基因重组大肠杆菌发酵糖枫木片热水抽提物水解液转化合成生物乙醇的研究
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摘要
自20世纪70年代石油危机以来,人们认为燃料乙醇的生产是缓解能源危机的重要途径之一。在木质原料的糖化液中,主要存在己糖和戊糖两种成分,自然界中没有一种微生物可以有效地转化这些糖类生成乙醇。大肠杆菌Escherichia coli可以利用己糖和戊糖,但没有大量生产乙醇的能力。在E. coli中构建特异性生产乙醇的代谢途径成为了基因工程改造乙醇生产菌的一个研究热点。
半纤维素是造纸工业的副产物,如果将其直接排放会产生巨大的环境污染,用稀酸将其水解后可以获得大量的木糖以及少量的甘露糖、鼠李糖、葡萄糖、半乳糖和阿拉伯糖。同时,由于生物质稀酸水解过程中,糖类物质会降解生成糠醛和5-羟甲基糠醛(HMF)等强烈抑制微生物生长和乙醇发酵的有毒物质。并且水解液经中和后含有一定浓度的Na2SO4和CaSO4等盐类物质,形成较高的渗透压,也对微生物生长形成抑制作用。
本文首先研究了pH和缓冲液用量、温度、接种量、种龄和溶氧水平等培养条件对基因重组菌株E.coli FBR5发酵纯木糖为碳源的培养基生产乙醇的影响。实验证明E.coliFBR5的适宜生长条件为：磷酸盐缓冲液用量为12 mL、培养温度为35℃、5%的接种量、12～14小时种龄和摇床转速180 r／min的微氧环境。
在纯木糖为碳源的培养基中,进行了各种微生物生长及发酵抑制剂的添加试验,可知E. coli FBR5对水解液中各种抑制剂的耐受极限浓度为：硫酸钠5～10 g／L；氯化钙10 g／L；硫酸亚铁0.50 g／L；糠醛2.0 g／L；苯酚0.2～0.4g／L。该结果表明,在本研究所制得的糖枫木片热水抽提物水解液中对大肠杆菌的生长起主要抑制作用的是过高浓度的盐类物质。
研究表明糖枫木片热水抽提所得低聚糖溶液经纳滤膜浓缩、稀硫酸水解、中和并经纳滤膜分级分离后可获得可发酵的混合糖溶液。但由于成分复杂以及一些抑制剂的存在,E. coli FBR5在以该水解液为碳源的培养基中生长受到严重抑制,乙醇产量很低。经过菌种驯化和筛选,获得了一株能够耐受一定浓度水解液毒性的突变株,命名为E. coli FBHW.稀硫酸水解液经过离子交换树脂处理可以有效降低毒性,菌株FBHW在脱毒水解液中可以快速生长,并取得22.6g／L的乙醇产量。考虑到脱毒处理会使生产工艺复杂,增加生产成本,本研究通过采用混合酸水解、混合碱中和的方法均衡水解液中各种离子浓度,降低其对微生物生长的抑制作用。在不经任何脱毒处理的混合酸水解液中,FBHW发酵乙醇产量提高到36.9 g／L。
事物都有两方面,一方面水解液中过高浓度的金属离子会对微生物生长产生抑制作用,另一方面某些金属离子也是微生物生长所必需的。本文运用ICP方法测定了LB肉汤培养基、菌株E. coli FBHW在LB肉汤培养基中生长所得细胞以及水解液中的各种元素含量。结果表明在2MAB水解液中除了Cu、K、Na和P外,其他元素均大大过量,尤其是各种微量元素如Fe、Ni、Zn、Mn等。本文尝试以氨水替代LB肉汤培养基作为微生物生长所需氮源。初步研究在该合成培养基中,大肠杆菌可以生长获得菌体,但乙醇产量较低。
Since the 1970's, renewable fuels and materials have attracted significant interest due to high petroleum prices and awareness of the depletion of fossil fuel reserves. Bioethanol production is considered to be a milestone for sustainable development. Hydrolyzate of biomas includes a number of pentoses and hexoses. There is no single organism yet shown to efficiently convert all these sugars to ethanol. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. E. coli is naturally able to use a wide spectrum of sugars. One of the research work has concentrated on engineering E. coli to selectively produce ethanol.
Hemicellulose is a by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer, which can be hydrolyzed into monosaccharides by a dilute acid process. In addition to aromatics, acetic acid, formic acid and methanol released from woody biomass during the acid process and the hydrolysis of carbohydrates also generates toxic compounds. During dilute acid hydrolysis of biomass, monosaccharided are dehydrated and various toxic compounds are formed that can inhibit microorganism growth and ethanol fermentation, such as furfural and 5-hydroxymethylfurfural (HMF). Lignin byproducts during biomass hydrolysis are also inhibitory to microorganisms. Na2SO4 and CaSO4 formed during neutralization of the hydrolyzate contribute to a high osmotic pressure. This mixture of inhibitors will likely be a great challenge to those fermenting strains.
In this study, the basic culture conditions of E. coli FBR5 were investigated. The optimal culture conditions are as following:12 mL phosphate buffer,35℃, inoculum size of 5% by volume,12～14 hour-old inoculum and 180 r/min of shake speed.
Inhibitors were added into the pure xylose media to investigate the tolerance of E. coli FBR5 to those inhibitors. It's found that FBR5 was inhibited in the present of 5～10 g/L Na2SO4,10 g/L CaCl2,0.50 g/L FeSO4,2.0 g/L furfural and 0.2～0.4 g/L phenol, respectively. The results indicate that the salts in hot-water wood extract hydrolyzate can be inhibitory to FBR5.
Sugar Maple hot-water extract hemicellulosic hydrolyzate was concentrated and fractionated by a Nano-filtration membrane process in our lab. In this study, E. coli FBR5 was challenged by concentrated hot-water wood extract hemicellulosic dilute sulfuric acid hydrolyzate. After repeated strain adaptation, a new improved strain:E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of dilute sulfuric acid hydrolyzate in the fermentation media, and xylose was completely utilized by the strain to produce ethanol. Dilute sulfuric acid hydrolyzate was detoxified by ion-exchange, and FBHW can be grown rapidly in the desalted hydrolyzate with a yield of 22.6 g/L ethanol. To avoid the increase of production cost by ion-exchange, mixed-acid hydrolysis was carried out. Then the mixed-acid hydrolyzate was neutralized by mixed-base. Due to the balance of ions in the hydrolyzate, its inhibition to microorganism decreased. FBHW was grown in the concentrated mixed-acid hydrolyzate without any treatment and yielded 36.9 g/L ethanol.
High concentration of metal ions could be inhibitory to microorganisms. On the other hand, some ions are essential to growth of microorganism. In this study, ICP method was used to analyze elements content of LB broth media, cells of E. coli FBHW and hydrolyzate. Except for Cu、K、Na and P, all the other metal ions are excess for FBHW to grow, especially for trace elements such as, Fe, Ni, Zn and Mn. To reduce the cost of medium, NH3·H2O was used as the only nitrogen source to replace LB in the hydrolyzate to grow E. coli FBHW. FBHW can be grown in the simplified medium with a low ethanol yield.
半纤维素是造纸工业的副产物,如果将其直接排放会产生巨大的环境污染,用稀酸将其水解后可以获得大量的木糖以及少量的甘露糖、鼠李糖、葡萄糖、半乳糖和阿拉伯糖。同时,由于生物质稀酸水解过程中,糖类物质会降解生成糠醛和5-羟甲基糠醛(HMF)等强烈抑制微生物生长和乙醇发酵的有毒物质。并且水解液经中和后含有一定浓度的Na2SO4和CaSO4等盐类物质,形成较高的渗透压,也对微生物生长形成抑制作用。
本文首先研究了pH和缓冲液用量、温度、接种量、种龄和溶氧水平等培养条件对基因重组菌株E.coli FBR5发酵纯木糖为碳源的培养基生产乙醇的影响。实验证明E.coliFBR5的适宜生长条件为：磷酸盐缓冲液用量为12 mL、培养温度为35℃、5%的接种量、12～14小时种龄和摇床转速180 r／min的微氧环境。
在纯木糖为碳源的培养基中,进行了各种微生物生长及发酵抑制剂的添加试验,可知E. coli FBR5对水解液中各种抑制剂的耐受极限浓度为：硫酸钠5～10 g／L；氯化钙10 g／L；硫酸亚铁0.50 g／L；糠醛2.0 g／L；苯酚0.2～0.4g／L。该结果表明,在本研究所制得的糖枫木片热水抽提物水解液中对大肠杆菌的生长起主要抑制作用的是过高浓度的盐类物质。
研究表明糖枫木片热水抽提所得低聚糖溶液经纳滤膜浓缩、稀硫酸水解、中和并经纳滤膜分级分离后可获得可发酵的混合糖溶液。但由于成分复杂以及一些抑制剂的存在,E. coli FBR5在以该水解液为碳源的培养基中生长受到严重抑制,乙醇产量很低。经过菌种驯化和筛选,获得了一株能够耐受一定浓度水解液毒性的突变株,命名为E. coli FBHW.稀硫酸水解液经过离子交换树脂处理可以有效降低毒性,菌株FBHW在脱毒水解液中可以快速生长,并取得22.6g／L的乙醇产量。考虑到脱毒处理会使生产工艺复杂,增加生产成本,本研究通过采用混合酸水解、混合碱中和的方法均衡水解液中各种离子浓度,降低其对微生物生长的抑制作用。在不经任何脱毒处理的混合酸水解液中,FBHW发酵乙醇产量提高到36.9 g／L。
事物都有两方面,一方面水解液中过高浓度的金属离子会对微生物生长产生抑制作用,另一方面某些金属离子也是微生物生长所必需的。本文运用ICP方法测定了LB肉汤培养基、菌株E. coli FBHW在LB肉汤培养基中生长所得细胞以及水解液中的各种元素含量。结果表明在2MAB水解液中除了Cu、K、Na和P外,其他元素均大大过量,尤其是各种微量元素如Fe、Ni、Zn、Mn等。本文尝试以氨水替代LB肉汤培养基作为微生物生长所需氮源。初步研究在该合成培养基中,大肠杆菌可以生长获得菌体,但乙醇产量较低。
Since the 1970's, renewable fuels and materials have attracted significant interest due to high petroleum prices and awareness of the depletion of fossil fuel reserves. Bioethanol production is considered to be a milestone for sustainable development. Hydrolyzate of biomas includes a number of pentoses and hexoses. There is no single organism yet shown to efficiently convert all these sugars to ethanol. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. E. coli is naturally able to use a wide spectrum of sugars. One of the research work has concentrated on engineering E. coli to selectively produce ethanol.
Hemicellulose is a by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer, which can be hydrolyzed into monosaccharides by a dilute acid process. In addition to aromatics, acetic acid, formic acid and methanol released from woody biomass during the acid process and the hydrolysis of carbohydrates also generates toxic compounds. During dilute acid hydrolysis of biomass, monosaccharided are dehydrated and various toxic compounds are formed that can inhibit microorganism growth and ethanol fermentation, such as furfural and 5-hydroxymethylfurfural (HMF). Lignin byproducts during biomass hydrolysis are also inhibitory to microorganisms. Na2SO4 and CaSO4 formed during neutralization of the hydrolyzate contribute to a high osmotic pressure. This mixture of inhibitors will likely be a great challenge to those fermenting strains.
In this study, the basic culture conditions of E. coli FBR5 were investigated. The optimal culture conditions are as following:12 mL phosphate buffer,35℃, inoculum size of 5% by volume,12～14 hour-old inoculum and 180 r/min of shake speed.
Inhibitors were added into the pure xylose media to investigate the tolerance of E. coli FBR5 to those inhibitors. It's found that FBR5 was inhibited in the present of 5～10 g/L Na2SO4,10 g/L CaCl2,0.50 g/L FeSO4,2.0 g/L furfural and 0.2～0.4 g/L phenol, respectively. The results indicate that the salts in hot-water wood extract hydrolyzate can be inhibitory to FBR5.
Sugar Maple hot-water extract hemicellulosic hydrolyzate was concentrated and fractionated by a Nano-filtration membrane process in our lab. In this study, E. coli FBR5 was challenged by concentrated hot-water wood extract hemicellulosic dilute sulfuric acid hydrolyzate. After repeated strain adaptation, a new improved strain:E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of dilute sulfuric acid hydrolyzate in the fermentation media, and xylose was completely utilized by the strain to produce ethanol. Dilute sulfuric acid hydrolyzate was detoxified by ion-exchange, and FBHW can be grown rapidly in the desalted hydrolyzate with a yield of 22.6 g/L ethanol. To avoid the increase of production cost by ion-exchange, mixed-acid hydrolysis was carried out. Then the mixed-acid hydrolyzate was neutralized by mixed-base. Due to the balance of ions in the hydrolyzate, its inhibition to microorganism decreased. FBHW was grown in the concentrated mixed-acid hydrolyzate without any treatment and yielded 36.9 g/L ethanol.
High concentration of metal ions could be inhibitory to microorganisms. On the other hand, some ions are essential to growth of microorganism. In this study, ICP method was used to analyze elements content of LB broth media, cells of E. coli FBHW and hydrolyzate. Except for Cu、K、Na and P, all the other metal ions are excess for FBHW to grow, especially for trace elements such as, Fe, Ni, Zn and Mn. To reduce the cost of medium, NH3·H2O was used as the only nitrogen source to replace LB in the hydrolyzate to grow E. coli FBHW. FBHW can be grown in the simplified medium with a low ethanol yield.
引文
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