Characterization of water-soluble lignin extractives from extractive ammonia (EA) pretreatment and their effects on yeast fermentation using synthetic biomass hydrolysate (SynH)

The DOE Great Lakes Bioenergy Research Center (GLBRC) has formulated a chemically-defined synthetic hydrolysate(SynH) to mimic real AFEX^TM corn stover(ACS) hydrolysate. The SynH, which is based on the chemical composition of real hydrolysate, is very useful in studying the inhibitory effects of degradation products generated during pretreatment. Extractive Ammonia (EA) is a newly-developed pretreatment technology that selectively extracts lignin in lignocellulosic biomass during pretreatment. Compared to AFEX, EA uses higher ammonia-to-biomass loading and lower water-loading, extracts 30-50% of the lignin from biomass depending on ammonia to biomass loading, generates a separate lignin extractive stream and also removes most degradation products from previously pretreated biomass. EA-pretreated corn stover(EA-CS) was found to have cellulose-III (allomorph of cellulose), reduced lignin content. It is highly digestible during enzyme hydrolysis and fermentation. In this work, we use SynH to study the inhibition of yeast fermentation by water-soluble lignin extractives(LEs) isolated from EA extractives. We found that SynH with 20g/L LEs mimics real hydrolysate in cell growth, sugar consumption and ethanol production. Nevertheless, a long lag phase was observed in the first 48hrs of fermentation, showing that additional inhibitors were produced during EA pretreatment. Ethyl acetate extraction was used to separate phenolic compounds from other components(sugars, buffers, etc.) in water-soluble LEs. These nitrogenous/phenolic compounds were then characterized and quantified using LC/GC-MS to determine the major inhibitors released during EA pretreatment. Some compounds identified in LE’s include coumaroyl amide, feruloyl amide and coumaroyl glycerol. These and other water-soluble degradation compounds act as key inhibitors in microbial fermentation.