溶藻微生物的分离及溶藻特性的初步研究
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摘要
微生物控藻技术用于防治蓝藻水华具有巨大的潜力，但国内外对溶藻微生物的研究基本上停留在实验室阶段，且多为溶藻细菌的分离、鉴定及溶藻机制的研究，对其他类型的溶藻微生物的研究甚少。本研究建立了溶藻微生物初步筛选体系，且分别从武汉东湖水体和东湖边的土壤中分离到两类特殊的溶藻微生物——类蛭弧菌Y及放线菌11。
类蛭弧菌Y可透过0.22μm滤膜，只有在其宿主菌的协助下才可以分泌溶藻物质。类蛭弧菌Y对多种蓝藻均有强烈的致死作用，且可以通过不断转接持续使其裂解，对采集的多种水样中的细菌也有强烈的致死作用。
在28℃，避光条件下，浓度为1.3×10~6 cell·mL~(-1)的铜绿微囊藻DS在添加10~4 cell·mL~(-1) Y后，4h，24h，48h后铜绿微囊藻DS的死亡率分别为12.1％，23.0％，100％，类蛭弧菌Y的生物量分别为10~2 cell·mL~(-1)，10~4 cell·mL~(-1)，10~6 cell·mL~(-1)。感染32h后，Y的生物量达到峰值(10~8 cell·mL~(-1))，藻细胞完全裂解后的2天内，类蛭弧菌Y的生物量急剧下跌至10~2 cell·mL~(-1)，随后的3-5天，则一直保持在10~2 cell·mL~(-1)的低浓度。
在室内8℃-32℃，黑暗、室内或室外强光日照、缺氧条件下均可以观察到类蛭弧菌Y对蓝藻的强烈地致死作用。类蛭弧菌Y对热和乙醇敏感，45℃水浴处理15min或低浓度(0.0001％)乙醇都可使其完全失活。Y可在软琼脂顶层平板上形成不断扩大的溶藻斑及溶菌斑。通过不断添加蓝藻来传代类蛭弧菌Y的方式可长久保持较高的杀藻活性，研究发现4℃保存有溶藻斑及溶菌斑的平板的方法可维持其杀藻活力1-2个月，传代几次即可恢复高杀藻活性。
我们通过比较建立了一种有效的筛选溶藻放线菌的方法——菌藻双层平板法。此方法简单易行，具有较高的准确性和灵敏度。通过菌藻双层平板法，我们从东湖边的土壤中分离得到的200株余株放线菌中，筛选到30种对野生微囊藻有显著抑制或杀死作用的菌株，6株具有较好的抗污染能力，其中菌株2-11在高氏一号液体培养基中，摇床培养72小时左右后，开始分泌溶藻物质，此时将培养物添加到不同浓度的野生铜绿微囊藻(叶绿素a含量为1.0μg·mL~(-1)和0.21μg·mL~(-1))的培养液中，24h后观察，藻死亡率分别为18.5％和25.0％。
In our study, a primary isolation system of some special algae lysing microorganisms was set up. Through the system, abdellovibrio like organism(BLO) Y and actinomycetes 11 were isolated from the East Lake and soil aside respectively in Wuhan, China.
Y , which could pass through the membrane filter of 0.22μm, secreted unknown algae-lysing substances by capable of killing the host bacteria. Y could kill a few cyanobacteria species and its algae-lysing ability could be maintained through continuous infection. But no algae-lysing effect was observed in eukaryotic algae culture.
As the result of infection of Y on Microcystis aeruginosa DS show that 4, 24,48h later, the mortality ratio of M.aeruginosa DS were 12.1%, 23.0%, 100% respectively. 32h later the concentration of Y reached its peak(about 10~8 cell-mL~(-1)), 2 days later, when algae cells were completely lysed, the concentration of Y sharply decreased to about 10~2 cell-mL~(-1) and maintained this concentration as long as 3 to 5 days.
The algae-lysing characters of Y under certain environmental factors were studied and the result indicated that presented obvious infection effect at temperature from8-32°C,dark, intense light irradiation, but was sensitive to heat and ethanol. (i.e. It would completely lose the activity after insulated at 45°C for 15 mins or exposured to ethanol at a very low concentration (0.0001%).) Also, the strong ability of algae-lysing ability of Y could be preserved only by continous addition of cyanobacteria into the colture. The continous expanded plaques were observed on plate of canobacteria or bacteria growing. Thus, the strain would be stored at 4°C and the viability of algae-lysing could be sustained for 1-2 month.
We constructed a method for effective isolation actinomycetes, which was simple, exact and reliable. Using this method ,we isolated more than 200 actinomycete stains from soil ,among which 30 actinomycete stains could form clear algae-inhibiting-zopm on wild M.aeruginosa plate. Among the 30 actinomycetes,6 strains had strong ability of anti-contamination against other fungi and bacteria ,1 stain(No.2-No.11) started to secrete algae-lysing substance after 72h incubation. And then, the atinomycete sample culture of 72 hours was mixed with culture of wild M.aeruginosa at higher and lower concentration (Chla range from 0.2 to 1μg·mL~(-1)). The mortality of M.aeruginosa after 24
类蛭弧菌Y可透过0.22μm滤膜，只有在其宿主菌的协助下才可以分泌溶藻物质。类蛭弧菌Y对多种蓝藻均有强烈的致死作用，且可以通过不断转接持续使其裂解，对采集的多种水样中的细菌也有强烈的致死作用。
在28℃，避光条件下，浓度为1.3×10~6 cell·mL~(-1)的铜绿微囊藻DS在添加10~4 cell·mL~(-1) Y后，4h，24h，48h后铜绿微囊藻DS的死亡率分别为12.1％，23.0％，100％，类蛭弧菌Y的生物量分别为10~2 cell·mL~(-1)，10~4 cell·mL~(-1)，10~6 cell·mL~(-1)。感染32h后，Y的生物量达到峰值(10~8 cell·mL~(-1))，藻细胞完全裂解后的2天内，类蛭弧菌Y的生物量急剧下跌至10~2 cell·mL~(-1)，随后的3-5天，则一直保持在10~2 cell·mL~(-1)的低浓度。
在室内8℃-32℃，黑暗、室内或室外强光日照、缺氧条件下均可以观察到类蛭弧菌Y对蓝藻的强烈地致死作用。类蛭弧菌Y对热和乙醇敏感，45℃水浴处理15min或低浓度(0.0001％)乙醇都可使其完全失活。Y可在软琼脂顶层平板上形成不断扩大的溶藻斑及溶菌斑。通过不断添加蓝藻来传代类蛭弧菌Y的方式可长久保持较高的杀藻活性，研究发现4℃保存有溶藻斑及溶菌斑的平板的方法可维持其杀藻活力1-2个月，传代几次即可恢复高杀藻活性。
我们通过比较建立了一种有效的筛选溶藻放线菌的方法——菌藻双层平板法。此方法简单易行，具有较高的准确性和灵敏度。通过菌藻双层平板法，我们从东湖边的土壤中分离得到的200株余株放线菌中，筛选到30种对野生微囊藻有显著抑制或杀死作用的菌株，6株具有较好的抗污染能力，其中菌株2-11在高氏一号液体培养基中，摇床培养72小时左右后，开始分泌溶藻物质，此时将培养物添加到不同浓度的野生铜绿微囊藻(叶绿素a含量为1.0μg·mL~(-1)和0.21μg·mL~(-1))的培养液中，24h后观察，藻死亡率分别为18.5％和25.0％。
In our study, a primary isolation system of some special algae lysing microorganisms was set up. Through the system, abdellovibrio like organism(BLO) Y and actinomycetes 11 were isolated from the East Lake and soil aside respectively in Wuhan, China.
Y , which could pass through the membrane filter of 0.22μm, secreted unknown algae-lysing substances by capable of killing the host bacteria. Y could kill a few cyanobacteria species and its algae-lysing ability could be maintained through continuous infection. But no algae-lysing effect was observed in eukaryotic algae culture.
As the result of infection of Y on Microcystis aeruginosa DS show that 4, 24,48h later, the mortality ratio of M.aeruginosa DS were 12.1%, 23.0%, 100% respectively. 32h later the concentration of Y reached its peak(about 10~8 cell-mL~(-1)), 2 days later, when algae cells were completely lysed, the concentration of Y sharply decreased to about 10~2 cell-mL~(-1) and maintained this concentration as long as 3 to 5 days.
The algae-lysing characters of Y under certain environmental factors were studied and the result indicated that presented obvious infection effect at temperature from8-32°C,dark, intense light irradiation, but was sensitive to heat and ethanol. (i.e. It would completely lose the activity after insulated at 45°C for 15 mins or exposured to ethanol at a very low concentration (0.0001%).) Also, the strong ability of algae-lysing ability of Y could be preserved only by continous addition of cyanobacteria into the colture. The continous expanded plaques were observed on plate of canobacteria or bacteria growing. Thus, the strain would be stored at 4°C and the viability of algae-lysing could be sustained for 1-2 month.
We constructed a method for effective isolation actinomycetes, which was simple, exact and reliable. Using this method ,we isolated more than 200 actinomycete stains from soil ,among which 30 actinomycete stains could form clear algae-inhibiting-zopm on wild M.aeruginosa plate. Among the 30 actinomycetes,6 strains had strong ability of anti-contamination against other fungi and bacteria ,1 stain(No.2-No.11) started to secrete algae-lysing substance after 72h incubation. And then, the atinomycete sample culture of 72 hours was mixed with culture of wild M.aeruginosa at higher and lower concentration (Chla range from 0.2 to 1μg·mL~(-1)). The mortality of M.aeruginosa after 24
引文
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