微囊藻抑制—AgBiO_3应急处置与酵母菌生态抑制法的研究
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摘要
随着现代社会经济的高速发展和城市人口密度的急剧增加,水体中污染物含量越来越高,富营养化状态日趋严重。藻类水华已经给水体、经济和人类健康带来了很大的危害,因此开发安全高效的藻华应急处理和长效生态抑藻技术已经成为富营养化控制中迫切需要解决的问题。当前抑制微生物的纳米材料的开发以及水体中有益菌胞外分泌物对微藻化感抑制作用的发现为人们提供了控制水华的新思路。
在藻华应急处理方面,纳米光催化材料能够利用太阳能有效抑制藻类生长,且安全高效无毒,是一种具有良好应用前景的环境污染治理新技术。常规光催化材料如TiO_2只能利用太阳能中不足5%的紫外光,在实际应用中作用有限,本研究中针对该问题以离子交换法制备了可见光响应型AgBiO_3纳米光催化材料,并将其应用于藻类爆发的应急控制。
同时,着眼于富营养水体的长期藻华污染防治方面,微生物化感抑藻是一种安全、不破坏水体生态环境、不需要外源添加的原位抑藻技术,是解决长效生态抑藻的有效手段。本研究在课题组前期工作基础上以自然环境中大量存在的酵母菌群为对象研究通过促进其胞外化感抑藻物质的产生达到长效抑藻的目的。在研究抑藻效果的基础上,初步探讨了纳米光催化材料及酵母菌胞外化感物质的抑藻机理,为其在水华防治应用中提供理论参考及相关技术支持。主要研究内容包括:
1、用离子交换法制备了具有可见光响应的催化剂AgBiO_3,晶体结构为六方晶格,晶胞常数是a= 5.641(1),c=16.118(2) (?),RWP=7.96,RP=6.12%;透射电镜分析其团聚体为直径接近200nm的游离状球体;光吸收阈值约为470nm,对应的带隙宽度为2.5eV;可见光下对甲基橙有良好的降解能力,并能有效分解亚甲蓝。
2、藻液初始浓度、AgBiO_3浓度、藻液初始pH值和光质是影响铜绿微囊藻受AgBiO_3胁迫时生长状况的主要因素。与AgNO_3和NaBiO_3相比,在可见光下AgBiO_3抑藻效果显著,有效抑制了铜绿微囊藻细胞的活性和叶绿素a含量,使正常藻细胞数目减少;细胞质膜分离,类囊体结构发生改变,气囊破裂。根据实验结果推测AgBiO_3的抑藻机理为其产生的自由基抑制铜绿微囊藻的光合作用,破坏细胞膜,使膜透性增大、电解质外渗,胞内物质流出,进而引发细胞凋亡。
3、通过对BG11培养基优化选出四种对AgBiO_3抑藻效果有极显著影响的因素:ZnSO_4、CaCl_2、CuSO_4和NaNO_3;其中ZnSO_4、CaCl_2、CuSO_4表现出正影响,NaNO_3表现出负影响。当它们浓度分别为:ZnSO_4 0.24 mg·L~(-1),CaCl_2 125.94 mg·L~(-1),CuSO_4 0.28 mg·L~(-1),NaNO_3 600 mg·L~(-1)时,AgBiO_3对铜绿微囊藻的4h抑制率达到最大为61.86%,比优化前的平均值42.79%增长了44.57%。
4、在不添加葡萄糖基质的条件下,混合酵母菌液并未对微囊藻产生抑制作用;而在接种混合酵母菌并添加葡萄糖的混合藻液中,微囊藻生长受到显著抑制,且经高温灭菌的葡萄糖酵母发酵液同样表现出了良好的抑藻活性,表明酵母菌抑藻活性可能是由酵母菌代谢葡萄糖分泌的具有高热稳定性的胞外分泌物表达的。
5、受酵母菌胞外化感物质抑制的藻细胞轮廓清晰,结构完整,而颜色减淡,推测该混合酵母菌的抑藻活性物质主要是通过破坏叶绿素而达到抑藻效果的。通过对受试水样的HPLC-SEC与三维荧光光谱分析,初步确定该抑藻活性物质是有较强紫外吸收作用的含有芳香环的酸性物质。经气相色谱-质谱分析,初步推测酵母菌抑藻活性物质可能为分子量为220.35的2, 6-二叔丁基-4-甲基苯酚和分子量为151.18的2-(3H)-苯并噻唑酮。将两种物质分别用于微囊藻生长实验中,它们都表现出很好的抑藻效果。
With the rapid development of social economy and sharp increase of population density,more and more pollutants in water were discharged into the water,water eutrophication is getting more severe. The bloom of algae in water has brought a large amount of adverse effects to water quality,local economies and human health. Therefore,restricting the overgrowth of algae has received increasing attention. There are many limitations in the traditional methods of inhibiting algae,so the development of new products and new ideas has become an urgent need to control eutrophication problems. The inhibitory effects of microalgae from extracellular matters of beneficial bacterium in water and the current nanosized photocatalyst have provided new ideas for controlling algal blooms.
Photocatalysis technology,which can effectively inhibit the growth of algae with the use of light as the energy source,has been showed to be potentially advantageous for environmental pollution prevention and a bright application future. However,common photocatalyst such as TiO_2 is only activated by UV light with wavelengths below 387 nm for its band gap,which means few of the solar energy could be used for bactericide,thus limited the application of TiO_2. In this study,a novel,visible light-responsive AgBiO_3 algaecide was prepared and characterized,which could restrict the growth of Microcystis aeruginosa and even kill them combined with the simulated solar radiation energy. Microbiological method with extracellular matters of beneficial bacterium is characterized as safety , undestroying with ecological environment of water,in-situ remediation technology with no addition,which is a future direction of eutrophication prevention and controlling. This study find out the preliminary mechanism of inhibition of macroalgae by extracellular matters and photocatalyst and provide a scientific basis for inhibit bloom of microalgae. The main contents of this paper are as follows:
(1)AgBiO_3 was prepared with the ion exchange method,which was the hexagonal lattice with parameters as a= 5.641(1) and c=16.118(2) (?) and the final R factors were R_(WP)=7.96 and R_P=6.12%. SEM photograph showed AgBiO_3 was mainly spherical shape with a diameter of approximately 50~200 nm. There is a strong absorption in the visible region and the absorption edge is approximately 470nm,the band gap calculated is approximately 2.5eV for AgBiO_3. Comparing to undoped TiO2,AgBiO_3 greatly improved photocatalytic activity,decomposing methyl orange dye solutions under visible light irradiation.
(2) Initial biomass density,concentration of AgBiO_3,initial pH and light quality were the main factors which effect the growth inhibition of Microcystis aeruginosa. Compared with AgNO_3 and NaBiO_3,AgBiO_3 had a stronger deleterious effect on cyanobacterium cultured in BG11with radiation of simulated natural light,while NaBiO_3 was the weakest inhibitor. Morphology analysis,determination of chlorophyll a and electrolyte leaking rate revealed that AgBiO_3 algaecide could damage the cyanobacterium wall and cell membrane irreversibly with their high photocatalytic activity,thus inhibited growth and proliferation of M. aeruginosa effectively.
(3) Culture conditions were optimized for improved growth-inhibition of Microcystis aeruginosa exposed to AgBiO_3. The optimised concentrations of medium components were as follows: ZnSO_4 0.24 mg·L~(-1),CaCl_2 125.94 mg·L~(-1),CuSO_4 0.28 mg·L~(-1) and NaNO_3 600 mg·L~(-1). The inhibition rate reached 61.86%,an increased by 44.57% over the mean value (42.79%) before optimisation。
(4) In the absence of growth substrate,the mixed liquid yeast did not produce growth inhibition of algae; however,under the mixed yeast with liquid glucose added,the algae chlorophyll concentration significantly decreased and continued to maintain at a low level,meanwhile,the extracellular matters of yeast sterilized in high temperature showed a good inhibition activity of algae. It indicated that the inhibition activity of yeast is due to extracellular matters which have high thermal stability.
(5) Microscope showed that the algae in the treatment sample had clear-cut contours,integrate structures and pale color compared to that in the control sample. It can be showed that active substance of the mixed yeast inhibited the growth of algae by means of destructing algal chlorophyll. By the analysis of HPLC-SEC and EEM of the samples,it is initially determined that the active substance inhibiting growth of algae is a kind of humic acid with aromatic rings which absorb ultraviolet rays strongly. By the analysis of GC/MS, active substance of mixed yeast inhibiting the growth of algae may be 2,6-Di-tert-butyl-4-methylphenol (the molecular weight of 220.35) and 2-(3H)-benzothiazole ketone (the molecular weight of 151.18).
在藻华应急处理方面,纳米光催化材料能够利用太阳能有效抑制藻类生长,且安全高效无毒,是一种具有良好应用前景的环境污染治理新技术。常规光催化材料如TiO_2只能利用太阳能中不足5%的紫外光,在实际应用中作用有限,本研究中针对该问题以离子交换法制备了可见光响应型AgBiO_3纳米光催化材料,并将其应用于藻类爆发的应急控制。
同时,着眼于富营养水体的长期藻华污染防治方面,微生物化感抑藻是一种安全、不破坏水体生态环境、不需要外源添加的原位抑藻技术,是解决长效生态抑藻的有效手段。本研究在课题组前期工作基础上以自然环境中大量存在的酵母菌群为对象研究通过促进其胞外化感抑藻物质的产生达到长效抑藻的目的。在研究抑藻效果的基础上,初步探讨了纳米光催化材料及酵母菌胞外化感物质的抑藻机理,为其在水华防治应用中提供理论参考及相关技术支持。主要研究内容包括:
1、用离子交换法制备了具有可见光响应的催化剂AgBiO_3,晶体结构为六方晶格,晶胞常数是a= 5.641(1),c=16.118(2) (?),RWP=7.96,RP=6.12%;透射电镜分析其团聚体为直径接近200nm的游离状球体;光吸收阈值约为470nm,对应的带隙宽度为2.5eV;可见光下对甲基橙有良好的降解能力,并能有效分解亚甲蓝。
2、藻液初始浓度、AgBiO_3浓度、藻液初始pH值和光质是影响铜绿微囊藻受AgBiO_3胁迫时生长状况的主要因素。与AgNO_3和NaBiO_3相比,在可见光下AgBiO_3抑藻效果显著,有效抑制了铜绿微囊藻细胞的活性和叶绿素a含量,使正常藻细胞数目减少;细胞质膜分离,类囊体结构发生改变,气囊破裂。根据实验结果推测AgBiO_3的抑藻机理为其产生的自由基抑制铜绿微囊藻的光合作用,破坏细胞膜,使膜透性增大、电解质外渗,胞内物质流出,进而引发细胞凋亡。
3、通过对BG11培养基优化选出四种对AgBiO_3抑藻效果有极显著影响的因素:ZnSO_4、CaCl_2、CuSO_4和NaNO_3;其中ZnSO_4、CaCl_2、CuSO_4表现出正影响,NaNO_3表现出负影响。当它们浓度分别为:ZnSO_4 0.24 mg·L~(-1),CaCl_2 125.94 mg·L~(-1),CuSO_4 0.28 mg·L~(-1),NaNO_3 600 mg·L~(-1)时,AgBiO_3对铜绿微囊藻的4h抑制率达到最大为61.86%,比优化前的平均值42.79%增长了44.57%。
4、在不添加葡萄糖基质的条件下,混合酵母菌液并未对微囊藻产生抑制作用;而在接种混合酵母菌并添加葡萄糖的混合藻液中,微囊藻生长受到显著抑制,且经高温灭菌的葡萄糖酵母发酵液同样表现出了良好的抑藻活性,表明酵母菌抑藻活性可能是由酵母菌代谢葡萄糖分泌的具有高热稳定性的胞外分泌物表达的。
5、受酵母菌胞外化感物质抑制的藻细胞轮廓清晰,结构完整,而颜色减淡,推测该混合酵母菌的抑藻活性物质主要是通过破坏叶绿素而达到抑藻效果的。通过对受试水样的HPLC-SEC与三维荧光光谱分析,初步确定该抑藻活性物质是有较强紫外吸收作用的含有芳香环的酸性物质。经气相色谱-质谱分析,初步推测酵母菌抑藻活性物质可能为分子量为220.35的2, 6-二叔丁基-4-甲基苯酚和分子量为151.18的2-(3H)-苯并噻唑酮。将两种物质分别用于微囊藻生长实验中,它们都表现出很好的抑藻效果。
With the rapid development of social economy and sharp increase of population density,more and more pollutants in water were discharged into the water,water eutrophication is getting more severe. The bloom of algae in water has brought a large amount of adverse effects to water quality,local economies and human health. Therefore,restricting the overgrowth of algae has received increasing attention. There are many limitations in the traditional methods of inhibiting algae,so the development of new products and new ideas has become an urgent need to control eutrophication problems. The inhibitory effects of microalgae from extracellular matters of beneficial bacterium in water and the current nanosized photocatalyst have provided new ideas for controlling algal blooms.
Photocatalysis technology,which can effectively inhibit the growth of algae with the use of light as the energy source,has been showed to be potentially advantageous for environmental pollution prevention and a bright application future. However,common photocatalyst such as TiO_2 is only activated by UV light with wavelengths below 387 nm for its band gap,which means few of the solar energy could be used for bactericide,thus limited the application of TiO_2. In this study,a novel,visible light-responsive AgBiO_3 algaecide was prepared and characterized,which could restrict the growth of Microcystis aeruginosa and even kill them combined with the simulated solar radiation energy. Microbiological method with extracellular matters of beneficial bacterium is characterized as safety , undestroying with ecological environment of water,in-situ remediation technology with no addition,which is a future direction of eutrophication prevention and controlling. This study find out the preliminary mechanism of inhibition of macroalgae by extracellular matters and photocatalyst and provide a scientific basis for inhibit bloom of microalgae. The main contents of this paper are as follows:
(1)AgBiO_3 was prepared with the ion exchange method,which was the hexagonal lattice with parameters as a= 5.641(1) and c=16.118(2) (?) and the final R factors were R_(WP)=7.96 and R_P=6.12%. SEM photograph showed AgBiO_3 was mainly spherical shape with a diameter of approximately 50~200 nm. There is a strong absorption in the visible region and the absorption edge is approximately 470nm,the band gap calculated is approximately 2.5eV for AgBiO_3. Comparing to undoped TiO2,AgBiO_3 greatly improved photocatalytic activity,decomposing methyl orange dye solutions under visible light irradiation.
(2) Initial biomass density,concentration of AgBiO_3,initial pH and light quality were the main factors which effect the growth inhibition of Microcystis aeruginosa. Compared with AgNO_3 and NaBiO_3,AgBiO_3 had a stronger deleterious effect on cyanobacterium cultured in BG11with radiation of simulated natural light,while NaBiO_3 was the weakest inhibitor. Morphology analysis,determination of chlorophyll a and electrolyte leaking rate revealed that AgBiO_3 algaecide could damage the cyanobacterium wall and cell membrane irreversibly with their high photocatalytic activity,thus inhibited growth and proliferation of M. aeruginosa effectively.
(3) Culture conditions were optimized for improved growth-inhibition of Microcystis aeruginosa exposed to AgBiO_3. The optimised concentrations of medium components were as follows: ZnSO_4 0.24 mg·L~(-1),CaCl_2 125.94 mg·L~(-1),CuSO_4 0.28 mg·L~(-1) and NaNO_3 600 mg·L~(-1). The inhibition rate reached 61.86%,an increased by 44.57% over the mean value (42.79%) before optimisation。
(4) In the absence of growth substrate,the mixed liquid yeast did not produce growth inhibition of algae; however,under the mixed yeast with liquid glucose added,the algae chlorophyll concentration significantly decreased and continued to maintain at a low level,meanwhile,the extracellular matters of yeast sterilized in high temperature showed a good inhibition activity of algae. It indicated that the inhibition activity of yeast is due to extracellular matters which have high thermal stability.
(5) Microscope showed that the algae in the treatment sample had clear-cut contours,integrate structures and pale color compared to that in the control sample. It can be showed that active substance of the mixed yeast inhibited the growth of algae by means of destructing algal chlorophyll. By the analysis of HPLC-SEC and EEM of the samples,it is initially determined that the active substance inhibiting growth of algae is a kind of humic acid with aromatic rings which absorb ultraviolet rays strongly. By the analysis of GC/MS, active substance of mixed yeast inhibiting the growth of algae may be 2,6-Di-tert-butyl-4-methylphenol (the molecular weight of 220.35) and 2-(3H)-benzothiazole ketone (the molecular weight of 151.18).
引文
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