Characterization of water-soluble lignin extractives from Extractive Ammonia (EA) pretreatment and their effects on yeast fermentation

DOE Great Lakes Bioenergy Research Center (GLBRC) has formulated a chemically defined synthetic hydrolysate to mimic real Ammonia Fiber Expansion pretreated corn stover (AFEX^TM-CS) hydrolysate. The synthetic hydrolysate, whose formula is based on the quantified chemical composition of real hydrolysate, is very useful in biological studies of inhibitory effects of degradation products generated during pretreatment. Extractive Ammonia (EA) is a newly developed pretreatment technology based on AFEX. Compared to AFEX, EA uses higher ammonia loading and lower water loading, extracts 30-45% of the lignin from lignocellulosic biomass, generates a lignin extractive stream and hence removes part of the degradation products from pretreated biomass. EA-pretreated corn stover (EA-CS) is more fermentable than AFEX-CS. In the present work, we investigate the inhibitory effect of water-soluble lignin extractives (LEs) from EA pretreatment on yeast fermentation in synthetic hydrolysate. The chemical compositions of water-soluble LEs are characterized using LC-MS and GC-MS to determine the major inhibitors removed during EA pretreatment and which thereby contributes to the higher fermentability of EA-CS. Several groups of target compounds including nitrogenous compounds, aliphatic acid and aromatic compounds were quantified after separation using size exclusion column chromatography and ultrafiltration of water-soluble LEs. We formulated the synthetic hydrolysate (sugars, nutrients and different combination of degradation products) that mimic the real AFEX hydrolysate to study their inhibitory effects on yeast fermentation and compared with control samples (sugars and nutrients only). These experiments have helped us to identify water-soluble degradation compounds that are key inhibitors in AFEX hydrolysate that inhibit fermentation microbes.