木质纤维素和菊芋生物质生产乳酸的生物炼制技术研究
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摘要
生物可降解聚乳酸材料需求量的显著增加极大地促进了乳酸生产行业的发展。利用价格低廉且资源丰富的农业废弃物作为原料进行生物发酵乳酸的生产技术更是受到了极大关注。这些生物质原料主要包括菊芋、玉米秸秆、小麦秸秆以及稻秆等。本论文主要对利用菊芋和纤维素生物质为原料生产乳酸的生物炼制工艺进行了深入系统的研究。
首先,从用于玉米秸秆原料进行乙醇发酵的体系中分离得到了一株非常特异的乳酸菌Pediococcus acidilacticiDQ2,并利用16S DNA测序和微生物实验方法对其进行菌种鉴定。研究证明,其具有超级耐热和耐受木质纤维素来源抑制物的特性。
其次,将这株新分离得到的乳酸菌P. acidilactici DQ2用于以菊芋块茎为原料的乳酸发酵工艺中。同时,在该研究过程中发现三种商业糖化酶存在较高的菊粉酶活性。对其根本原因进行研究,进一步鉴定发现在工业糖化酶中存在两种具有菊粉酶活性的蛋白。利用Genencor公司生产的一种GA-L New工业糖化酶对菊芋块茎进行糖化能够获得较高的果糖糖化得率。然后,以P. acidilactici DQ2作为乳酸发酵菌,GA-L New作为菊粉酶,利用菊芋块茎进行同步糖化和乳酸发酵(SSF)72h,能够得到111.5g/L的乳酸,其得率为0.46g/g DM,并且生产速率可达1.55g/L/h。对常规菊粉酶和以商业糖化酶替代菊粉酶进行的发酵过程中添加酶所占的成本进行了比较,发现利用商业糖化酶替代菊粉酶能够有效降低该发酵工艺成本。上述结果提出了一条利用价格低廉的商业糖化酶水解菊芋块茎进行乳酸发酵的现实可行的生产工艺。
最后,通过最新研发的干法生物炼制工艺,以玉米秸秆为原料,进行P. acidilacticiDQ2的乳酸发酵。主要考察了生物脱毒菌Amorphotheca resinae ZN1对预处理原料中抑制物的去除能力,以及生物脱毒作用对后续P. acidilactici DQ2发酵乳酸的影响。在预处理后的玉米秸秆中残留的不同浓度的抑制物对乳酸的发酵性能存在严重影响。通过7-10天的生物脱毒过程能够完全去除原料中的抑制物,从而使同步糖化和乳酸发酵过程的乳酸浓度和得率显著提高。同时,利用盘磨对预处理后的玉米秸秆进行磨浆处理,并考察了磨浆过程对乳酸发酵的影响。以经过上述磨浆和深度生物脱毒处理的玉米秸秆作为原料,在带有螺带式搅拌桨的5L发酵罐中,固含量为25%DM、pH5.5、温度48℃C条件下进行SSF,最终乳酸浓度可达74.2g/L,相应得率为59.76%,并且生产速率可达1.03g/L/h。该结果表明以生物质原料资源链条中的农业木质纤维素废弃物作为原料进行纤维素乳酸的生产过程是一条实际可行的生物炼制工艺。
通过以上结果可以得出以下结论,本论文分离鉴定了一株耐热耐抑制物乳酸发酵菌P. acidilactici DQ2,并研究开发了一种利用该乳酸菌分别以菊芋和纤维素生物质为原料进行高浓度乳酸生产的生物炼制工艺。这些研究将为以后通过价格低廉资源丰富的农业生物质生产高附加值的化工产品的产业化发展铺平道路。
The sharp increase in manufacturing biodegradable polylactic acid (PLA) has significantly stimulated lactic acid production from low value and abundant agricultural residues materials such as inulin, corn stover, wheat straw, and rice straw. In this thesis, the biorefinery processing technology for fermentative production of lactic acid using inulin and lignocellulose biomass as the raw materials wasintensively studied.
First, an interesting lactic acid bacteria strain, Pediococcus acidilacticiDQ2, was isolated from the corn stover slurry of ethanol fermentation. P. acidilactici DQ2was identified using16S DNA sequencing and microbial methods. The unique properties of P. acidilaclici DQ2, the excellent thermo-and inhibitor-tolerance, were demonstrated and carefully investigated.
Then, the newly isolatedP. acidilactici DQ2strain was applied to the lactic acid fermentation using Jerusalem artichoke tubers (Jat) as the raw materials. A high inulinase activity was found in three commercially available glucoamylase enzymes. Its origin was investigated and two proteins in the commercial glucoanylases were identified as the potential enzymes showing inulinase activity. One of the commercial glucoamylases, GA-L New from Genencor, was used for Jerusalem artichoke tubers (Jat) hydrolysis and a high hydrolysis yield of fructose was obtained. The simultaneous saccharification and lactic acid fermentation (SSF) of Jat was carried out using GA-L New as the inulinase and P. acidilactici DQ2as the fermenting strain. A high lactic acid titer, yield, and productivity of111.5g/L,0.46g/g dry material (DM), and1.55g/L/h, respectively, were obtained within72hours. The enzyme cost using the commercial glucoamylase as inulinase was compared to that using the typical inulinase and a large profit margin was identified. The results provided a practical way of Jat application for lactic acid production using cheap commercial glucoamylase enzyme.
Finally, corn stover was used as the raw materials for lactic acid production using the same strain P. acidilactici DQ2through the newly developed dry biorefinery processing technology. The effect of the inhibitor removal intensity by the biodetoxification strain Amorphotheca resinae ZN1on the lactic acid fermentation of P. acidilactici DQ2was carefully investigated. Different amount of inhibitor residues in the pretreated corn stover materials severely affected the performance of lactic acid fermentation. The complete removal of all the inhibitor substances after7-10days of bio-detoxification led to the higher lactic acid yield and titer in the simultaneous saccharification and lactic acid fermentation process. The effect of disk milling of the pretreated corn stover on the lactic acid fermentation was also investigated. After the deep biodetoxification and disk milling, the SSF at48℃, pH5.5, and25%(w/w) solids loading in5L of helical agitatedbioreactor resulted in the lactic acid titer, yield, and productivity of74.2g/L,59.76%, and1.03g/L/h, respectively. The result provided a practical process option for cellulosic lactic acid production using agro-lignocellulose residual in the chain of biomaterial resources.
Conclusively, the biorefinery processing technology of inulin and lignocellulose biomass for production of lactic acid were established with high lactic acid titer and yield using new microorganisms and new processing technology in this thesis. These studies paved the way of industrial application of value added chemicals from cheap and abundant agricultural biomass.
首先,从用于玉米秸秆原料进行乙醇发酵的体系中分离得到了一株非常特异的乳酸菌Pediococcus acidilacticiDQ2,并利用16S DNA测序和微生物实验方法对其进行菌种鉴定。研究证明,其具有超级耐热和耐受木质纤维素来源抑制物的特性。
其次,将这株新分离得到的乳酸菌P. acidilactici DQ2用于以菊芋块茎为原料的乳酸发酵工艺中。同时,在该研究过程中发现三种商业糖化酶存在较高的菊粉酶活性。对其根本原因进行研究,进一步鉴定发现在工业糖化酶中存在两种具有菊粉酶活性的蛋白。利用Genencor公司生产的一种GA-L New工业糖化酶对菊芋块茎进行糖化能够获得较高的果糖糖化得率。然后,以P. acidilactici DQ2作为乳酸发酵菌,GA-L New作为菊粉酶,利用菊芋块茎进行同步糖化和乳酸发酵(SSF)72h,能够得到111.5g/L的乳酸,其得率为0.46g/g DM,并且生产速率可达1.55g/L/h。对常规菊粉酶和以商业糖化酶替代菊粉酶进行的发酵过程中添加酶所占的成本进行了比较,发现利用商业糖化酶替代菊粉酶能够有效降低该发酵工艺成本。上述结果提出了一条利用价格低廉的商业糖化酶水解菊芋块茎进行乳酸发酵的现实可行的生产工艺。
最后,通过最新研发的干法生物炼制工艺,以玉米秸秆为原料,进行P. acidilacticiDQ2的乳酸发酵。主要考察了生物脱毒菌Amorphotheca resinae ZN1对预处理原料中抑制物的去除能力,以及生物脱毒作用对后续P. acidilactici DQ2发酵乳酸的影响。在预处理后的玉米秸秆中残留的不同浓度的抑制物对乳酸的发酵性能存在严重影响。通过7-10天的生物脱毒过程能够完全去除原料中的抑制物,从而使同步糖化和乳酸发酵过程的乳酸浓度和得率显著提高。同时,利用盘磨对预处理后的玉米秸秆进行磨浆处理,并考察了磨浆过程对乳酸发酵的影响。以经过上述磨浆和深度生物脱毒处理的玉米秸秆作为原料,在带有螺带式搅拌桨的5L发酵罐中,固含量为25%DM、pH5.5、温度48℃C条件下进行SSF,最终乳酸浓度可达74.2g/L,相应得率为59.76%,并且生产速率可达1.03g/L/h。该结果表明以生物质原料资源链条中的农业木质纤维素废弃物作为原料进行纤维素乳酸的生产过程是一条实际可行的生物炼制工艺。
通过以上结果可以得出以下结论,本论文分离鉴定了一株耐热耐抑制物乳酸发酵菌P. acidilactici DQ2,并研究开发了一种利用该乳酸菌分别以菊芋和纤维素生物质为原料进行高浓度乳酸生产的生物炼制工艺。这些研究将为以后通过价格低廉资源丰富的农业生物质生产高附加值的化工产品的产业化发展铺平道路。
The sharp increase in manufacturing biodegradable polylactic acid (PLA) has significantly stimulated lactic acid production from low value and abundant agricultural residues materials such as inulin, corn stover, wheat straw, and rice straw. In this thesis, the biorefinery processing technology for fermentative production of lactic acid using inulin and lignocellulose biomass as the raw materials wasintensively studied.
First, an interesting lactic acid bacteria strain, Pediococcus acidilacticiDQ2, was isolated from the corn stover slurry of ethanol fermentation. P. acidilactici DQ2was identified using16S DNA sequencing and microbial methods. The unique properties of P. acidilaclici DQ2, the excellent thermo-and inhibitor-tolerance, were demonstrated and carefully investigated.
Then, the newly isolatedP. acidilactici DQ2strain was applied to the lactic acid fermentation using Jerusalem artichoke tubers (Jat) as the raw materials. A high inulinase activity was found in three commercially available glucoamylase enzymes. Its origin was investigated and two proteins in the commercial glucoanylases were identified as the potential enzymes showing inulinase activity. One of the commercial glucoamylases, GA-L New from Genencor, was used for Jerusalem artichoke tubers (Jat) hydrolysis and a high hydrolysis yield of fructose was obtained. The simultaneous saccharification and lactic acid fermentation (SSF) of Jat was carried out using GA-L New as the inulinase and P. acidilactici DQ2as the fermenting strain. A high lactic acid titer, yield, and productivity of111.5g/L,0.46g/g dry material (DM), and1.55g/L/h, respectively, were obtained within72hours. The enzyme cost using the commercial glucoamylase as inulinase was compared to that using the typical inulinase and a large profit margin was identified. The results provided a practical way of Jat application for lactic acid production using cheap commercial glucoamylase enzyme.
Finally, corn stover was used as the raw materials for lactic acid production using the same strain P. acidilactici DQ2through the newly developed dry biorefinery processing technology. The effect of the inhibitor removal intensity by the biodetoxification strain Amorphotheca resinae ZN1on the lactic acid fermentation of P. acidilactici DQ2was carefully investigated. Different amount of inhibitor residues in the pretreated corn stover materials severely affected the performance of lactic acid fermentation. The complete removal of all the inhibitor substances after7-10days of bio-detoxification led to the higher lactic acid yield and titer in the simultaneous saccharification and lactic acid fermentation process. The effect of disk milling of the pretreated corn stover on the lactic acid fermentation was also investigated. After the deep biodetoxification and disk milling, the SSF at48℃, pH5.5, and25%(w/w) solids loading in5L of helical agitatedbioreactor resulted in the lactic acid titer, yield, and productivity of74.2g/L,59.76%, and1.03g/L/h, respectively. The result provided a practical process option for cellulosic lactic acid production using agro-lignocellulose residual in the chain of biomaterial resources.
Conclusively, the biorefinery processing technology of inulin and lignocellulose biomass for production of lactic acid were established with high lactic acid titer and yield using new microorganisms and new processing technology in this thesis. These studies paved the way of industrial application of value added chemicals from cheap and abundant agricultural biomass.
引文
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