Characterization of N-methyl-N’-nitro-N-nitrosoguanidine (NTG)-treated Zymomonas mobilis mutant: its potential ability for ethanol production on acid stress condition

• Main Authors: Puspa Julistia Puspita, 張立婷
• Other Authors: Chia-Wen Hsieh, Ph.D
• Format: Others
• Online Access: http://ndltd.ncl.edu.tw/handle/56043539247603865200

碩士 === 國立嘉義大學 === 生化科技學系研究所 === 100 === Zymomonas mobilis is an ethanol producer that has the highest ethanol yield on sugar complex-containing glucose. It becomes one of the most promising microorganisms having the potential to replace yeast for ethanol production (ethanol yields up to 97%). The lignocellulosic pretreatment is needed to increase accessibility of lignocellulosic for effi-cient ethanol production. However, pretreatment methods will create some inhibitor com-pounds such as acetic acid that reduces ethanol production. Therefore, improvement of ace-tate-tolerant microorganism, such as Z. mobilis is necessary. Furthermore, some mechanisms in Z. mobilis to survive under stress condition must be figured out. Thus, the aims in this study were to characterize AcR/2-12 as an acetate-tolerant mutant that has the ability to resist sodium acetate (NaAc) and to investigate mechanisms of this mutant to protect itself from acetic acid toxicity by two-dimensional electrophoresis-based proteomic approaches. The acetate-tolerant mutant Z. mobilis AcR/2-12 was generated from Z. mobilis ZM481 by N-methyl-N’-nitro-N-nitrosoguanidine (NTG). As reported in this study, the effect of the increasing NaAc level related to pH. The growth of AcR/2-12 and ZM481 for 24 h at pH 5 was inhibited by NaAc concentration above 100 mM. However, the mutant strain AcR/2-12 on fermentation medium contained 100 g/l glucose and supplemented with 195 mM NaAc at pH 5 was still able to grow well and produced 60 g/l ethanol with the ethanol yield of 0.62 g/g (Yproduct/substrate) in 148 h. Meanwhile, without pH control, AcR/2-12 was able to tolerate NaAc up to 250 mM and ZM481 was significantly inhibited at 195 mM NaAc. Under the same condition without pH control, the glucose was completely consumed in 24 h by AcR/2-12 and 57 g/l ethanol was produced with the ethanol yield of 0.58 g/g (Yp/s). Direct comparison of altered intensities by at least twofold (increased/decreased) between the same strain in the difference condition was performed by two-dimensional electrophoresis and proved that AcR/2-12 possessed mechanisms to protect itself from ru-ining acetic acid. Due to stress condition a total 35 protein spots were found. These protein expressions were significantly different between the mutant AcR/2-12 and wild-type ZM481.