光合细菌对Cu~（2+）的响应及其净水研究
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摘要
水体重金属污染已成为全球性环境污染问题,并且严重影响人类健康和生命。工业废液如经生物材料处理后再排放,可减少对环境的危害。光合细菌是地球上出现最早、自然界中普遍存在、具有原始光能合成体系的原核生物,是在厌氧条件下进行不放氧光合作用的细菌的总称,广泛分布于自然界的土壤、水田、沼泽、湖泊、江海等处,主要分布于水生环境中光线能透射到的缺氧区。对于受到重金属污染的排放物,使用微生物进行处理有环保和经济的价值。本文主要内容是在水体中分离光合细菌并鉴定,对不同浓度Cu2+对光合细菌的影响和光合细菌对养殖场排放污水的净化进行了研究。
(一)从信阳市南湾水库大坝下层水域取水样及附近鱼塘底泥取样。使用液体富集培养基再加灭菌液体石蜡,隔绝氧气培养,重复进行富集培养2-3次后进行纯化分离。得到不同形状颜色的纯化菌2株。经过在不同光照氧气供给的培养条件下,测定2种菌株的生长曲线,其中红色菌株在显微镜下观察,及生化试验后初步鉴定为一株光合细菌。
根据16S rDNA的保守序列设计的常用光合细菌引物。PCR扩增DNA片段大小约为400bp,挑选菌液测序。结果在BLAST比对与Rhodopseudomonas sp的序列同源性达到了100%,证实了分离到的菌种为沼泽红假单胞菌。
(二)在培养至对数期的菌液中添加不同浓度的Cu2+,培养48h。测其生长曲线,利用连续激发式荧光仪测定叶绿素荧光值Fv／Fm,测定不同浓度Cu2对供试培养液光合速率的影响。拍摄透射电镜照片分析其显微结构变化。在高浓度Cu2+处理下,光合细菌生长受到明显抑制,大部分光合片层依然可以分辨,但是一些细胞内部类囊体膨大,整体被膜结构严重受损甚至消失,使内部基粒片层散于细胞内。测定Cu2+对光合细菌的抗氧化物酶活性的影响,发现与对照组相比,SOD, CAT, POD酶活性都有显著提高的变化。
(三)在信阳市郊区某小型猪场(每年饲养猪在200头左右)1个氧化塘中(不含固体排泄物)投放大量培养的沼泽红假单胞菌菌液及水浮莲测其净水效果。经过1年的实验结果表明：流出污水的TP浓度最低为7.1mg／L。总氮浓度最低降低了26%,最高降低了60.8%；流出污水中TN浓度最低为120mg／L。COD浓度最低降低了37.5%最高降低了58.7%。COD浓度和原水相比,最低降低了36.8%,最高降低了65.1%,流出污水中COD浓度最低为670mg／L。进过光合细菌处理后,BOD5最低降低了46%,最高降低了56.4%。结果显示分离的沼泽红假单胞菌对养猪场污水水体理化性质有明显的改善。
The heavy metal pollution in aquatic system has become a global environment problem, and it is severely influence the physical health of people. It will decrease the influence on the environment if the industrial effluents be disposed by biomaterial before discharge into rivers. Photosynthetic bacterium(PSB) is the most original prokaryotes which widespread exist on the earth and nature, and has a synthesis of the original solar energy system, a general call of bacteria which will not react the photosynthetic oxygen under the anaerobic conditions.It is widely existed naturally in soil, paddy fields, marshes, lakes, rivers and sea. PSB mainly exists in the anoxic areas, the place where the sunshine can transmission into deep of water.The main content is that a stain of PSB was separated and identitied, the effects of treatment with Cu2+ and water quality improved of the PSB were analysised in this article.
(一)The water sample was gotten from the upper waters of the Dam of NanWan reservoir in XinYang and the sample sediment from the pound nearby, The liquid enrichment medium was used to culture, and cultivate it isolated from oxygen. And take it pure separate after two times enrichment cultures. Then get three kinds of pure strains in different shapes and colors, give them the different conditions of sunshine oxygen and to test the growth carve of them, then get the conclusion that two of them which grow most quickly in the sunshine anaerobic conditions. The red strain is observed under the microscope, after biochemical confirm it to be a PSB. Next, take the common use PSB consensus primer that designed by which according to 16sRNA consensus sequence. The primer resource is 16rDNA, F5'-TGGTYTGAGAGGATGRYCA*, R5'-CGAATTTCACCTCTACACTCG, PCR Amplification chip estimate about 384bp.The amplification parameters of PCR is 94℃2min,94℃30 s,55℃30 s,72℃30 s,30 cycles,72℃5 min totally have 30 cycles. Take 5μL PCR product to test the electrophoresis on the 1.5% of agarose.PCR Amplification chip estimated as 400bp, and pick the PMMO to sequence. The sequence identity result has reach to 100% on the comparison between Blast alignment and Rhodopseudanonas palustris. The result proved that separated Microorganism is Rhodopseudanonas palustris.
(二)The bacterium reached the logarithmic phage, cultured for 48 h, Cu2+ was added into the culture. The growth curve were tested of it, used the continuously pumped fluorescence to test the Fv/Fm, oxygen electrode was used to test the influence of the different effective microorganism Cu2+ to the tested medium respiratory rate. To analyze the microscopic structure change by taking the photos of TEM imagine of SWCNT. Under the effect of high inhibitory concentration, the growth of the PSB was inhibited obviously. Most of the photosynthetic lamellae still can distinguish, some of the inner class cysts of cell expand. The overall envelope structure was damaged seriously even dissolved and made the lamellae within the grand spread inner of the cell. The influence of the C2+ to the antioxidant activities of PSB and to compare with the control group were determined. Every kind of the enzyme active changed obvious.
(三)The oxidation pond(not included solid wastes), in a small farm of XinYang countryside, is thrown in a large amount of cultivated Pseudomonas and water lettuce in order to test the effects of water purification. One year later, the experimental results is showed that the TN density of effluent was 120mg/L,COD concentration was reduced by 37.5% at lowest,60.8% at highest. COD concentration compared with raw water, was reduced by 36.8% at lowest,65.1% at highest, and COD concentration in the outflowed water was 670 mg/L at lowest,56.4% at highest. The resultes suggested the chemical characters of water bodies of polluted water were improved obviously by photosynthetic bacteria.
(一)从信阳市南湾水库大坝下层水域取水样及附近鱼塘底泥取样。使用液体富集培养基再加灭菌液体石蜡,隔绝氧气培养,重复进行富集培养2-3次后进行纯化分离。得到不同形状颜色的纯化菌2株。经过在不同光照氧气供给的培养条件下,测定2种菌株的生长曲线,其中红色菌株在显微镜下观察,及生化试验后初步鉴定为一株光合细菌。
根据16S rDNA的保守序列设计的常用光合细菌引物。PCR扩增DNA片段大小约为400bp,挑选菌液测序。结果在BLAST比对与Rhodopseudomonas sp的序列同源性达到了100%,证实了分离到的菌种为沼泽红假单胞菌。
(二)在培养至对数期的菌液中添加不同浓度的Cu2+,培养48h。测其生长曲线,利用连续激发式荧光仪测定叶绿素荧光值Fv／Fm,测定不同浓度Cu2对供试培养液光合速率的影响。拍摄透射电镜照片分析其显微结构变化。在高浓度Cu2+处理下,光合细菌生长受到明显抑制,大部分光合片层依然可以分辨,但是一些细胞内部类囊体膨大,整体被膜结构严重受损甚至消失,使内部基粒片层散于细胞内。测定Cu2+对光合细菌的抗氧化物酶活性的影响,发现与对照组相比,SOD, CAT, POD酶活性都有显著提高的变化。
(三)在信阳市郊区某小型猪场(每年饲养猪在200头左右)1个氧化塘中(不含固体排泄物)投放大量培养的沼泽红假单胞菌菌液及水浮莲测其净水效果。经过1年的实验结果表明：流出污水的TP浓度最低为7.1mg／L。总氮浓度最低降低了26%,最高降低了60.8%；流出污水中TN浓度最低为120mg／L。COD浓度最低降低了37.5%最高降低了58.7%。COD浓度和原水相比,最低降低了36.8%,最高降低了65.1%,流出污水中COD浓度最低为670mg／L。进过光合细菌处理后,BOD5最低降低了46%,最高降低了56.4%。结果显示分离的沼泽红假单胞菌对养猪场污水水体理化性质有明显的改善。
The heavy metal pollution in aquatic system has become a global environment problem, and it is severely influence the physical health of people. It will decrease the influence on the environment if the industrial effluents be disposed by biomaterial before discharge into rivers. Photosynthetic bacterium(PSB) is the most original prokaryotes which widespread exist on the earth and nature, and has a synthesis of the original solar energy system, a general call of bacteria which will not react the photosynthetic oxygen under the anaerobic conditions.It is widely existed naturally in soil, paddy fields, marshes, lakes, rivers and sea. PSB mainly exists in the anoxic areas, the place where the sunshine can transmission into deep of water.The main content is that a stain of PSB was separated and identitied, the effects of treatment with Cu2+ and water quality improved of the PSB were analysised in this article.
(一)The water sample was gotten from the upper waters of the Dam of NanWan reservoir in XinYang and the sample sediment from the pound nearby, The liquid enrichment medium was used to culture, and cultivate it isolated from oxygen. And take it pure separate after two times enrichment cultures. Then get three kinds of pure strains in different shapes and colors, give them the different conditions of sunshine oxygen and to test the growth carve of them, then get the conclusion that two of them which grow most quickly in the sunshine anaerobic conditions. The red strain is observed under the microscope, after biochemical confirm it to be a PSB. Next, take the common use PSB consensus primer that designed by which according to 16sRNA consensus sequence. The primer resource is 16rDNA, F5'-TGGTYTGAGAGGATGRYCA*, R5'-CGAATTTCACCTCTACACTCG, PCR Amplification chip estimate about 384bp.The amplification parameters of PCR is 94℃2min,94℃30 s,55℃30 s,72℃30 s,30 cycles,72℃5 min totally have 30 cycles. Take 5μL PCR product to test the electrophoresis on the 1.5% of agarose.PCR Amplification chip estimated as 400bp, and pick the PMMO to sequence. The sequence identity result has reach to 100% on the comparison between Blast alignment and Rhodopseudanonas palustris. The result proved that separated Microorganism is Rhodopseudanonas palustris.
(二)The bacterium reached the logarithmic phage, cultured for 48 h, Cu2+ was added into the culture. The growth curve were tested of it, used the continuously pumped fluorescence to test the Fv/Fm, oxygen electrode was used to test the influence of the different effective microorganism Cu2+ to the tested medium respiratory rate. To analyze the microscopic structure change by taking the photos of TEM imagine of SWCNT. Under the effect of high inhibitory concentration, the growth of the PSB was inhibited obviously. Most of the photosynthetic lamellae still can distinguish, some of the inner class cysts of cell expand. The overall envelope structure was damaged seriously even dissolved and made the lamellae within the grand spread inner of the cell. The influence of the C2+ to the antioxidant activities of PSB and to compare with the control group were determined. Every kind of the enzyme active changed obvious.
(三)The oxidation pond(not included solid wastes), in a small farm of XinYang countryside, is thrown in a large amount of cultivated Pseudomonas and water lettuce in order to test the effects of water purification. One year later, the experimental results is showed that the TN density of effluent was 120mg/L,COD concentration was reduced by 37.5% at lowest,60.8% at highest. COD concentration compared with raw water, was reduced by 36.8% at lowest,65.1% at highest, and COD concentration in the outflowed water was 670 mg/L at lowest,56.4% at highest. The resultes suggested the chemical characters of water bodies of polluted water were improved obviously by photosynthetic bacteria.
引文
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[3]Mahan C A, Majidi V, Holeomb JA.Anal. Chem., Evaluation of the metal uptake of several algae strains in a mulaticomponent matrix utilizing inductively couled plasma enissiom spectrometry. [J]. Analytical Chemistry 1989,(61):624.
[4]Marcel Dekker, Sadip M. Toxic Metal Chemistry in Marine Environments. [J].New York,1992:154.
[5]FujikiM. The transitional condition of MinamataBay and the neighbouring sea Polluted by faetory waste water coniaining mereury.In 6thlnternational. [J].Conference on water Pollation Researeh,1972(12):32-33.
[6]Grotsis-Skretas O, Christaki V. Physiologiealresponses of two marine Phytoptanktonic speeiesto Cu and Hg. [J].MaP Tech Rep Ser.1992,(69):151-155.
[7]Baker M D, Mayfield C 1, Inniss WE. Toxieityof P H, heavy metals and bisulfate to a freshwater green algae. [J].ChemosPhere,1983,12(1):35-37.
[8]章程.四种重金属对海洋钙质角毛藻的毒性.[J].台湾海峡,1999(3)：303-304
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