锰氧化细菌的分离鉴定及其锰氧化能力的研究

发布时间：2018-04-28 00:51

本文选题：锰氧化细菌 + 分离鉴定　；参考：《哈尔滨工业大学》2014年硕士论文

【摘要】：锰在地壳中的含量仅此于铁，是地壳中的主要成分。广泛存在于自然界中，是人体所必需的微量元素之一，但是如果摄入锰量过多会引起神经性疾病，危害身体健康。我国饮用水要求规定：对锰含量的最高允许浓度为0.1mg/L。且在生活用水和工业用水过程中，锰含量超标也是影响水质的主要因素之一，如影响水的色度和气味等。近年来，，生物除锰工艺得到了发展和广泛应用，与传统化学除锰法相比，生物氧化除锰具有处理效果好，处理效果稳定，运行所需费用低等明显优势。水相和土壤环境中广泛存在着锰氧化微生物，可高效地将Mn(II)氧化成Mn(IV)，在锰的生物地球化学循环中具有重要的作用。有些锰氧化细菌具有极强的Mn(IV)的生成率，比化学催化快10万倍左右。因此，锰氧化细菌在生物除锰研究中具有较高的应用价值，可用于含锰量高的水处理和土壤中重金属污染的治理。但是，目前对于锰氧化菌除锰机制的研究还不够深入。本实验利用三种不同富集培养基对不同环境样品进行富集，通过LBB指示剂法筛选富集效果好的样品作为分离样品。同时利用LBB指示剂法筛选具有锰氧化能力的菌株，待分离纯化后通过生理生化及分子生物学等鉴定方法进行分析鉴定。并测定分离菌株生长曲线、及接种量、Mn2+浓度、pH值等对生长曲线的影响及研究分离菌株的锰氧化特性。本研究以土壤和河流底泥等为分离样品，首先，利用PYCM培养基、K培养基和PC培养基进行富集和分离。对分离获得的10株细菌通过LBB指示剂进行了对Mn(II)的氧化能力检测。然后通过MIDI微生物鉴定系统、Biolog分析、生理生化分析和16S rDNA测序分析，初步鉴定菌种MB1为地衣芽胞杆菌（Bacillus licheniformis），MB2为巨大芽孢杆菌（Bacillus-megaterium）。实验结果表明不同接种量对除锰菌MB1和MB2的生长曲线总体来说影响不大。MB1的最适接种量为5%，MB2的最适接种量为15%。当培养基的pH值为5.0-7.0时，MB1和MB2菌体均在此范围内可以生长的很好，当pH值为7.0时，菌体生长状态达到最好，培养基中生物量最大，说明在此条件下菌体能够很快适应环境快速进入对数生长期，且对数期时间较其它pH值条件下菌体对数期时间更长。之后，随着培养基碱性增强，生物量又逐渐减小，直至pH值为9.0时菌体的生长被抑制。当pH值为9.0时，碱性环境中锰氧化菌的生理活动受到影响。在pH值为6.0-8.0的条件下，MB1和MB2对Mn2+氧化率均在35%以上。对除锰菌的耐锰能力进行分析，研究了不同Mn2+浓度对菌体生长的影响，当浓度高于12mmol/L时，MB1菌体的生长速度很慢甚至几乎不生长，表明当Mn2+浓度过高时会抑制细菌的生长。MB2菌株在含Mn2+培养基中的菌量比不含Mn2+的小，但当培养基在12mmol/L范围以内变化差距不大，表明MB2菌对高浓度Mn2+的耐受性比MB1菌要好。通过原子吸收法测定菌株除锰率，在K培养基中培养7天后，菌种MB1的锰氧化率为61.7%，菌种MB2的锰氧化率为90%。研究初始pH值（5-9）的K培养基中，pH值对锰氧化率的影响，在pH值为7.0时，MB1和MB2的生物氧化率最高，可达到80%左右。当pH为5.0和6.0时，菌株MB1和MB2对Mn2+氧化率不到60%。说明酸性环境影响了锰氧化菌的生长，可能是由于酸性环境使培养基的组成发生改变，从而导致锰氧化率下降。通过研究锰氧化菌在生长过程中对pH值的影响表明锰氧化菌使培养基pH值升高。对除锰菌MB1和MB2的除锰特性进行分析，证明本实验中所筛选到的锰氧化细菌在对Mn2+进行氧化过程中包含生物氧化和化学氧化的共同作用。该菌为进一步研究锰氧化机制和实现强化除锰提供了重要的菌种。
[Abstract]:Manganese content in the crust is only iron, it is the main component of the earth's crust. It is widely found in nature and is one of the essential trace elements necessary for the human body. However, excessive intake of manganese can cause neurogenic diseases and harm the health of the body. The requirement for drinking water in our country is that the maximum allowable concentration of manganese content is 0.1mg/L. and in domestic water use. In the process of water use, manganese content is also one of the main factors affecting the water quality, such as the color and odor of water. In recent years, the biological manganese removal process has been developed and widely used. Compared with the traditional chemical manganese removal method, biological oxidation removal of manganese has the advantages of good treatment effect, stable treatment effect and low cost of operation. Manganese oxide microorganism is widely used in water and soil environment, which can efficiently oxidize Mn (II) into Mn (IV) and play an important role in the biogeochemical cycle of manganese. Some manganese oxidizing bacteria have a very strong Mn (IV) formation rate, 100 thousand times faster than chemical catalysis. Therefore, manganese oxide bacteria have a better effect in the study of biological manganese removal. High application value can be used in the treatment of high manganese content water and the treatment of heavy metal pollution in soil. However, the research on manganese removal mechanism of manganese oxidizing bacteria is not enough.
In this experiment, three different enrichment medium were used to enrich the samples of different environment. The samples with good effect were screened by LBB indicator method. At the same time, the strains with manganese oxidation ability were screened by LBB indicator method. After separation and purification, the samples were identified by physiological and biochemical and molecular biology identification methods. The effects of growth curve, inoculum size, Mn2+ concentration and pH value on the growth curve and the manganese oxidation characteristics of the isolates were also determined.
In this study, the samples were separated from soil and river sediment. First, the PYCM medium, K medium and PC medium were enriched and separated. The oxidation capacity of Mn (II) was detected by the LBB indicator for 10 strains of isolated bacteria. Then, the MIDI microbial identification system, Biolog analysis, physiological and biochemical analysis and 16S rDNA sequencing were carried out. Preliminary analysis showed that the strain MB1 was Bacillus licheniformis (Bacillus licheniformis) and MB2 was Bacillus MEG (Bacillus-megaterium).
The experimental results showed that the optimum inoculation for the growth curve of manganese bacteria MB1 and MB2 was 5%, and the optimum inoculation of MB2 was 15%. when the pH value of the medium was 5.0-7.0, MB1 and MB2 could grow well in this range. When the pH value was 7, the growth state of the bacteria reached the best, in the medium. The biomass is the largest, indicating that the bacteria can quickly adapt to the environment and quickly enter the logarithmic growth period, and the logarithmic time is longer than the logarithmic period of the mycelium under the other pH values. Then, the biomass is gradually reduced as the base of the medium increases, and the growth of the bacteria is inhibited when the pH value is 9. When the value of pH is 9, the alkaline environment is in the alkaline environment. The physiological activities of the medium manganese oxidizing bacteria were affected. Under the condition of pH 6.0-8.0, the oxidation rate of Mn2+ and MB1 was above 35%. The manganese resistance of manganese removal bacteria was analyzed. The effect of different Mn2+ concentration on the growth of the bacteria was studied. When the concentration was higher than 12mmol/L, the growth rate of MB1 bacteria was slow or almost no growth, indicating that when Mn2+ is Mn2+. When the concentration was too high, the bacterial growth of.MB2 strain in the Mn2+ medium was smaller than that of Mn2+, but the gap in the medium of 12mmol/L was little, indicating that the tolerance of MB2 bacteria to high concentration Mn2+ was better than that of MB1 bacteria.
The manganese removal rate of strain was measured by atomic absorption spectrometry. The manganese oxidation rate of the strain MB1 was 61.7% after 7 days in the K culture medium. The manganese oxidation rate of the strain MB2 was 90%. in the initial pH value (5-9) K medium. The effect of pH value on the oxidation rate of manganese was 7, and the oxidation rate of MB1 and MB2 was up to 80% when pH value was 7. When pH was 5 and 6, The oxidation rate of Mn2+ by strain MB1 and MB2 indicates that the oxidation rate of Mn2+ affects the growth of manganese oxide bacteria, which may be due to the change of the composition of the medium in acid environment and the decrease of manganese oxidation rate. The effect of Manganese Oxidizing Bacteria on the pH value in the growth process indicates that manganese oxidizing bacteria increase the medium pH value of the manganese oxide bacteria and the manganese removing bacteria MB1. The manganese removal characteristics of MB2 have been analyzed. It is proved that the manganese oxide bacteria screened in this experiment include the common effect of biological oxidation and chemical oxidation during the oxidation of Mn2+.
This strain provides an important strain for further study of manganese oxidation mechanism and enhanced manganese removal.

【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：TU991.2;X172

【参考文献】