High-Throughput Screening of Inhibitory Compounds on Growth and Ethanol Production of Saccharomyces cerevisiae

Abstract

A high-throughput screening experiment to establish the individual and mixture effects of various common inhibitors found in lignocellulosic hydrolysates on the growth kinetics and ethanol production of Saccharomyces cerevisiae was carried out. Fermentations were performed utilizing 96-well microtiter plates to allow for carrying out fermentations in parallel, around which a central composite design of experiments was used to select inhibitor concentrations in each well. The individual and interaction effects of six common inhibitors were quantified using response surface fits of the growth rate, as determined by optical absorbance measurements and final cell density. For growth rate, 4-hydroxy-methylbenzaldehyde (phenol aldehyde) was found to be the strongest inhibitor of growth rate over the concentration range studied, while m-cresol (phenolic) had the least effect on growth rate and the largest inhibitory effect on final cell density. Both positive and negative interactions between inhibitors were found to affect both growth rate and maximum cell density. For example, both furfural and guaiacol when combined with m-cresol were found to have a positive effect on cell growth (less inhibitory), while guaiacol and m-cresol had a negative interaction with 4-hydroxy-methylbenzaldehyde. At all conditions studied, S. cerevisiae produced identical or higher ethanol yields compared to the inhibitor-free control fermentation which was attributed to the effects of physiological stress cause by the some inhibitors. These results quantify both the interactions of various inhibitors as well as their individual effects in a rapid and easy-to-perform experiment which can be easily expanded to include further inhibitors. Such a design can also be used for rapid and efficient screening of different pretreatments and feedstocks in the emerging field of lignocellulosic biofuels.