Abstract
BackgroundConsiderable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and co-fermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. Here, we investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw.ResultsMore than 50% cell viability was lost during the first 24 h of high-gravity SSCF. No beneficial effects of adding selected nutrients were observed in shake flask SSCF. Ethanol concentrations greater than 50 g L−1 led to significant loss of viability and prevented further fermentation in SSCF. The benefits of feeding preadapted yeast cells were marginal at later stages of SSCF. Yeast flocculation did not improve the viability but simplified cell harvest and improved the feasibility of the cell feeding strategy in demo scale. Cultivation at 30 °C instead of 35 °C increased cell survival significantly on solid media containing ethanol and inhibitors. Similarly, in multifeed SSCF, cells maintained the viability and fermentation capacity when the temperature was reduced from 35 to 30 °C during the process, but hydrolysis yields were compromised. By combining the yeast feeding and temperature change, an ethanol concentration of 65 g L−1, equivalent to 70% of the theoretical yield, was obtained in multifeed SSCF on pretreated wheat straw. In demo scale, the process with flocculating yeast and temperature profile resulted in 5% (w/w) ethanol, equivalent to 53% of the theoretical yield.ConclusionsMultifeed SSCF was further developed by means of a flocculating yeast and a temperature-reduction profile. Ethanol toxicity is intensified in the presence of lignocellulosic inhibitors at temperatures that are beneficial to hydrolysis in high-gravity SSCF. The counteracting effects of temperature on cell viability and hydrolysis call for more tolerant microorganisms, enzyme systems with lower temperature optimum, or full optimization of the multifeed strategy with temperature profile.
Highlights
Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging
Ethanol is a major inhibitor in high‐gravity lignocellulosic ethanol production In the fermentation of pretreated wheat straw by S. cerevisiae KE6-12.A in the multifeed simultaneous saccharification and cofermentation (SSCF) process, the concentration of viable cells decreased to almost zero during the process, regardless of whether more or less toxic pretreated wheat straw were used, and regardless of the scales of the process (Fig. 1a, b)
To investigate whether the observed decrease in viability during SSCF was due to some nutrient limitation, yeast extract (2 g kg−1), vitamin solution (1 mL kg−1), trace metal (TM) solution (2 mL kg−1) [51], biotin (1 mg kg−1), or inorganic salts [7.5 g kg−1 (NH4)2SO4, 3.5 g kg−1 KH2PO4 and 0.7 g kg−1 MgSO4·7H2O] were added to shake flask SSCF at a low content of water insoluble solids (WIS, 7% w/w)
Summary
Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and cofermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. We investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw. The yeast Saccharomyces cerevisiae is one of the most-used fermenting microorganisms for its efficient glucose utilization and high stress tolerance in general. Proper implementation of rapidly sedimenting flocculating cells in bioprocesses gives the possibility to omit energy-intensive centrifugation steps [27]
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