本文選題：活性污泥 + 菌株篩選　； 參考：《濟(jì)南大學(xué)》2017年碩士論文

【摘要】：聚羥基脂肪酸酯(polyhydroxyalkanoates,PHA)是由細(xì)菌合成的一種胞內(nèi)聚酯,普遍存在于自然界細(xì)菌體內(nèi)。現(xiàn)如今傳統(tǒng)塑料制品所帶來(lái)的白色污染和石油資源面臨枯竭的問(wèn)題日益嚴(yán)重,因此PHA作為可替代傳統(tǒng)塑料的環(huán)境友好型塑料受到了越來(lái)越多的關(guān)注。但是受制于其高昂的生產(chǎn)成本一直沒(méi)得到廣泛應(yīng)用。有研究發(fā)現(xiàn)活性污泥中含有大量的PHA生產(chǎn)菌,這為降低PHA的生產(chǎn)成本以及剩余污泥的資源化利用提供了前提條件。本文從能夠處理環(huán)氧丙烷皂化廢水的活性污泥中篩選到了一株P(guān)HA高產(chǎn)菌株。利用Biolog微生物鑒定法和16S rDNA測(cè)序方法對(duì)該菌株進(jìn)行菌種鑒定,最終確定為泡囊短波單胞菌(Brevundimonas vesicularis)。我們將其命名為Brevundimonas vesicularis UJN1。在此基礎(chǔ)上對(duì)UJN1的PHA合酶基因phaC進(jìn)行了PCR擴(kuò)增,然后對(duì)此基因進(jìn)行了生物信息學(xué)分析,預(yù)測(cè)了UJN1的PHA合酶的基本理化性質(zhì)和作用機(jī)理。隨后針對(duì)Brevundimonas vesicularis UJN1進(jìn)行了培養(yǎng)基和發(fā)酵條件的優(yōu)化,以提高其PHA的產(chǎn)量,降低生產(chǎn)成本。本文主要研究?jī)?nèi)容及結(jié)果如下:1.PHA生產(chǎn)菌株的篩選。對(duì)從工業(yè)污水處理廠取得的新鮮污泥進(jìn)行馴化培養(yǎng),采用尼羅蘭染色法對(duì)馴化后的污泥進(jìn)行初步篩選,得到19株可能合成PHA的菌株。將這19株菌進(jìn)行發(fā)酵培養(yǎng),分別測(cè)定其菌體干重,并用氣相色譜法檢測(cè)每株菌的PHA產(chǎn)量,選取其中生長(zhǎng)情況較好和PHA產(chǎn)量最高的菌株,命名為UJN1。2.UJN1的菌種鑒定。將復(fù)篩所得的UJN1菌株進(jìn)行生理生化實(shí)驗(yàn),結(jié)果表明其為革蘭氏陰性的非腸道菌。選用GN2微生物鑒定板對(duì)此菌株進(jìn)行了Biolog微生物鑒定。隨后又進(jìn)行了16S rDNA序列測(cè)定。通過(guò)BLAST序列比對(duì)表明此菌株與泡囊短波單胞菌有99%的同源性,由此確定了菌株UJN1為泡囊短波單胞菌(Brevundimonas vesicularis)。3.Brevundimonas vesicularis UJN1 PHA合酶(編碼基因?yàn)閜haC)的生物信息學(xué)分析。設(shè)計(jì)了phaC引物,通過(guò)PCR方法克隆得到Brevundimonas vesicularis UJN1 phaC基因序列,并將其成功測(cè)序。對(duì)phaC基因的開(kāi)放閱讀框、編碼蛋白序列的理化性質(zhì)、疏水性、跨膜區(qū)域、信號(hào)肽、結(jié)構(gòu)域分析和蛋白質(zhì)結(jié)構(gòu)進(jìn)行了生物信息學(xué)分析。結(jié)果表明該基因ORF長(zhǎng)1731 bp,編碼576個(gè)氨基酸,所編碼蛋白質(zhì)的等電點(diǎn)為5.40。在組成蛋白質(zhì)的20種氨基酸中,丙氨酸(Ala)所占的比例最高,達(dá)到14.2%;不穩(wěn)定指數(shù)(II)為36.13,為穩(wěn)定蛋白且有較好的親水性。菌株UJN1的PHA合酶可歸屬為I型,我們簡(jiǎn)單的分析了這一PHA合酶的生理生化特征、預(yù)測(cè)了其合成PHA的機(jī)理,這為這一PHA合酶的進(jìn)一步功能鑒定,表達(dá)調(diào)控和轉(zhuǎn)基因克隆等研究提供了研究基礎(chǔ)和理論依據(jù)。4.Brevundimonas vesicularis UJN1生產(chǎn)PHA的條件優(yōu)化。對(duì)篩得的PHA合成菌株Brevundimonas vesicularis UJN1進(jìn)行了培養(yǎng)基成分(碳源、氮源、碳氮比)和發(fā)酵條件(培養(yǎng)時(shí)間、初始pH、發(fā)酵溫度、搖床轉(zhuǎn)速、接種量、裝液量等)的優(yōu)化。同時(shí)在完成單因素影響實(shí)驗(yàn)的前提下,進(jìn)行了多因素交互影響實(shí)驗(yàn)以獲得最適的發(fā)酵條件。實(shí)驗(yàn)確定了最適的碳源蔗糖與氮源氯化銨的比例為100/1.04,培養(yǎng)基初始pH為6.7,最適培養(yǎng)溫度為33.4℃。經(jīng)過(guò)48 h的發(fā)酵培養(yǎng)能夠得到占細(xì)菌干重34.1%的PHA。結(jié)果顯示發(fā)酵優(yōu)化提高了PHA的合成效率,為利用Brevundimonas vesicularis UJN1大規(guī)模合成PHA奠定了理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X703;X172

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 向沙;陳洪波;李小明;楊麒;趙建偉;賈利濤;劉芳芳;李娟娟;;溶解氧對(duì)好氧/延長(zhǎng)閑置SBR除磷性能的影響[J];環(huán)境科學(xué)學(xué)報(bào);2015年04期

2 馮廣達(dá);陳美標(biāo);羊宋貞;朱紅惠;;用于PCR擴(kuò)增的細(xì)菌DNA提取方法比較[J];華南農(nóng)業(yè)大學(xué)學(xué)報(bào);2013年03期

3 張志華;;Biolog微生物鑒定系統(tǒng)在絲狀真菌鑒定碳源同化方面的應(yīng)用[J];安徽農(nóng)業(yè)科學(xué);2013年02期

4 任中佳;王亞宜;耿軍軍;;活性污泥胞內(nèi)物質(zhì)合成影響因素的研究進(jìn)展[J];環(huán)境污染與防治;2011年04期

5 洪慶華;石璐;孫井梅;秦璐璐;盧楠;;革蘭氏染色三步法應(yīng)用試驗(yàn)探討[J];實(shí)驗(yàn)室研究與探索;2010年11期

6 薛林貴;趙旭;景春娥;常思靜;;尼羅藍(lán)在篩選PHB高產(chǎn)菌株中的應(yīng)用研究[J];生物技術(shù)通報(bào);2010年03期

7 陳國(guó)強(qiáng);;生物塑料聚羥基脂肪酸酯PHA發(fā)展近況[J];塑料制造;2009年07期

8 許旭萍;陳接鋒;;球衣菌胞內(nèi)聚β-羥基丁酸提取方法的研究[J];生物技術(shù)通報(bào);2006年03期

9 程池;楊梅;李金霞;姚粟;胡海蓉;;Biolog微生物自動(dòng)分析系統(tǒng)——細(xì)菌鑒定操作規(guī)程的研究[J];食品與發(fā)酵工業(yè);2006年05期

10 李篤中,何品晶;污泥性質(zhì)、膠羽結(jié)構(gòu)與處置[J];科技導(dǎo)報(bào);2004年09期

相關(guān)碩士學(xué)位論文 前1條

1 楊?yuàn)檴?聚羥基脂肪酸酯產(chǎn)生菌的篩選及其發(fā)酵條件的優(yōu)化[D];山東輕工業(yè)學(xué)院;2012年

，

本文編號(hào)：1784029