| 多轮次筛选选育高酚酸耐受性丁醇生产菌 |
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| 引用本文： | 经玉洁,黄小倩,田璐毅,丁欢欢,卜京,施超越,李汉广.多轮次筛选选育高酚酸耐受性丁醇生产菌[J].农业工程学报,2023,39(7):236-243. |
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| 作者姓名： | 经玉洁 黄小倩 田璐毅 丁欢欢 卜京 施超越 李汉广 |
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| 作者单位： | 江西农业大学生物科学与工程学院/应用微生物研究所，南昌 330045 |
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| 基金项目： | 国家自然科学基金项目（21466014）；江西省自然科学基金项目（20202BABL203042）；江西省研究生创新专项资金项目（YC2021-S350，YC2022-S433） |
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| 摘 要： | 纤维原料预处理过程中会产生酚酸等抑制菌株生长的物质，为选育出高丁醇产量及高耐受酚酸胁迫丁醇生产菌株，该研究利用多因子复合筛选策略筛选出一株能够合成足够还原力与对丁醇耐受性较好的菌株Clostridium beijerinckii W6。通过丁醇胁迫适应性进化获得丁醇耐受菌W6-1，其丁醇和总溶剂产量相较于菌株W6分别提高了14.01%和16.85%。通过紫外诱变处理菌株W6-1并结合理性筛选模型最终获得丁醇产量较高菌株W6-2，其丁醇及总溶剂产量分别可达到（9.51±0.06）和（15.32±0.11）g/L。最后将菌株W6-2通过酚酸胁迫适应性进化得到突变菌W6-3，其能耐受1.0 g/L酚酸胁迫环境，且丁醇和总溶剂产量相较于菌株W6-2分别提高了18.17%和17.49%。当以葛渣水解液为底物进行丙酮丁醇发酵时，突变菌W6-3的丁醇产量达（8.54±0.31）g/L，相较于菌株W6-2提高了26.71%。经多轮次诱变及适应性进化处理获得的突变菌的酚酸耐受性及发酵性能均有较大提高，该文所采用的多轮次筛选方法可以为其他快速筛选优良生产菌提供可靠的理论参考。
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| 关 键 词： | 菌株 发酵 复合筛选 酚酸胁迫 水解液 |
| 收稿时间： | 2022/9/28 0:00:00 |
| 修稿时间： | 2023/3/9 0:00:00 |
Screening of butanol producing strain with high phenolic acid tolerance by the approach of multi-repeating stress acclimation |
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| JING Yujie,HUANG Xiaoqian,TIAN Luyi,DING Huanhuan,BU Jing,SHI Chaoyue,LI Hanguang.Screening of butanol producing strain with high phenolic acid tolerance by the approach of multi-repeating stress acclimation[J].Transactions of the Chinese Society of Agricultural Engineering,2023,39(7):236-243. |
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| Authors: | JING Yujie HUANG Xiaoqian TIAN Luyi DING Huanhuan BU Jing SHI Chaoyue LI Hanguang |
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| Institution: | College of Biological Science and Engineering/Institute of Applied Microbiology, Jiangxi Agricultural University, Nanchang 330045, China |
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| Abstract: | Carbon-neutral fuels (such as ethanol and butanol) have gradually drawn much attention in recent years, due to the ever-increasing perception of global warming and environmental protection. Among them, butanol can be expected to serve as one of the promising candidates for biofuels, such as less corrosive, higher octane number, lower solubility in water, and higher energy content, compared with ethanol. However, the high feedstock cost and low productivity can be still a challenge in the acetone-butanol-ethanol (ABE) fermentation, due to the product''s (especially butanol) resistance or toxicity to the current butanol-producing strains. The overall economics of bio-butanol production can be enhanced using the abundant source, and low-price of materials (such as lignocellulose). Nevertheless, it must be appropriately pretreated, when these materials are used as the feedstock for the ABE fermentation. Various compounds can be formed during pretreatment. Moreover, the phenolic acids that are derived from lignin degradation can be the most toxicity inhibitor for the butanol-producing strains. In this study, some excellent strains were screened for the high tolerant phenolic acids stress and high butanol production. A multi-factor screening strategy was carried out to obtain the strain W6, in order to synthesize the sufficient reducing power and high tolerance to butanol. Furthermore, the strain W6 was then domesticated to further enhance the butanol tolerance via the appropriate concentration of butanol stress. As such, strain W6-1 was obtained, where the butanol and total solvent production were (8.22 ± 0.21) and (11.72 ± 0.26) g/L, respectively, which were 14.01% and 16.85% higher than that of strain W6. After that, strain W6-1 was treated to improve the production of butanol using UV mutagenesis combined with the multi-factor screening model. The high butanol production strain W6-2 was then selected after treatment. The butanol and total solvent production reached (9.51 ± 0.06) and (15.32 ± 0.11) g/L, resulting in an increase of 15.69% and 30.72%, respectively, compared with the strain W6-1. Finally, the high phenolic acid-tolerant strain W6-3 was achieved by the adaptive evolution strategy with phenolic acid stress condition. At the end of fermentation, the biomass increased by 50.00%, whereas, the butanol and solvent production of the mutant strain W6-3 increased by 18.17% and 17.49%, respectively, compared with the strain W6-2. When strain W6-3 was in the 0.5 g/L of phenol acid stress environment, the butanol production were 12.11% higher than that of strain W6-2, respectively. Once the phenolic acid stress concentration reached 1.0 g/L, there was no growth in the pre-domestication strain W6-2. However, the total solvent production of (3.42 ± 0.42) g/L was obtained for the mutant strain W6-3, indicating excellent phenol acid tolerance. Taking the puerariae slag hydrolysate as the fermentation feedstock, the production of butanol was (8.54 ± 0.31) g/L in the mutant strains W6-3, which was 26.71% higher than that of the strain W6-2. The phenolic acid tolerance and fermentation performance of the mutant strain were greatly improved after multiple rounds of mutagenesis and adaptive evolution. This finding can provide a reliable theoretical reference to rapidly screen the excellent producing trains, in order to fully meet the requirements of fermentation performance. |
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| Keywords: | strains fermentation compound screening phenolic acid stress hydrolysate |
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