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

Abstract

BackgroundA previously developed mathematical model of low solids thermophilic simultaneous saccharification and fermentation (tSSF) with Avicel was unable to predict performance at high solids using a commercial cellulase preparation (Spezyme CP) and the high ethanol yield Thermoanaerobacterium saccharolyticum strain ALK2. The observed hydrolysis proceeded more slowly than predicted at solids concentrations greater than 50 g/L Avicel. Factors responsible for this inaccuracy were investigated in this study.ResultsEthanol dramatically reduced cellulase activity in tSSF. At an Avicel concentration of 20 g/L, the addition of ethanol decreased conversion at 96 hours, from 75% in the absence of added ethanol down to 32% with the addition of 34 g/L initial ethanol. This decrease is much greater than expected based on hydrolysis inhibition results in the absence of a fermenting organism. The enhanced effects of ethanol were attributed to the reduced, anaerobic conditions of tSSF, which were shown to inhibit cellulase activity relative to hydrolysis under aerobic conditions. Cellulose hydrolysis in anaerobic conditions was roughly 30% slower than in the presence of air. However, this anaerobic inhibition was reversed by exposing the cellulase enzymes to air.ConclusionThis work demonstrates a previously unrecognized incompatibility of enzymes secreted by an aerobic fungus with the fermentation conditions of an anaerobic bacterium and suggests that enzymes better suited to industrially relevant fermentation conditions would be valuable. The effects observed may be due to inactivation or starvation of oxygen dependent GH61 activity, and manipulation or replacement of this activity may provide an opportunity to improve biomass to fuel process efficiency.

Engineered Pentafunctional Minicellulosome for Simultaneous Saccharification and Ethanol Fermentation in Saccharomyces cerevisiae

“…However, the overall ethanol titer still went up to 2.7 g/liter, thus demonstrating that the low levels of oxygen was sufficient for some LPMO activity. The improvement in ethanol titers also corroborated the synergistic effects of adding LPMO and CDH to traditional cellulases, as reported in previous in vitro studies (12)(13)(14)(15). Due to the current limitations of the experimental setup, the oxygen levels were not regulated and measured in the present study.…”

“…However, there is still plenty of room for improvement in the ethanol titers. As demonstrated by many recent studies, the addition of oxidative LPMOs to hydrolytic cellulase cocktails led to significant enhancement to the breakdown of cellulose (12)(13)(14)(15). In a recent study published while the present manuscript was being prepared, the integration of bacterial LPMOs to cellulosomes in vitro resulted in 2.6-fold enhancement in the production of soluble sugars compared to free enzymes without the LPMOs (29).…”

“…While the traditional enzyme cocktails were comprised solely of hydrolytic enzymes, LPMO is an oxidative enzyme that requires the presence of oxygen for its function (12)(13)(14)(15). In the present study, instead of adopting strict anaerobic conditions typically used in fermentation, we introduced oxygen by leaving a small headspace for air in our sealed tubes.…”

“…On top of the commonly utilized cellulases, recent studies have found that the addition of lytic polysaccharide monooxygenases (LPMOs; formally known as family 61 glycoside hydrolases) and cellobiose dehydrogenases (CDHs) to enzyme cocktails of CBH, EG, and BGL led to significant improvements in cellulosic degradation (12)(13)(14)(15). LPMO-encoding genes are abundant in biomassconverting microorganisms.…”

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

“…The use of anaerobic or facultatively anaerobic bacteria eliminates the need for expensive aerated industrial reactors. For SSF, facultative anaerobes have the advantage over strict anaerobes that reduction of the medium is not necessary, as reduced medium has been shown to inhibit saccharolytic enzymes (6).…”