Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings

Abstract

Background: Economically feasible cellulosic ethanol production requires that the process can be operated at high solid loadings, which currently imparts technical challenges including inefficient mixing leading to heat and mass transfer limitations and high concentrations of inhibitory compounds hindering microbial activity during simultaneous saccharification and fermentation (SSF) process. Consequently, there is a need to develop cost effective processes overcoming the challenges when working at high solid loadings.

Results: In this study we have modified the yeast cultivation procedure and designed a SSF process to address some of the challenges at high water insoluble solids (WIS) content. The slurry of non-detoxified pretreated spruce when used in a batch SSF at 19% (w/w) WIS was found to be inhibitory to Saccharomyces cerevisiae Thermosacc that produced 2 g l-1 of ethanol. In order to reduce the inhibitory effect, the non-washed solid fraction containing reduced amount of inhibitors compared to the slurry was used in the SSF. Further, the cells were cultivated in the liquid fraction of pretreated spruce in a continuous culture wherein the outflow of cell suspension was used as cell feed to the SSF reactor in order to maintain the metabolic state of the cell. Enhanced cell viability was observed with cell, enzyme and substrate feed in a SSF producing 40 g l-1 ethanol after 96 h corresponding to 53% of theoretical yield based on available hexose sugars compared to 28 g l-1 ethanol in SSF with enzyme and substrate feed but no cell feed resulting in 37% of theoretical yield at a high solids loading of 20% (w/w) WIS content. The fed-batch SSF also significantly eased the mixing, which is usually challenging in batch SSF at high solids loading.

Conclusions: A simple modification of the cell cultivation procedure together with a combination of yeast, enzyme and substrate feed in a fed-batch SSF process, made it possible to operate at high solids loadings in a conventional bioreactor. The proposed process strategy significantly increased the yeast cell viability and overall ethanol yield. It was also possible to obtain 4% (w/v) ethanol concentration, which is a minimum requirement for an economical distillation process.

Figure 1

Evaluation of fermentation performance of Thermosacc in a batch simultaneous saccharification and fermentation (SSF) process using spruce slurry. Increase in glucose concentration (diamonds) was observed during the initial prehydrolysis for 24 h at 50°C. After 24 h, the temperature was reduced to 35°C and followed by the addition of yeast cell suspension, minute traces of ethanol production (circles) were observed. The enzyme and yeast loading was 7.5 FPU gWIS^-1 and 5 g L^-1, respectively. FPU, filter paper units; WIS, water insoluble solids.

Figure 2

Evaluation of fermentation performance of Thermosacc in the liquid fraction of pretreated slurry. Glucose (diamonds), mannose (triangles), and galactose (squares) consumption, and ethanol (circles) production in anaerobic fermentation of liquid fraction diluted to 90% (v/v) (solid lines) and 60% (v/v) (dotted lines) with 3 g L^-1 of yeast loading. The graph represents the mean of two biological replicates. Error bars are omitted for the sake of clarity.

Figure 3

Comparison of ethanol production during batch and fed-batch simultaneous saccharification and fermentation (SSF) using non-washed solids as substrate. Ethanol concentration (circles) during batch SSF (dashed-dotted lines), fed-batch SSF with substrate and enzyme feed (dashed lines), fed-batch SSF with yeast and substrate feed (dotted lines) and fed-batch SSF with yeast, enzymes and substrate feed (solid lines). The graph represents the mean of two biological replicates. Error bars are omitted for the sake of clarity. The total water insoluble solids (WIS), enzyme loading and yeast loading were 20% (w/w), 7.5 filter paper units (FPU) g WIS^-1 and 5 g L^-1, respectively, in all the experiments.

Figure 4

Comparison of colony forming units (CFU) during the batch and fed-batch SSF using non-washed solids as substrate. CFU (triangles) during batch SSF (dashed-dotted lines), fed-batch SSF with substrate and enzyme feed (dashed lines), fed-batch SSF with yeast and substrate feed (dotted lines) and fed-batch SSF with yeast, enzymes and substrate feed (solid lines). The graph represents the mean of two biological replicates and two technical replicates with error bars indicating standard deviation. The total WIS, enzyme loading and yeast loading were 20% (w/w), 7.5 FPU gWIS^-1 and 5 g L^-1, respectively, in all the experiments.