Greetham D, Wimalasena T, Kerruish D W M, Brindley S, Ibbett R N, Linforth R L, Tucker G, Phister T G, Smart K A
Bioenergy and Brewing Science, School of Biosciences, Sutton Bonington Campus, University of Nottingham, Loughborough, Leicestershire, LE12 6RD, UK,
J Ind Microbiol Biotechnol. 2014 Jun;41(6):931-45. doi: 10.1007/s10295-014-1431-6. Epub 2014 Mar 25.
Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.
植物细胞壁分解产生的抑制剂会阻碍糖高效转化为乙醇。本研究的目的是开发一种针对产乙醇酵母菌株的快速且可靠的抑制剂敏感性检测方法。该检测方法包括定制的96孔板，其中单独或组合含有抑制剂，其形式与表型微阵列Omnilog读数仪（美国加利福尼亚州海沃德市的Biolog公司）兼容。检测中的氧化还原报告基因允许在需氧和/或厌氧条件下分析抑制剂敏感性。通过斑点平板上的生长情况和评估存活率的耐受性检测对检测结果进行验证。该检测方法能够确定抑制剂的个体效应和协同效应。观察到乙酸和甲酸的同时存在显著抑制了所评估的酵母菌株，不过通过缓冲至中性pH值，这种影响可得到部分缓解。树干毕赤酵母、念珠菌属和季也蒙毕赤酵母在木质纤维素水解产物中通常存在的浓度下，对短链弱酸的敏感性增加。酿酒酵母在这些浓度下对短链弱酸表现出耐受性。然而，与酿酒酵母相比，树干毕赤酵母、念珠菌属和季也蒙毕赤酵母对5-羟甲基糠醛（HMF）的耐受性增强。结果表明，本研究中开发的表型微阵列检测方法是一种有价值的工具，可用于鉴定对木质纤维素水解产物中存在的抑制性化合物具有理想抗性的酵母菌株。
