工业废水活性污泥中PHA生产菌种的筛选与发酵优化及PHA合酶的生物信息学分析

发布时间：2018-04-21 20:44

本文选题：活性污泥 + 菌株筛选　；参考：《济南大学》2017年硕士论文

【摘要】：聚羟基脂肪酸酯(polyhydroxyalkanoates,PHA)是由细菌合成的一种胞内聚酯,普遍存在于自然界细菌体内。现如今传统塑料制品所带来的白色污染和石油资源面临枯竭的问题日益严重,因此PHA作为可替代传统塑料的环境友好型塑料受到了越来越多的关注。但是受制于其高昂的生产成本一直没得到广泛应用。有研究发现活性污泥中含有大量的PHA生产菌,这为降低PHA的生产成本以及剩余污泥的资源化利用提供了前提条件。本文从能够处理环氧丙烷皂化废水的活性污泥中筛选到了一株PHA高产菌株。利用Biolog微生物鉴定法和16S rDNA测序方法对该菌株进行菌种鉴定,最终确定为泡囊短波单胞菌(Brevundimonas vesicularis)。我们将其命名为Brevundimonas vesicularis UJN1。在此基础上对UJN1的PHA合酶基因phaC进行了PCR扩增,然后对此基因进行了生物信息学分析,预测了UJN1的PHA合酶的基本理化性质和作用机理。随后针对Brevundimonas vesicularis UJN1进行了培养基和发酵条件的优化,以提高其PHA的产量,降低生产成本。本文主要研究内容及结果如下:1.PHA生产菌株的筛选。对从工业污水处理厂取得的新鲜污泥进行驯化培养,采用尼罗兰染色法对驯化后的污泥进行初步筛选,得到19株可能合成PHA的菌株。将这19株菌进行发酵培养,分别测定其菌体干重,并用气相色谱法检测每株菌的PHA产量,选取其中生长情况较好和PHA产量最高的菌株,命名为UJN1。2.UJN1的菌种鉴定。将复筛所得的UJN1菌株进行生理生化实验,结果表明其为革兰氏阴性的非肠道菌。选用GN2微生物鉴定板对此菌株进行了Biolog微生物鉴定。随后又进行了16S rDNA序列测定。通过BLAST序列比对表明此菌株与泡囊短波单胞菌有99%的同源性,由此确定了菌株UJN1为泡囊短波单胞菌(Brevundimonas vesicularis)。3.Brevundimonas vesicularis UJN1 PHA合酶(编码基因为phaC)的生物信息学分析。设计了phaC引物,通过PCR方法克隆得到Brevundimonas vesicularis UJN1 phaC基因序列,并将其成功测序。对phaC基因的开放阅读框、编码蛋白序列的理化性质、疏水性、跨膜区域、信号肽、结构域分析和蛋白质结构进行了生物信息学分析。结果表明该基因ORF长1731 bp,编码576个氨基酸,所编码蛋白质的等电点为5.40。在组成蛋白质的20种氨基酸中,丙氨酸(Ala)所占的比例最高,达到14.2%;不稳定指数(II)为36.13,为稳定蛋白且有较好的亲水性。菌株UJN1的PHA合酶可归属为I型,我们简单的分析了这一PHA合酶的生理生化特征、预测了其合成PHA的机理,这为这一PHA合酶的进一步功能鉴定,表达调控和转基因克隆等研究提供了研究基础和理论依据。4.Brevundimonas vesicularis UJN1生产PHA的条件优化。对筛得的PHA合成菌株Brevundimonas vesicularis UJN1进行了培养基成分(碳源、氮源、碳氮比)和发酵条件(培养时间、初始pH、发酵温度、摇床转速、接种量、装液量等)的优化。同时在完成单因素影响实验的前提下,进行了多因素交互影响实验以获得最适的发酵条件。实验确定了最适的碳源蔗糖与氮源氯化铵的比例为100/1.04,培养基初始pH为6.7,最适培养温度为33.4℃。经过48 h的发酵培养能够得到占细菌干重34.1%的PHA。结果显示发酵优化提高了PHA的合成效率,为利用Brevundimonas vesicularis UJN1大规模合成PHA奠定了理论基础。
[Abstract]:Polyhydroxyalkanoates (PHA) is a kind of intracellular polyester synthesized by bacteria, which is commonly found in the natural bacteria. Nowadays, the problem of the white pollution and the depletion of petroleum resources in the traditional plastic products is becoming more and more serious. Therefore, the more PHA is the environment-friendly plastic that can replace the traditional plastic. More attention has been paid. But the high cost of production has not been widely used. It is found that there are a large number of PHA producing bacteria in activated sludge, which provides the precondition for reducing the production cost of PHA and the utilization of residual sludge in the activated sludge. A strain of high yield PHA was selected. The strain was identified by Biolog microbial identification method and 16S rDNA sequencing method. Finally, it was identified as Brevundimonas vesicularis (Brevundimonas vesicularis). Based on the Brevundimonas vesicularis UJN1., we amplified UJN1 PHA synthase gene phaC. After the bioinformatics analysis of the gene, the basic physicochemical properties and mechanism of the PHA synthase of UJN1 were predicted. Then the culture medium and fermentation conditions of Brevundimonas vesicularis UJN1 were optimized in order to improve the production of PHA and reduce the production cost. The main contents and results of this paper are as follows: 1.PHA production strain The fresh sludge obtained from the industrial sewage treatment plant was domesticated, and the acclimated sludge was preliminarily screened by Nile blue staining, and 19 strains of PHA were obtained. The 19 strains were fermented to determine the dry weight of the bacteria, and the PHA yield of each strain was detected by gas chromatography. The strain with the best long condition and the highest yield of PHA was identified as the strain of UJN1.2.UJN1. The physiological and biochemical tests of the UJN1 strain of the rescreened strain showed that it was a gram-negative non intestinal bacteria. The microbiological identification of the strain was carried out by the GN2 microbial identification board. Then, the 16S rDNA sequence was also carried out, and BLA was carried out by BLA. The ST sequence alignment showed that the strain had 99% homology with the vesicular monocyclic bacteria. Thus, the strain UJN1 was identified as the bioinformatics analysis of the.3.Brevundimonas vesicularis UJN1 PHA synthase (Brevundimonas vesicularis) of the vesicle short wave monomonas (Brevundimonas vesicularis). The phaC primers were designed and the Brevundimonas gland was cloned by the PCR method. The esicularis UJN1 phaC gene sequence was sequenced and sequenced successfully. The bioinformatics analysis of the open reading frame of the phaC gene, the physical and chemical properties of the encoded protein sequence, the hydrophobicity, the transmembrane region, the signal peptide, the domain analysis and the protein structure were analyzed. The results showed that the gene ORF was 1731 BP long, encoded 576 amino acids, and the encoded protein was equal. Of the 20 amino acids consisting of 5.40. in protein, the proportion of alanine (Ala) is the highest, up to 14.2%, and the instability index (II) is 36.13, which is a stable protein and has a good hydrophilic property. The PHA synthase of strain UJN1 can belong to I type. We briefly analyzed the physiological and biochemical characteristics of the PHA synthase, and predicted the mechanism of its synthesis of PHA. This provides a basic and theoretical basis for the study of the further functional identification of the PHA synthase, expression regulation and transgenic cloning, such as the.4.Brevundimonas vesicularis UJN1 production PHA. The cultured PHA synthesis strain Brevundimonas vesicularis UJN1 was used as a nutrient group (carbon, nitrogen, carbon and nitrogen ratio) and the fermentation bar. At the same time, under the premise of the single factor influence experiment, the optimum fermentation conditions were obtained under the premise of the single factor influence experiment. The experiment confirmed that the optimum ratio of carbon source sucrose and nitrogen source ammonium chloride was 100/1.04, and the initial pH of the medium was 6.7. The optimum culture temperature was 33.4 C. After 48 h fermentation, the results of PHA. that accounted for 34.1% of the dry weight of bacteria showed that the optimization of fermentation improved the synthesis efficiency of PHA and laid the theoretical foundation for the large-scale synthesis of PHA by Brevundimonas vesicularis UJN1.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X703;X172

【参考文献】