,and ammonium (NH4+-N) were positively correlated with DMS,DMTS,β-cyclocitral,and β-ionone.The experimental results suggested that preventing the anaerobic decomposition of cyanobacterial blooms is an important strategy against the recurrence of a malodor crisis in Lake Taihu.     

南京玄武湖微囊藻水华种类组成的研究

2005-08～2006-10采集蓝藻水华样品, 通过形态学特征和ITS序列分子标记的方法对南京玄武湖水华的优势种类进行了鉴定. 结果表明,玄武湖水华主要由微囊藻构成, 通过形态特征鉴定主要种类为铜绿微囊藻(M. aeruginosa)、M. novacekii 和惠氏微囊藻(M. wesenbergii). 3种藻的生物量在水华中的相对比例分别为30%～45%、35%～40%和10%～15%. 采用分子标记的方法对２种优势种类进行了分子鉴定. PCR扩增ITS序列并进行测序, 通过基因序列比对和基于ITS序列的系统发生树分析, 进一步确定了优势种类为M. aeruginosa和M. novacekii; 通过检测产毒基因mcyB来确认2株优势微囊藻的产毒特性, PCR扩增结果均出现了大约为780 bp的特异性片段, 表明这2种藻均为产毒种. 对水华提取物的高效液相色谱(HPLC)测定表明, 玄武湖水华中微囊藻毒素的种类主要是MC-LR和MC-RR.     

Responses of alkaline phosphatase activity to wind-driven waves in a large, shallow lake: Implications for phosphorus availability and algal blooms

Phosphorus is a vital nutrient for algal growth, thus, a better understanding of phosphorus availability is essential to mitigate harmful algal blooms in lakes. Wind waves are a ubiquitous characteristic of lake ecosystems. However, its effects on the cycling of organic phosphorus and its usage by phytoplankton remain poorly elucidated in shallow eutrophic lakes. A mesocosm experiment was carried out to investigate the responses of alkaline phosphatase activity fractions to wind waves in large, shallow, eutrophic Lake Taihu. Results showed that wind-driven waves induced the release of alkaline phosphatase and phosphorus from the sediment, and dramatically enhanced phytoplanktonic alkaline phosphatase activity. However, compared to the calm conditions, bacterial and dissolved alkaline phosphatase activity decreased in wind-wave conditions. Consistently, the gene copies of Microcystis phoX increased but bacterial phoX decreased under wind-wave conditions. The ecological effects of these waves on phosphorus and phytoplankton likely accelerated the biogeochemical cycling of phosphorus and promoted phytoplankton production in Lake Taihu. This study provides an improved current understanding of phosphorus availability and the phosphorus strategies of plankton in shallow, eutrophic lakes.     

Quantification of microcystin-producing and non-microcystin producing Microcystis populations during the 2009 and 2010 blooms in Lake Taihu using quantitative real-time PCR

Lake Taihu, a large, shallow hypertrophic freshwater lake in eastern China, has experienced lake-wide toxic cyanobacterial blooms annually during summer season in the past decades. Spatial changes in the abundance of hepatotoxin microcystin-producing and non-microcystin producing Microcystis populations were investigated in the lake in August of 2009 and 2010. To monitor the densities of the total Microcystis population and the potential microcystin-producing subpopulation, we used a quantitative real-time PCR assay targeting the phycocyanin intergenic spacer (PC-IGS) and the microcystin synthetase gene (mcyD), respectively. On the basis of quantification by real-time PCR analysis, the abundance of potential toxic Microcystis genotypes and the ratio of the mcyD subpopulation to the total Microcystis varied significantly, from 4.08× 10⁴ to 5.22× 10⁷ copies/mL, from 5.7% to 65.8%, respectively. Correlation analysis showed a strong positive relationship between chlorophyll-a, toxic Microcystis and total Microcystis; the abundance of toxic Microcystis correlated positively with total phosphorus and ortho-phosphate concentrations, but negatively with TN:TP ratio and nitrate concentrations. Meanwhile the proportion of potential toxic genotypes within Microcystis population showed positive correlation with total phosphorus and ortho-phosphate concentrations. Our data suggest that increased phosphorus loading may be a significant factor promoting the occurrence of toxic Microcystis bloom in Lake Taihu.     

铜绿微囊藻生长代谢的密度依赖性特征及分子机制

蓝藻水华是淡水湖库普遍面临的水环境问题,目前水华形成机制研究大多关注外界环境因素而忽略了藻类自身及群体的关键调节作用.因此,本文以初始细胞密度为单一变量,考察了不同初始细胞密度条件下蓝藻生长、营养物质利用、叶绿素、藻毒素、胞外分泌物随时间的变化情况.结果发现,初始细胞密度会影响铜绿微囊藻的环境适应性和生长情况,当接种密度≥1×10⁶ cell·mL^-1,铜绿微囊藻适应期消失,初始细胞密度从1×10⁵ cell·mL^-1增加至1×10⁷ cell·mL^-1,最高细胞密度增加了68%,生长速率提高了21%,并且叶绿素变化趋势与生长情况一致.胞外分泌物随初始细胞密度增加而逐渐增加,有利于蓝藻细胞聚集成膜.当初始细胞密度较低（1×10⁵ cell·mL^-1）时,单细胞藻毒素分泌量反而增加,以提高蓝藻的环境适应能力.在高初始密度（1×10⁷ cell·mL^-1）条件下,铜绿微囊藻主要通过上调丙酮酸代谢和碳代谢,促进细胞生长增殖.因此,细胞密度是影响铜绿微囊藻生长代谢的重要因素,在蓝藻水华形成过程中可能发挥重要作用,水华防治应当在细胞密度较低的阶段进行.     

中肋骨条藻高效溶藻菌FDHY-C3的分离鉴定及溶藻作用研究

从福建长乐海域的表层海水中分离获得一株对中肋骨条藻具有高效溶藻作用的溶藻菌FDHY-C3,通过形态学、生理生化及16S rDNA序列分析表明,菌株FDHY-C3属于交替单胞菌属(Alteromonas)。该菌株对常见的甲藻（dinoflagellate）、硅藻(diatom)以及针胞藻（raphidophyte）类的多种赤潮藻都有较高的溶藻作用,对中肋骨条藻溶藻效果尤为明显,72 h可达98.18%。溶藻菌FDHY-C3通过胞外分泌物间接溶藻;溶藻物质的溶藻特性不受反复冻融的影响,但是受酸碱性及温度的影响;溶藻活性物质具有被乙醇和乙酸乙酯沉淀的特性,为蛋白类物质,分子量为10～14 kD。     

海洋细菌N3对几种赤潮藻的溶藻效应

从深圳大鹏湾南澳赤潮暴发海域的表层沉积物中分离得到1株海洋溶藻细菌(编号N3),以三角褐指藻(Phaeodactylumtricornutum)和常见赤潮生物锥状斯氏藻(Scrippsiella trochoidea)、海洋原甲藻(Prorocentrum micans)和中肋骨条藻(Skeletonemacostatum)为实验藻种,利用液体感染法研究了该株溶藻细菌的溶藻效应及溶藻方式.结果表明,菌株N3对中肋骨条藻和三角褐指藻均无溶藻活性,而菌悬液的体积分数为2%和10%时分别对锥状斯氏藻和海洋原甲藻有较好的溶藻效果,但溶藻现象不同.菌株N3可使锥状斯氏藻的细胞变形,胞内物质分布不均匀并最终死亡;使海洋原甲藻的藻细胞膨胀变形,细胞膜内物质聚集于一端,最终藻细胞破裂死亡.菌株N3对锥状斯氏藻和海洋原甲藻的溶藻方式相同,均以直接溶藻为主.菌株N3的菌悬液以2%、1%和0.1%的体积分数接种到锥状斯氏藻藻液中时,细菌的密度整体上呈下降趋势,2%处理组中藻细胞在120 h内全部死亡,而1%和0.1%处理组藻细胞的增长率略低于对照组.菌株N3的菌悬液以10%、5%和1%的体积分数接种到海洋原甲藻藻液中时,细菌的密度呈下降趋势,10%和5%处理组中藻细胞至120 h时死亡率分别为78%和70%,而1%处理组的藻细胞与对照组无显著差异.电镜观察、生理生化鉴定及16S rRNA鉴定结果表明,菌株N3为芽胞杆菌属(Bacillus sp.).     

A flow cytometer based protocol for quantitative analysis of bloom-forming cyanobacteria (Microcystis) in lake sediments

A quantitative protocol for the rapid analysis of Microcystis cells and colonies in lake sediment was developed using a modified flow cytometer, the CytoSense. For cell enumeration, diluted sediment samples containing Microcystis were processed with sonication to disintegrate colonies into single cells. An optimized procedure suggested that 5 mg dw (dry weight)/mL dilution combined with 200 W × 2 min sonication yielded the highest counting efficiency. Under the optimized determination conditions, the quantification limit of this protocol was 3.3×10⁴ cells/g dw. For colony analysis, Microcystis were isolated from the sediment by filtration. Colony lengths measured by flow cytometry were similar to those measured by microscopy for the size range of one single cell to almost 400 μupm in length. Moreover, the relationship between colony size and cell number was determined for three Microcystis species, including Microcystis flos-aquae, M. aeruginosa and M. wessenbergii. Regression formulas were used to calculate the cell numbers in different-sized colonies. The developed protocol was applied to field sediment samples from Lake Taihu. The results indicated the potential and applicability of flow cytometry as a tool for the rapid analysis of benthic Microcystis. This study provided a new capability for the high frequency monitoring of benthic overwintering and population dynamics of this bloom-forming cyanobacterium.     

水稻秸秆浸泡液对铜绿微囊藻生理特性的影响

室内利用流式细胞仪对暴露于不同浓度水稻秸秆浸泡液下铜绿微囊藻的细胞生长、细胞膜完整性、膜电位、酯酶活性进行了为期15 d的检测,研究了水稻秸秆浸泡液对铜绿微囊藻生理特性的影响.结果发现,浸泡5 d的水稻秸秆液可以抑制藻的生长,呈明显的浓度抑制型变化;PI荧光检测显示暴露于浸泡液的各组细胞(>98%)的细胞膜保持高度完整;FDA荧光检测显示与对照组相比,第1、4d处理组酯酶活性增强和减小的细胞都有增加,但活性下降的细胞数量明显多于活性增强的细胞,第7 d酯酶活性下降的细胞数量明显增加,而增强的细胞数量基本不变,第10、15 d酯酶活性正常的细胞数量增加显著,而酯酶活性下降的细胞数量明显减少,增强的细胞变化幅度较小;DIOC6(3)荧光检测显示膜电位在前7 d变化显著,第10、15d变化程度减弱,与酯酶活性变化趋势一致.分析表明,浸泡液存在促进和抑制藻细胞生长的两种作用,抑制作用占据优势,随着暴露时间延长,促进作用消失,抑制作用有所下滑,浸泡液对藻细胞生长具有抑制性而非致死性的作用.     

Decomposition and phosphorus release from four di erent size fractions of Microcystis spp. taken from Lake Taihu, China

Decomposition of Microcystis is accompanied by the release of phosphorus, during bacteria play an important role. A series of experiments were undertaken to evaluate the e ect of bacteria on the decomposition of Microcystis taken from Lake Taihu, China, a lake that is su ering from dense Microcystis blooms. The 16 experiments involved four size fractions of colonial Microcystis with or without the addition of lake sediment and Gram-negative bacterial inhibitor NaN3. The highest decomposition rates were recorded for the smallest size Microcystis fraction (< 25 m) with the addition of the sediment. The lowest decomposition rates were recorded for the smallest Microcystis fraction without the sediment, but with the addition of Gram-negative bacterial inhibitor NaN3. The higher decomposition rates in the treatments with NaN3 and sediment suggest that Gram-positive bacteria in the sediment are important for the decomposition process. Additionally, higher concentrations of total dissolved phosphorus (TDP) in the treatments with NaN3 suggest that more phosphorus accumulates in the Gram-negative bacterial cells around the colony, which may be an important source of phosphorus for Microcystis cells. In the no-sediment treatments, the ratios of TDP concentration to initial TP concentration were 64%–82%. The results of this experiment suggest that both Gram-negative and Gram-positive bacteria play an important role in the decomposition of Microcystis cells and the release of phosphorus from Microcystis colonies.     

铜绿微囊藻溶藻菌EA-1的分离鉴定及溶藻特性

从广州流花湖分离获得一株溶藻菌株EA-1,16S rDNA分析表明菌株EA-1属于肠杆菌属（Enterobacter）.研究了肠杆菌EA-1对铜绿微囊藻的溶藻效果和溶藻机制.结果表明,对数期EA-1具有最佳溶藻效果,投加比例为10%,初始叶绿素a含量为1.43mg/L时,EA-1能实现3d内完全除藻,叶绿素a含量为2.39mg/L时,共培养6d后,抑制率为84.1%±1.3%.EA-1通过分泌胞外溶藻物质间接溶藻,生理生化响应表明,EA-1无菌滤液胁迫下,藻细胞膜脂过氧化损伤严重,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和抗坏血酸过氧化物酶（APX）活性先急剧上升后下降.三维荧光光谱（EEM）表明溶藻产物为类腐殖酸,类富里酸和类蛋白类物质.扫描电子显微镜（SEM）显示藻细胞出现褶皱,内陷和萎缩现象.透射电子显微镜（TEM）显示藻细胞破坏过程为：首先,胶质层与细胞壁分离,光合片层变得松散和不规则,内含物被部分降解.随后,光合片层被彻底破坏,DNA核物质和多聚磷酸盐等营养物质颗粒被降解,藻细胞内部结构被完全破坏,藻细胞死亡.     

太湖蓝藻水华期间水体和底泥中产毒微囊藻与非产毒微囊藻种群丰度研究

运用实时荧光定量PCR(Quantitative real-time PCR,QPCR)技术研究了太湖蓝藻水华期间不同湖区水体和底泥中产毒微囊藻与总微囊藻种群丰度.结果表明,湖区间产毒微囊藻种群和总微囊藻种群丰度明显不同:在水体中,竺山湾(N5)和梅梁湾(N2)产毒微囊藻和总微囊藻种群丰度高于贡湖湾(N4)和湖心(S4...     

Decomposition and phosphorus release from four different size fractions of Microcystis spp. taken from Lake Taihu, China

Decomposition of Microcystis is accompanied by the release of phosphorus,during bacteria play an important role.A series of experiments were undertaken to evaluate the effect of bacteria on the decomposition of Microcystis taken from Lake Taihu,China,a lake that is suffering from dense Microcystis blooms.The 16 experiments involved four size fractions of colonial Microcystis with or without the addition of lake sediment and Gram-negative bacterial inhibitor NAN_3.The highest decomposition rates were reco...