Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50 °C with Thermoanaerobacterium saccharolyticum ALK2

Abstract

Studies were undertaken to understand phenomena operative during simultaneous saccharification and fermentation (SSF) of a model cellulosic substrate (Avicel) at 50°C with enzymatic hydrolysis mediated by a commercial cellulase preparation (Spezyme CP) and fermentation by a thermophilic bacterium engineered to produce ethanol at high yield, Thermoanaerobacterium saccharolyticum ALK2. Thermal inactivation at 50 °C, as shown by the loss of 50% of enzyme activity over 4 days in the absence of ethanol, was more severe than at 37 °C, where only 25% of enzyme activity was lost. In addition, at 50 °C ethanol more strongly influenced enzyme stability. Enzyme activity was moderately stabilized between ethanol concentrations of 0 and 40 g/L, but ethanol concentrations above 40 g/L accelerated enzyme inactivation, leading to 75% loss of enzymatic activity in 80 g/L ethanol after 4 days. At 37 °C, ethanol did not show a strong effect on the rate of enzyme inactivation. Inhibition of cellulase activity by ethanol, measured at both temperatures, was relatively similar, with the relative rate of hydrolysis inhibited 50% at ethanol concentrations of 56.4 and 58.7 g/L at 50 and 37 °C, respectively. A mathematical model was developed to test whether the measured phenomena were sufficient to quantitatively describe system behavior and was found to have good predictive capability at initial Avicel concentrations of 20 and 50 g/L.

Bioconversion of lignocellulose: inhibitors and detoxification

“…Hydrolytic enzymes are inhibited by their products, i.e. sugars such as cellobiose and glucose [18], by fermentation products such as ethanol [19,20], and by phenolic compounds [21]. …”

- Synthetic Biology: A Promising Technology for Biofuel Production

“…Hydrolytic enzymes are inhibited by their products, i.e. sugars such as cellobiose and glucose [18], by fermentation products such as ethanol [19,20], and by phenolic compounds [21].…”

Ethanol and anaerobic conditions reversibly inhibit commercial cellulase activity in thermophilic simultaneous saccharification and fermentation (tSSF)

“…While these end products and solvents inhibit cellulase activity [3-5], this inhibition does not account for the total loss of activity seen under these high solid conditions [1]. Other factors that have been implicated in the observed slowdown in hydrolysis include enzyme inactivation [6-8], substrate inhibition [9,10], mass transfer [11,12], interference by lignin [13], loss of synergism and unproductive binding [14], inhibitors carried over from the feedstock [15] and changes in adsorption [1]. Yet as Kristensen and co-workers [1] demonstrated, none of these fully explained the declining activity across a spectrum of hydrolysis and SSF conditions, including a range of substrates and enzyme loadings.…”