Ethanol production from non-detoxified hardwood spent sulfite liquor in submerged fed-batch culture using advanced yeasts

Abstract

To improve process feasibility, it is essential to use hardwood spent sulfite liquor (HSSL) as the main feedstock for bioethanol production, without prior detoxification. In addition, operating at large-scale under cost-effective conditions such as a small inoculum size (< 1 g/L), pH 5, using industrially acceptable nutrients, and without sugar addition, will require the use of harsh, concentrated HSSL streams. The potential of non-detoxified HSSL as a feedstock for ethanol production using two recombinant Saccharomyces cerevisiae strains, CelluX™4 and TFA7, was assessed. The inhibitory effect of non-detoxified HSSL was mitigated, and the ethanol titer increased from 4.1 to 7.9 g/L when pulse fed-batch was used instead of batch production, with CelluX™4 performing best. Both strains made use of the xylose isomerase (XI) pathway, with strain TFA7 engineered for increased tolerance against inhibitors. By administering concentrated HSSL in pulses to shake-flask cultures, the ethanol titer could be increased by approximately 50–90% when compared to simple batch cultures supplemented with 20%, 40%, and 60% (v/v) dilutions of HSSL. CelluX™4 was used in non-aerated, non-sterile 5-L bioreactor fermentations with a low cell concentration (< 1 g/L), pH 5, and 5 g/L corn steep liquor (CSL) as the nitrogen source. In comparison, undiluted HSSL was fed continuously to obtain a final 65% (v/v) HSSL supplementation, which corresponded to a total sugar concentration of 70.8–80.8 g/L. Despite the use of harsher, concentrated feedstock and inexpensive process conditions, the reactor fed-batch fermentations obtained ethanol yields of 0.35–0.43 g/g, which, based on a maximum theoretical ethanol yield of 0.51 g/g of hexoses or pentoses, corresponds to yield efficiencies of 68.6 and 84.3%. This illustrates an improvement on the highest titers reported in the literature for non-detoxified HSSL. The use of the advanced industrial S. cerevisiae strain, CelluX™4, combined with a fed-batch strategy, offers an inexpensive and straightforward process with real upscaling potential for industrial HSSL fermentations.