微生物法治理滇池水厂藻渣的基础研究
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摘要
近年来滇池水域严重污染,水体富营养化加剧,常常形成持续周期漫长的水华。水华的爆发不仅带来了严重的生态灾害,同时也给滇池自来水厂带来困扰:在高浓度含藻水的处理过程中,由于藻渣量大而难于处理,经常发生设备堵塞故障。滇池水华藻渣的控制和治理已成为滇池治理的关键课题。目前,除藻方式大体包括三类,一类是单纯的物理除藻,其效果甚微;另一类是化学除藻,该方法虽然效果显著,但容易产生其它的负面影响或造成水质的二次污染;第三类方法就是生物除藻,该种除藻方式具有更为广阔的前景。它充分利用生态系统的食物链或植物群落里的化感作用有效抑制藻类生长。本研究以滇池水华藻渣为研究对象,主要目的是拟通过微生物溶藻作用清除藻渣,处理产生的水经过滇池自来水厂后续处理净化后供水。主要研究内容和结论如下:
首先,从滇池中取含高浓度藻渣的水样和未爆发水华区域的水样进行藻类多样性调查并从水样中分离后续实验藻种;其次,将滇池水样中的溶藻菌株进行分离纯化、鉴定及酶活测定;再次,利用分离到的溶藻细菌进行溶藻特征试验、溶藻条件优化试验以及溶藻菌之间协同作用试验;最后,提出了清除藻渣的工程应用构想。
经过研究,得到以下结论:
(1)无论是春季还是秋季,铜绿微囊藻都是滇池水样中的优势藻。不同季节相比,春季藻类的多样性均明显优于秋季;同一时期滇池水华爆发区域的藻渣样品和未爆发水华区域的水样相比,水华爆发区域藻渣样品中的藻类多样性明显劣于未爆发水华区域的水样。本研究使用在细菌培养基中添加葡萄糖的方法,成功分离到一株藻种,为今后相关的研究提供了实验材料。经研究发现,该藻种叶绿素a在95%冷酒精中680nm处的吸光度值可以代表该藻的细胞数量。
(2)本实验分别采用以果胶为唯一碳源的液体选择培养基和以藻渣为唯一碳源的固体选择培养基筛选溶藻菌,并设计初步溶藻实验对菌株进行复筛,成功筛选出12株对滇池藻渣具有明显的溶藻效果的菌株。对12株溶藻菌株进行形态学、生理生化以及分子生物学鉴定,结果显示,本研究分离出的12株溶藻菌株分别属于5个属,即类芽孢杆菌属Paenibacillus、芽孢杆菌属Bacillus、假单胞菌属Pseudomonas、梭形杆菌属Lysinibacillus和微小杆菌属Exiguobacterium。对10株菌的酶活进行了测定,结果表示:利用果胶为单一碳源的液体培养基分离出的z02、z05、z08均具有较高的果胶酶活性。此外,通过固体培养基分离出的z03、z07也有很高的果胶酶活性,z02与z07的酶活大致相同但是溶藻效果却有一定差异,酶活性不高的g22、g00、z01、z04、z06也有很好的溶藻效果,说明除果胶酶外还有其它的溶藻物质或溶藻方式存在,细菌的溶藻方式多样,溶藻物质的种类非常丰富。
(3)10株溶藻菌溶藻特征试验结果显示,菌液初始浓度越高,溶藻现象发生的越快、越剧烈。而到了试验末期则不同,这可能是由于菌液浓度高,有机营养的含量也很高,而藻渣中的优势藻铜绿微囊藻可以利用有机营养迅速繁殖,菌液浓度越高藻的繁殖越快。因此,中、低加菌浓度在实验后期的溶藻效果有可能优于高加菌浓度的溶藻效果。温度37℃、摇床转速150r/min、加藻量在2.5%-5%、初始pH为7-8时,是z05溶解藻渣的最佳实验条件; z05菌株传代在三代以内不会造成溶藻效果的下降。在溶解藻渣方面,z03与z05之间有互相促进作用。
(4)本研究在前期研究的基础上,进一步提出了利用微生物法清除藻渣的工程应用构想,为滇池水厂藻渣处理工艺提供了重要的经验与参考。本研究经进一步研究和试验完善后,其研究成果有望实现工程应用。
In recent years, the water pollution in Dianchi Lake becomes serious, and the eutrophication is intensifying. It often leads to a along period of alga-bloom that in a continuous cycle. The outbreak of the alga-bloom not only leads to serious ecological disaster, but also brings many difficulties to the waterworks of Dianchi Lake. In the treatment process of the water with high concentrations of alga, the incident of equipment plug often occurred because the volume of algal residue is very large and difficult to deal with. The control and management of the algal residue in Dianchi Lake has become a key issue. At present, there are three Methods in the removal of the alga. The first one is purely physical, and its effect is minimal. The second one is chemical, it may produce negative impacts and cause re-contamination, although the effect is remarkable. The third one is biological, and it has a more broadly prospect. It takes advantage of the food chain of ecosystems and the allelopathy of plant communities, to effectively inhibit the growth of alga. The object of this study is the algal residues in Dianchi Lake, and the main purpose is to remove the algal residue by microorganisms. The product of the clean water from the process will be supplied for Kuming, after followed-up treatment of the waterworks of Dianchi Lake. Main contents and conclusions are as follows:
First of all, algal diversity of water samples which was taken from the algal residue with high concentration of alga and the water samples of the areas where the alga-bloom was not out-breaking were investigated; one of algal species was separated for the follow-up experiment. Secondly, the lytic bacteria strains were isolated and purified from Dianchi Lake, the enzymes’activity of these bacteria was identified. Then, the characteristic test of alga-lying using isolated bacteria, the test on the optimization of alga-lysing and the synergy effect between the alga-lysing bacteria, were carried out. Finally, the engineering applied idea was proposed for removal of algal residue.
After the study, the conclusions are as follows:
(1)Whether it is spring or autumn, Microcystis aeruginosa is the dominant algae in Dianchi Lake. Compared in different seasons, the diversity of alga are much better in spring than in autumn; in the same reason, the diversity of alga in water samples which was taken from the algal residue is better than the samples of the areas where the alga-bloom was not out-breaking. In this study, one of algal species was successfully isolated by adding glucose into the medium for bacteria, This can provide a kind of materials for the follow-up test It can be found from the study that the value of absorbance at 680nm of Chlorophyll a of the algal species in 95% cold alcohol can represent the number of the cells of this algal species.
(2) In this experiment, the alga-lying bacteria were selected by using liquid selection medium with pectin as sole carbon source and solid selection medium with algal residue as the sole carbon source, and the rescreening experiment was designed to reselect alga-lysing strains. Then, 12 strains of alga-lysing bacteria were successfully selected from Dianchi Lake, obviously have strong dissolution of algae. The identification results on morphology、physiological ecology and molecular biology of these strains show that these 12 strains belong to five generas, respectively as Paenibacillus、Bacillus、Pseudomonas、Lysinibacillus and Exiguobacterium. The identification results of enzymes’activity of these bacteria indicated that: z02、z05、z08 which were selected by using liquid selection medium with pectin as sole carbon source have higher pectinase activity. In addition, z03、z07 which were selected by using solid selection medium with algal residue as the sole carbon source also have high pectinase activity. z02 and z07 have the same effect in the pectinase activity, but they have some differences in alga-lysing effect. g22、g00、z01、z04、z06 which pectinase activity are not high, also have a good alga-lysing effect. These study indicate that there are other things or manners existing which can kill algal cells except pectinase, the forms of alga-lysing by bacteria are variety, the type of lysing material is very rich.
(3) The alga-lysing test results of these 10 bacteria show that, the higher the initial concentration of bacteria is, the faster and more intense lytic phenomenon happens. By the end of the test the situation is different, this may be due to high concentrations of bacteria, a high content of organic nutrients, while the dominant algae Microcystis aeruginosa in algal residue can multiply rapidly by using organic nutrients. The higher the concentration of bacteria is, the faster the alga breed. Therefore, the alga-lysing effect of the medium and low concentration of bacteria may be better than the high concentration of bacteria. Temperature of 37℃、rotation speed of 150r/min、algal residue content of 2.5% -5%、the initial pH of 7-8、are the best experimental conditions by z05 to dissolve the algal residue. The decline of lytic effect will not be caused when z05 passaged less than three generations. In areas of dissolving the algal residue, z03 and z05 can promote each other.
(4) In this study, the engineering applied idea was proposed for removal of algal residue using microbial method, based on preliminary studies. It provides a lot of experiences and references for the waterworks of Dianchi Lake in treatment process for removing the alga. Improved by further research and testing, the results of this study can be expected to achieve the level of engineering applications.
首先,从滇池中取含高浓度藻渣的水样和未爆发水华区域的水样进行藻类多样性调查并从水样中分离后续实验藻种;其次,将滇池水样中的溶藻菌株进行分离纯化、鉴定及酶活测定;再次,利用分离到的溶藻细菌进行溶藻特征试验、溶藻条件优化试验以及溶藻菌之间协同作用试验;最后,提出了清除藻渣的工程应用构想。
经过研究,得到以下结论:
(1)无论是春季还是秋季,铜绿微囊藻都是滇池水样中的优势藻。不同季节相比,春季藻类的多样性均明显优于秋季;同一时期滇池水华爆发区域的藻渣样品和未爆发水华区域的水样相比,水华爆发区域藻渣样品中的藻类多样性明显劣于未爆发水华区域的水样。本研究使用在细菌培养基中添加葡萄糖的方法,成功分离到一株藻种,为今后相关的研究提供了实验材料。经研究发现,该藻种叶绿素a在95%冷酒精中680nm处的吸光度值可以代表该藻的细胞数量。
(2)本实验分别采用以果胶为唯一碳源的液体选择培养基和以藻渣为唯一碳源的固体选择培养基筛选溶藻菌,并设计初步溶藻实验对菌株进行复筛,成功筛选出12株对滇池藻渣具有明显的溶藻效果的菌株。对12株溶藻菌株进行形态学、生理生化以及分子生物学鉴定,结果显示,本研究分离出的12株溶藻菌株分别属于5个属,即类芽孢杆菌属Paenibacillus、芽孢杆菌属Bacillus、假单胞菌属Pseudomonas、梭形杆菌属Lysinibacillus和微小杆菌属Exiguobacterium。对10株菌的酶活进行了测定,结果表示:利用果胶为单一碳源的液体培养基分离出的z02、z05、z08均具有较高的果胶酶活性。此外,通过固体培养基分离出的z03、z07也有很高的果胶酶活性,z02与z07的酶活大致相同但是溶藻效果却有一定差异,酶活性不高的g22、g00、z01、z04、z06也有很好的溶藻效果,说明除果胶酶外还有其它的溶藻物质或溶藻方式存在,细菌的溶藻方式多样,溶藻物质的种类非常丰富。
(3)10株溶藻菌溶藻特征试验结果显示,菌液初始浓度越高,溶藻现象发生的越快、越剧烈。而到了试验末期则不同,这可能是由于菌液浓度高,有机营养的含量也很高,而藻渣中的优势藻铜绿微囊藻可以利用有机营养迅速繁殖,菌液浓度越高藻的繁殖越快。因此,中、低加菌浓度在实验后期的溶藻效果有可能优于高加菌浓度的溶藻效果。温度37℃、摇床转速150r/min、加藻量在2.5%-5%、初始pH为7-8时,是z05溶解藻渣的最佳实验条件; z05菌株传代在三代以内不会造成溶藻效果的下降。在溶解藻渣方面,z03与z05之间有互相促进作用。
(4)本研究在前期研究的基础上,进一步提出了利用微生物法清除藻渣的工程应用构想,为滇池水厂藻渣处理工艺提供了重要的经验与参考。本研究经进一步研究和试验完善后,其研究成果有望实现工程应用。
In recent years, the water pollution in Dianchi Lake becomes serious, and the eutrophication is intensifying. It often leads to a along period of alga-bloom that in a continuous cycle. The outbreak of the alga-bloom not only leads to serious ecological disaster, but also brings many difficulties to the waterworks of Dianchi Lake. In the treatment process of the water with high concentrations of alga, the incident of equipment plug often occurred because the volume of algal residue is very large and difficult to deal with. The control and management of the algal residue in Dianchi Lake has become a key issue. At present, there are three Methods in the removal of the alga. The first one is purely physical, and its effect is minimal. The second one is chemical, it may produce negative impacts and cause re-contamination, although the effect is remarkable. The third one is biological, and it has a more broadly prospect. It takes advantage of the food chain of ecosystems and the allelopathy of plant communities, to effectively inhibit the growth of alga. The object of this study is the algal residues in Dianchi Lake, and the main purpose is to remove the algal residue by microorganisms. The product of the clean water from the process will be supplied for Kuming, after followed-up treatment of the waterworks of Dianchi Lake. Main contents and conclusions are as follows:
First of all, algal diversity of water samples which was taken from the algal residue with high concentration of alga and the water samples of the areas where the alga-bloom was not out-breaking were investigated; one of algal species was separated for the follow-up experiment. Secondly, the lytic bacteria strains were isolated and purified from Dianchi Lake, the enzymes’activity of these bacteria was identified. Then, the characteristic test of alga-lying using isolated bacteria, the test on the optimization of alga-lysing and the synergy effect between the alga-lysing bacteria, were carried out. Finally, the engineering applied idea was proposed for removal of algal residue.
After the study, the conclusions are as follows:
(1)Whether it is spring or autumn, Microcystis aeruginosa is the dominant algae in Dianchi Lake. Compared in different seasons, the diversity of alga are much better in spring than in autumn; in the same reason, the diversity of alga in water samples which was taken from the algal residue is better than the samples of the areas where the alga-bloom was not out-breaking. In this study, one of algal species was successfully isolated by adding glucose into the medium for bacteria, This can provide a kind of materials for the follow-up test It can be found from the study that the value of absorbance at 680nm of Chlorophyll a of the algal species in 95% cold alcohol can represent the number of the cells of this algal species.
(2) In this experiment, the alga-lying bacteria were selected by using liquid selection medium with pectin as sole carbon source and solid selection medium with algal residue as the sole carbon source, and the rescreening experiment was designed to reselect alga-lysing strains. Then, 12 strains of alga-lysing bacteria were successfully selected from Dianchi Lake, obviously have strong dissolution of algae. The identification results on morphology、physiological ecology and molecular biology of these strains show that these 12 strains belong to five generas, respectively as Paenibacillus、Bacillus、Pseudomonas、Lysinibacillus and Exiguobacterium. The identification results of enzymes’activity of these bacteria indicated that: z02、z05、z08 which were selected by using liquid selection medium with pectin as sole carbon source have higher pectinase activity. In addition, z03、z07 which were selected by using solid selection medium with algal residue as the sole carbon source also have high pectinase activity. z02 and z07 have the same effect in the pectinase activity, but they have some differences in alga-lysing effect. g22、g00、z01、z04、z06 which pectinase activity are not high, also have a good alga-lysing effect. These study indicate that there are other things or manners existing which can kill algal cells except pectinase, the forms of alga-lysing by bacteria are variety, the type of lysing material is very rich.
(3) The alga-lysing test results of these 10 bacteria show that, the higher the initial concentration of bacteria is, the faster and more intense lytic phenomenon happens. By the end of the test the situation is different, this may be due to high concentrations of bacteria, a high content of organic nutrients, while the dominant algae Microcystis aeruginosa in algal residue can multiply rapidly by using organic nutrients. The higher the concentration of bacteria is, the faster the alga breed. Therefore, the alga-lysing effect of the medium and low concentration of bacteria may be better than the high concentration of bacteria. Temperature of 37℃、rotation speed of 150r/min、algal residue content of 2.5% -5%、the initial pH of 7-8、are the best experimental conditions by z05 to dissolve the algal residue. The decline of lytic effect will not be caused when z05 passaged less than three generations. In areas of dissolving the algal residue, z03 and z05 can promote each other.
(4) In this study, the engineering applied idea was proposed for removal of algal residue using microbial method, based on preliminary studies. It provides a lot of experiences and references for the waterworks of Dianchi Lake in treatment process for removing the alga. Improved by further research and testing, the results of this study can be expected to achieve the level of engineering applications.
引文
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