氧化铁鞘细菌分离鉴定与保藏及其氧化机制的研究
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摘要
本文采用试管静止富集培养及生态模拟溢流装置富集培养，结合平板划线、稀释涂布、平板滴加法，成功地从150份水样中分离到94株鞘细菌。研究结果表明，当水样中鞘细菌数量较少时，采用生态模拟溢流装置富集培养分离效果较好。而当水样中鞘细菌数量较多时，直接采用平板划线、平板滴加法就可取得较好的分离效果。若水样中阳性菌较多时，可在富集培养基中加0.5u/ml青霉素钠盐，对于排除杂菌干扰，提高出菌率有明显的效果。
利用Winogradsky液体试管进行产铁氧化酶鞘细菌的快速初筛，从中筛选出24株产铁氧化酶能力较高的菌株，再经三角瓶液体培养基复筛，获得产酶活性最高的菌株FC9901。试验结果表明，初筛用试管法，以形成铁氧化物沉淀的程度、时间及颜色变化情况来定性表示菌株氧化铁能力的高低，与复筛定量测定酶活的结果相对应，说明Winogradsky液体试管法可以作为试验菌株对铁氧化能力的判断依据。
采用多相鉴定方法对菌株FC9901细胞形态、培养特征、生理生化反应及各种碳源利用情况进行了研究，根据《伯杰氏细菌鉴定手册》第九版，把该菌株鉴定为第十四群鞘细菌类（sheathed bacteria）球衣菌属（Sphaerotilus）浮游球衣菌（Sphaerotilus natans）。对几种鞘细菌保藏方法进行比较研究，表明蒸馏水液体保藏法保藏时间长且方法简便，是一种有效经济简便的保藏方法。
对该菌株氧化铁的生化机制进行了研究。结果表明，该菌株的营养类型为化能有机营养型，其能源主要来自有机碳源的氧化，并不能从Fe2+ 氧化过程中获取能量。活性定位研究和pH、温度、抑制剂试验结果表明，该铁氧化活性物质是酶，而且是一种胞外酶。铁的氧化属于酶学催化过程，并遵守酶的动力学原理。对菌株的最适产酶培养基及产酶条件进行了研究。结果表明，菌株最适产酶培养基（g/L）为：柠檬酸铁铵10，NaNO3 1.2， MgSO4·7H2O 0.5，K2HPO4·7H2O 0.5，CaCl2 0.015，ZnSO4?7H2O 0.0005。最适产酶条件为：温度30℃，起始pH7.0，接种量2%，150ml三角瓶装50ml，150rpm振荡培养72h。最适产酶培养基和产酶条件验证试验结果表明其产酶比基础产酶培养基和产酶条件提高了30%。
采用硫酸铵分级沉淀和透析法对该铁氧化酶进行了初步纯化，同时研究了该酶的酶学性质。该酶最适pH和最适温度分别为7.5和30℃。pH过高或过低都会对该酶的酶活产生较大影响；该酶在30℃室温下可稳定保存150min而活性不会损失。但该酶对高温较敏感，在50℃下加热5min时，活性损失可达65%。金属离子Ca2+、Mg2+、Zn2+对酶有一定的激活和稳定作用；Hg2+对酶有强烈的抑制作用，Cu2+和 Al3+对酶有较强的抑制作用，K+、Fe2+和Na+对酶活力影响不大。
94 strains sheathed bacteria were successfully isolated from 150 water samples by two enrichment methods combined with three isolation methods. the experiment showed that when the quantity of sheathed bacteria in water sample is low, enrichment method is better for isolation, whereas when the quantity of sheathed bacteria in water sample is high, plate streaking and plate dropping maybe more effective. If there is more Gram-positive bacteria in water sample, putting 0.5u/L Penicillin in the enrichment medium is good for isolation.
Rapid screening of sheathed bacteria with Fe2+ oxidizing activity was done by Winogradsky liquid tube method, and 24 strains were selected .In the 24 strains mentioned above, FC9901 was the best strain which can produce oxidizing enzyme with the highest activity by further screening. The study showed that rapid screening result is consistent with further screening result. It showed that Winogradsky liquid tube method can be used as a basis to judge oxidizing ability of strain.
The morphological, culture and physiological features of the strain were studied by poly- characterization method. and based on Bergy,s Manual of Determinative Bacteriology(9thed), FC9901 was identified as Sphaerotilus natans in the Sphaerotilus genus. The study on some preservation methods of sheathed bacteria showed that distilled-water preservation method is a effective and simple method.
Study on the Fe2+ oxidizing mechanism of sheathed bacteria showed that the trophy kind of sheathed bacteria is chemoorganotrophs. The energy is come from oxidize of organic, not gain energy from Fe2+ oxidation .Fe2+ oxidizing activity in the spent culture medium was sensitive to pH, heat, NaN3, pronase, SDS, NaCl, HgCl2. It showed that Fe2+oxidation is a kind of enzyme catalyzed process. The study on localization of activity showed that Fe2+ oxidizing activity is a extra cellular enzyme. Based on above experiment ,We study the optimal medium and condition of enzyme production. The optimum medium consisted of 10g citrate per litre ,1.2g NaNO3 per litre ,0.5gMgSO4 per litre,0.5g K2HPO4?7H2O perlitre ,0.005g CaCl2per litre,0.0005gZnSO4?7H2O per litre. The optimum condition of shaking flask producing enzyme were the temperature 30℃ ,initial pH 7.0, medium volume 50ml medium /150ml flask, inoculum's concentration is 2%, rate of shaking is 150rpm,culture period 72 hours. The enzyme producing activity could reached maximum level, oxidation rate could reached 91.5% after modification of medium and condition. It was increased 30% to first.
Crude oxidizing enzyme was obtained from ammonium sulfate precipitation and dialyze. Biochemical properties of the enzyme were examined as follows, the optimum temperature and pH for oxidizing activity was 30℃ and7.5.The enzyme was easy to lose activity in strong acid or strong alkaline circumstance . The enzyme could keep its original activity after 150min at 30℃.But it was sensitive to high temperature. About 65% activity of the original activity is lost after 5 min at 50℃. Different metal ions showed different effects on the oxidizing activity :Ca2+ 、Mg2+ 、Zn2+ increased the oxidizing activity ,whereas Cu2+、Hg2+ 、Al3+ cause inhibition .K+ 、Na+ 、Fe2+ effected on the activity is not obvious.
利用Winogradsky液体试管进行产铁氧化酶鞘细菌的快速初筛，从中筛选出24株产铁氧化酶能力较高的菌株，再经三角瓶液体培养基复筛，获得产酶活性最高的菌株FC9901。试验结果表明，初筛用试管法，以形成铁氧化物沉淀的程度、时间及颜色变化情况来定性表示菌株氧化铁能力的高低，与复筛定量测定酶活的结果相对应，说明Winogradsky液体试管法可以作为试验菌株对铁氧化能力的判断依据。
采用多相鉴定方法对菌株FC9901细胞形态、培养特征、生理生化反应及各种碳源利用情况进行了研究，根据《伯杰氏细菌鉴定手册》第九版，把该菌株鉴定为第十四群鞘细菌类（sheathed bacteria）球衣菌属（Sphaerotilus）浮游球衣菌（Sphaerotilus natans）。对几种鞘细菌保藏方法进行比较研究，表明蒸馏水液体保藏法保藏时间长且方法简便，是一种有效经济简便的保藏方法。
对该菌株氧化铁的生化机制进行了研究。结果表明，该菌株的营养类型为化能有机营养型，其能源主要来自有机碳源的氧化，并不能从Fe2+ 氧化过程中获取能量。活性定位研究和pH、温度、抑制剂试验结果表明，该铁氧化活性物质是酶，而且是一种胞外酶。铁的氧化属于酶学催化过程，并遵守酶的动力学原理。对菌株的最适产酶培养基及产酶条件进行了研究。结果表明，菌株最适产酶培养基（g/L）为：柠檬酸铁铵10，NaNO3 1.2， MgSO4·7H2O 0.5，K2HPO4·7H2O 0.5，CaCl2 0.015，ZnSO4?7H2O 0.0005。最适产酶条件为：温度30℃，起始pH7.0，接种量2%，150ml三角瓶装50ml，150rpm振荡培养72h。最适产酶培养基和产酶条件验证试验结果表明其产酶比基础产酶培养基和产酶条件提高了30%。
94 strains sheathed bacteria were successfully isolated from 150 water samples by two enrichment methods combined with three isolation methods. the experiment showed that when the quantity of sheathed bacteria in water sample is low, enrichment method is better for isolation, whereas when the quantity of sheathed bacteria in water sample is high, plate streaking and plate dropping maybe more effective. If there is more Gram-positive bacteria in water sample, putting 0.5u/L Penicillin in the enrichment medium is good for isolation.
Rapid screening of sheathed bacteria with Fe2+ oxidizing activity was done by Winogradsky liquid tube method, and 24 strains were selected .In the 24 strains mentioned above, FC9901 was the best strain which can produce oxidizing enzyme with the highest activity by further screening. The study showed that rapid screening result is consistent with further screening result. It showed that Winogradsky liquid tube method can be used as a basis to judge oxidizing ability of strain.
The morphological, culture and physiological features of the strain were studied by poly- characterization method. and based on Bergy,s Manual of Determinative Bacteriology(9thed), FC9901 was identified as Sphaerotilus natans in the Sphaerotilus genus. The study on some preservation methods of sheathed bacteria showed that distilled-water preservation method is a effective and simple method.
Study on the Fe2+ oxidizing mechanism of sheathed bacteria showed that the trophy kind of sheathed bacteria is chemoorganotrophs. The energy is come from oxidize of organic, not gain energy from Fe2+ oxidation .Fe2+ oxidizing activity in the spent culture medium was sensitive to pH, heat, NaN3, pronase, SDS, NaCl, HgCl2. It showed that Fe2+oxidation is a kind of enzyme catalyzed process. The study on localization of activity showed that Fe2+ oxidizing activity is a extra cellular enzyme. Based on above experiment ,We study the optimal medium and condition of enzyme production. The optimum medium consisted of 10g citrate per litre ,1.2g NaNO3 per litre ,0.5gMgSO4 per litre,0.5g K2HPO4?7H2O per
Crude oxidizing enzyme was obtained from ammonium sulfate precipitation and dialyze. Biochemical properties of the enzyme were examined as follows, the optimum temperature and pH for oxidizing activity was 30℃ and7.5.The enzyme was easy to lose activity in strong acid or strong alkaline circumstance . The enzyme could keep its original activity after 150min at 30℃.But it was sensitive to high temperature. About 65% activity of the original activity is lost after 5 min at 50℃. Different metal ions showed different effects on the oxidizing activity :Ca2+ 、Mg2+ 、Zn2+ increased the oxidizing activity ,whereas Cu2+、Hg2+ 、Al3+ cause inhibition .K+ 、Na+ 、Fe2+ effected on the activity is not obvious.
引文
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[95]Takada.M; H. Matsuoka.. et al., Biosynthesis of poly(3-hydroxybutyrate-co -3-hydroxyralerate) by mutant of sphaerotilus natans., Applied Microbiology and Biotechnology, 1995, 44(2) : 37-42.
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[100]Abeliovich,Aharon.,Avoiding ochre deposits in soil drainage pipes .,Agric Water manage.,1985,10(4)327-334.
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[105]Lodi,A.C,et al ,Cadmium,Zinc,Copper,Sliver and Chromium removal from wasterwater by sphaerotilus natans., Bioprocess Engineering ,1998,19(3):197-203. [106]Stoessel,Fred ., On the ecology of ciliates and filamentous bacteria.,Aquat Sci, 1989,51(3):235-248.
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福建师范大学硕士学位论文
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[91]朴真三等,自来水厂细菌固定化除锰及其水质条件研究,环境科学,1998,19(5)：37－40.
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[97]许晓路,活性污泥丝状菌膨胀起因分析及控制措施,环境科
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[100]Abeliovich,Aharon.,Avoiding ochre deposits in soil drainage pipes .,Agric Water manage.,1985,10(4)327-334.
[101]Slowik et al ,Bioaccummulation of copper and lead by sphaerotilus natans.,Acta Microbiol Pol ,1981,30(2):183-194.
[102]Sidorowicz,et al.,Bioaccummulation of copper by polysaprobic zone bacteria sphaeotilus natans., Pol Arch Hydrobiol .,1979,26(4):583-586.
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福建师范大学硕士学位论文