生物联合加工菊芋生产乙醇的酿酒酵母菌株构建

发布时间：2018-08-25 18:33

【摘要】：利用生物质资源通过生物炼制生产燃料乙醇是近年来研究的热点。生物联合加工策略(Consolidated bioprocessing,CBP)集成产酶、酶解和发酵,是具有良好发展前景的生物燃料生产技术。菊芋可在干旱和盐碱地等边际土地生长且生物质产量高,是可选择的能源作物,但利用菊芋发酵生产乙醇的研究报道很少。菊芋的主要生物量来自块茎和秸秆,块茎主要成分是菊粉,是提取附加值较高菊粉产品的主要原料,而秸秆可用于生产燃料乙醇。本研究的目的是基于原料组成特点,选育具有高发酵活性的酿酒酵母CBP菌株并优化工艺过程的操作单元,提高乙醇发酵水平。首先研究了菊芋秸秆乙醇发酵。结果表明:2%(w/v)氢氧化钠预处理可以有效去除木质素,纤维素含量由39.4%提高到58.2%,且保留16.4%的菊粉。20%秸秆补料批次糖化总还原糖为115.8 g/L。通过细胞表面展示在耐高温工业酿酒酵母安琪酵母中整合菊粉酶基因,构建CBP菌株Saccharomyces cerevisiae MK01,其水解秸秆中菊粉性能良好,发酵液中乙醇浓度为38.3 g/L,得率为0.361 g/g,为理论得率的71%。其次,针对菊芋大规模种植块茎产量可能过剩的问题,采用S.cerevisiaeMK01以块茎为底物进行CBP乙醇发酵的研究。结果表明:温度控制在38-42℃,c,S.cerevisiae MK01生长没有明显差异,但在42 ℃时菊粉酶活力提高,乙醇发酵过程加快,72 h内消耗198.2 g/L菊粉水解糖,产生89.3 g/L乙醇;以185.7 g/L菊芋块茎水解总还原糖为底物,72 h内产生85.2 g/L乙醇,乙醇得率为0.459 g/g,达理论得率的90%。最后,探讨了有利于工业生产环境的耐高温CBP菌株选育,并对其耐高温分子机理进行了研究。通过导入人工转录因子,获得了一株表面展示纤维素酶且耐高温的CBP菌株。与亲本相比工程菌株可以在43℃条件下生长,与对照菌株相比,二者在胞内海藻糖含量、活性氧自由基积累和超氧化物歧化酶活力方面存在明显差异。工程菌株磷酸膨化纤维素的降解率提高了 45.8%,此外在43 ℃时以菊芋秸杆为底物的乙醇发酵周期缩短了 24 h。比较转录组学分析表明,工程菌株细胞壁完整性、PKC1-MPK1代谢通路及γ-氨基丁酸代谢旁路(GABA shunt)关键酶基因表达上调,表明人工转录因子通过对代谢的整体调控改善了细胞膜组成,进而改善了高温耐性。这些研究进展为菊芋秸秆和块茎综合利用生产燃料乙醇,奠定了良好技术基础。
[Abstract]:Using biomass resources to produce fuel ethanol through biological refining is a hot topic in recent years. Consolidated bioprocessing,CBP integrated enzyme production, enzymatic hydrolysis and fermentation is a promising biofuel production technology. Jerusalem artichoke (Jerusalem artichoke) is an alternative energy crop which can grow in marginal land such as arid and saline-alkali land and has high biomass yield. However, there are few reports on the production of ethanol by using Jerusalem artichoke fermentation. The main biomass of Jerusalem artichoke comes from tuber and straw. The main component of tuber is chrysanthemum powder, which is the main raw material to extract higher added value inulin product, and straw can be used to produce fuel ethanol. The aim of this study was to select the yeast CBP strain with high fermentative activity and optimize the operation unit of the process based on the characteristics of raw material composition to improve the ethanol fermentation level. Firstly, ethanol fermentation of Jerusalem artichoke straw was studied. The results showed that sodium hydroxide pretreatment (w / v) could effectively remove lignin, increase cellulose content from 39.4% to 58.2%, and retain 16.4% inulin. 20% straw batch saccharification total reducing sugar was 115.8 g / L. The inulinase gene was integrated on the cell surface of yeast An Qi, a thermotolerant industrial Saccharomyces cerevisiae, and CBP strain Saccharomyces cerevisiae MK01, was constructed. The hydrolyzed straw showed good inulinase performance. The ethanol concentration in fermentation broth was 38.3 g / L, and the yield was 0.361 g / g, which was 71g / g of the theoretical yield. Secondly, aiming at the possible excess of tuber yield in large scale planting of Jerusalem artichoke, S.cerevisiaeMK01 was used as substrate for CBP ethanol fermentation. The results showed that there was no significant difference in the growth of S.cerevisiae MK01 at 38-42 鈩，

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