Probing the nature of AFEX-pretreated corn stover derived decomposition products that inhibit cellulase activity

Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifugation, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products to evaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates that caused no appreciable enzyme inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn stover extractives using a 10 kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24–28% of the total observed enzyme inhibition while a 3 kDa MWCO membrane removed 60–65%, suggesting significant inhibition is caused by oligomeric materials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifugation removed 34–43% of the inhibition; ultra-filtration with a 5 kDa membrane removed 44–56% of the inhibition and when this ultra-filtrate was subjected to SPE a total of 69–70% of the inhibition were removed. Mass spectrometry found several phenolic compounds among the hydrophobic inhibition removed by SPE adsorption.