本文選題：大腸桿菌 + 琥珀酸　； 參考：《湖北工業(yè)大學(xué)》2017年碩士論文

【摘要】：琥珀酸是一種非常重要的C4平臺(tái)化合物是合成很多重要產(chǎn)品的中間化合物,以琥珀酸為基礎(chǔ)的化學(xué)衍生產(chǎn)品在工業(yè)、農(nóng)業(yè)、食品、醫(yī)療等領(lǐng)域有著巨大的應(yīng)用價(jià)值。發(fā)酵法產(chǎn)琥珀酸以可再生能源為原料通過微生物轉(zhuǎn)化的方法生產(chǎn)琥珀酸作為一種綠色環(huán)保的方法較傳統(tǒng)的以石油為原料生產(chǎn)琥珀酸有著巨大的優(yōu)勢。本文在實(shí)驗(yàn)室已構(gòu)建的大腸桿菌工程菌WS100的基礎(chǔ)上,通過λRed同源重組系統(tǒng),敲除其丙酮醛合成酶基因(mgsA),再通過無氧管培養(yǎng)得到菌株WS102,進(jìn)一步消除副產(chǎn)物乳酸的代謝。通過NBS培養(yǎng)基發(fā)酵罐驗(yàn)證,得到的發(fā)酵液琥珀酸含量64.9 g/L,琥珀酸的質(zhì)量收率為75.1%,且發(fā)酵液中無副產(chǎn)物乳酸檢出,而WS100琥珀酸收率72.3%。在此基礎(chǔ)上進(jìn)行WS102利用二氧化碳發(fā)酵琥珀酸的研究,研究發(fā)現(xiàn)全階段以二氧化碳?xì)怏w發(fā)酵琥珀酸并不能很好的產(chǎn)琥珀酸,前期的厭氧條件容易造成代謝失衡,產(chǎn)生大量副產(chǎn)物乙酸,在NBS培養(yǎng)基中發(fā)酵琥珀酸產(chǎn)量和收率分別為52.65 g/L和68.9%。因此,通過單因素的比較設(shè)計(jì)一條微好氧-厭氧的發(fā)酵工藝,通過在前期添加KHCO_3保證菌體良好生長,在穩(wěn)定期之前以K_2CO_3提供固定“CO_2”所需C源,通過攪拌使前期保持微好氧的狀態(tài),在穩(wěn)定期以后通二氧化碳?xì)怏w進(jìn)行琥珀酸發(fā)酵的工藝。通過在NBS培養(yǎng)基中分批發(fā)酵得到琥珀酸產(chǎn)量為58.3g/L,琥珀酸的質(zhì)量收率為73.1%,生產(chǎn)強(qiáng)度為0.972 g/L·h。在此基礎(chǔ)上進(jìn)行分批補(bǔ)料發(fā)酵,琥珀酸產(chǎn)量為76.5 g/L,琥珀酸的生產(chǎn)強(qiáng)度和質(zhì)量收率分別為1.063 g/L·h和76.5%。本文最后以鈣鹽法分離提取發(fā)酵液中的琥珀酸,工藝路線為首先將發(fā)酵液通過簡單的加熱過濾除去大顆粒物質(zhì),然后通過陶瓷膜過濾去除絕大部分菌體,通過鈣鹽沉淀,活性炭脫色以及離子交換的方法分離提取發(fā)酵液中的琥珀酸。并且對(duì)鈣化工藝做了優(yōu)化,優(yōu)化鈣化反應(yīng)條件為在80℃條件下添加等體積的30%氯化鈣溶液反應(yīng)30 min。最終得到琥珀酸結(jié)晶99.6 g,琥珀酸收率為56.8%。
[Abstract]:Succinic acid is a very important C4 platform compound which is a intermediate compound for the synthesis of many important products. Succinic acid based chemical derivatives have great application value in the fields of industry, agriculture, food, medical and other fields. Succinic acid is produced by succinic acid to produce succinic acid using renewable energy as raw material through microbial transformation. As a green environmental protection method, it has a great advantage over the traditional production of succinic acid with oil as raw material. On the basis of the constructed Escherichia coli WS100 in the laboratory, this paper knocks out its acetone aldehyde synthase gene (mgsA) through the homologous recombination system of lambda Red, and then through the anaerobic tube culture to obtain the strain WS102 and further eliminate the pair. The metabolism of lactic acid was verified by NBS culture medium fermentation tank, the content of succinic acid was 64.9 g/L, the yield of succinic acid was 75.1%, and no by-product lactic acid was detected in the fermentation broth, while the WS100 succinic acid yield 72.3%. was based on WS102 utilization of carbon dioxide in the study of succinic acid, and the study found that the whole stage was two oxygen. Succinic acid fermented by carbon gas can not produce succinic acid well. The anaerobic conditions in the early stage can easily lead to metabolic imbalance and produce a large number of by-products acetic acid. The yield and yield of succinic acid fermented in the NBS medium are 52.65 g/L and 68.9%. respectively. The addition of KHCO_3 ensures good growth of the bacteria and provides the C source for the fixed "CO_2" before the stabilization period. The process of succinic acid fermentation by carbon dioxide gas is carried out after the stabilization period by stirring to keep the state of microaerobic at the early stage. The yield of succinic acid is 58.3g/L, succinic acid by the wholesale fermentation in the NBS medium. The yield was 73.1% and the production intensity was 0.972 g/L. H. on this basis. The yield of succinic acid was 76.5 g/L. The yield and yield of succinic acid were 1.063 g/L. H and 76.5%. respectively. Finally, succinic acid in the fermentation liquid was separated by calcium salt method. The large bulk material was removed by filtration, and most of the bacteria were removed through ceramic membrane filtration. Succinic acid was separated and extracted by calcium salt precipitation, decolorization of activated carbon and ion exchange, and the calcification process was optimized. The optimum calcification reaction condition was 30% calcium chloride solution of equal volume at 80 C under the condition of adding 3. 0 min. finally obtained succinic acid crystallization 99.6 g, succinic acid yield was 56.8%.
【學(xué)位授予單位】：湖北工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ921;Q933

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 楊如惠;楊效軍;;丁二酸生產(chǎn)工藝技術(shù)進(jìn)展[J];合成技術(shù)及應(yīng)用;2015年02期

2 郝夕祥;趙祥穎;田延軍;劉建軍;;CO_2固定途徑在L-蘋果酸積累中的作用[J];山東食品發(fā)酵;2011年01期

3 王金主;袁建國;王元秀;李峰;;發(fā)酵法生產(chǎn)琥珀酸的研究進(jìn)展[J];食品與藥品;2010年01期

4 張曉云;葉勤;;大腸桿菌乙酸產(chǎn)生及其控制研究[J];生物技術(shù)通報(bào);2009年10期

5 張玉秀;王姣;王丹;齊峰;;大腸桿菌產(chǎn)琥珀酸基因工程研究進(jìn)展[J];中國生物工程雜志;2009年07期

6 王慶昭;趙學(xué)明;;琥珀酸發(fā)酵菌種研究進(jìn)展[J];生物工程學(xué)報(bào);2007年04期

7 吳昊;姚忠;姜岷;劉輝;陳可泉;韋萍;;丁二酸發(fā)酵液的膜分離過程研究[J];食品與發(fā)酵工業(yè);2007年05期

8 王慶昭,吳巍,趙學(xué)明;生物轉(zhuǎn)化法制取琥珀酸及其衍生物的前景分析[J];化工進(jìn)展;2004年07期

9 詹曉北,朱一暉,Donghai Wang;琥珀酸發(fā)酵生產(chǎn)工藝及其產(chǎn)品市場[J];食品科技;2003年02期

10 鄺生魯;貢長生;;琥珀酸的開發(fā)與應(yīng)用前景[J];化工時(shí)刊;1991年01期

相關(guān)博士學(xué)位論文 前1條

1 李宜奎;好氧—厭氧產(chǎn)琥珀酸大腸桿菌的構(gòu)建及全階段發(fā)酵設(shè)計(jì)的研究[D];山東大學(xué);2014年

相關(guān)碩士學(xué)位論文 前5條

1 華渤文;產(chǎn)琥珀酸大腸桿菌工程菌的構(gòu)建及其發(fā)酵研究[D];湖北工業(yè)大學(xué);2014年

2 郝夕祥;黃曲霉SFW-7產(chǎn)L-蘋果酸的研究[D];山東農(nóng)業(yè)大學(xué);2011年

3 曲潔;蘋果酸脫氫酶及異檸檬酸脫氫酶擴(kuò)增對(duì)E.coli產(chǎn)琥珀酸的影響[D];天津大學(xué);2009年

4 孫超;發(fā)酵丁二酸的提取工藝研究[D];江南大學(xué);2008年

5 楊昆;產(chǎn)丁二酸菌種篩選及發(fā)酵工藝條件優(yōu)化的研究[D];江南大學(xué);2008年

，

本文編號(hào)：1959826