Carbonyl inhibition and detoxification in microbial fermentation of biomass hydrolysates

Abstract

Bioconversion of lignocellulosic biomass to biofuels has great potential to supplement petroleum-derived fuels. One of the major barriers to bioconversion is the release of considerable amount of carbonyl degradation compounds in the pretreatment of biomass, which because of their high reactivity toward biological nucleophiles inhibit the subsequent microbial fermentation. The development of a cost-effective detoxification approach and identification of the reaction mechanisms would alleviate the issue. Overliming has been widely used to detoxify biomass hydrolysates. However, the chemical mechanisms were not very well understood. My initial work therefore was to explore possible detoxification mechanisms by using a carbonyl model compound o-phthalaldehyde. At 1 mM, o-phthalaldehyde completely inhibited ethanol production by Saccharomyces cerevisiae, but interestingly, the inhibition disappeared under alkaline conditions (pH~10) at 60°C for 2 h in the presence of a reducing sugar. Non-reducing sugar had no effect. LC/MS analysis of the detoxification mixture revealed an aldol condensation reaction between o-phthalaldehyde and a reducing sugar. The reducing sugar converted to its enolate ion under alkaline conditions, which then reacted with one of the aldehyde groups through nucleophilic addition. Loss of one aldehyde group could be the key for the detoxification. In following work amino acids were used to detoxify the biomass hydrolysates for ethanol production by S. cerevisiae. I found cysteine was one of the amino acids that effectively detoxified loblolly pine hydrolysates. Ethanol production rate at 6 h increased from 0.18 in the untreated hydrolysate to 1.77 g/L/h and the final yield from 0.02 to 0.42 g/g, significant increases in both production rate and yield. The extraordinary detoxification by cysteine was probably due to its reactive thiol group that, in addition to its amine group, reacted with aldehydes to form thiazolidine derivatives. Meanwhile, the amine group could attack the carbon of aldehydes/ketones via electrophilic substitution to form imines. To understand the mechanism of aromatic aldehyde inhibition on yeast fermentation, I further investigated the structure-inhibition relationships using thirteen benzaldehydes. The results indicated that fermentation inhibition of benzaldehydes appeared related to their ortho-group (CHO > OH > OCH3) and position of the OH group in the benzene ring (ο-OH > m-OH > p-OH). Correlating the molecular descriptors to inhibition efficiency revealed a strong association between Log P and inhibitory activity.