Towards production of organic acids from industrial lignocellulosic substrates

Abstract

To further establish a vital bioeconomy, bioprocesses must use substrates which are both cost-effective and sustainable. Second-generation substrates such as lignocellulosic biomass meet both criteria. To convert them into value-added products effectively, optimized microbial strains and processes are needed. The utilization of D-xylose, an abundant carbon source in second-generation feedstock, is of special interest in this context. To optimize the industrial relevant production host Corynebacterium glutamicum for utilization of D-xylose, a novel automated and miniaturized adaptive laboratory evolution approach was developed. This process is based on repetitive batch cultivations in microtiter plates, enabling an automated process with a high information content. By applying this process to a previously engineered C. glutamicum strain capable of utilizing D-xylose via the Weimberg pathway, the significantly improved strain WMB2evo was obtained. Genome sequencing and reengineering led to also well-performing rationally engineered strains. Coutilization capabilities of the novel strains were tested and a 13C-labelling experiment confirmed the functionality of the Weimberg pathway. The capability of the adapted C. glutamicum WMB2evo to utilize carbon sources from industrial hydrolysates was demonstrated using spent sulfite liquor. Challenges in biomass quantification during microcultivation experiments were met by developing a suitable pretreatment procedure. Bioreactor cultivations showed the utilization of carbon sources from both untreated and calcium hydroxide pretreated hydrolysates. On the product side, the industrial relevant organic acids a-ketoglutaric acid and succinic acid could be found when cultivating C. glutamicum WMB2evo under microaerobic conditions. An optimal degree of oxygen limitation was found, and different feeding strategies were tested, aiming for processes with high titers and productivities. Phenotyping of D-xylose utilizing C. glutamicum strains revealed that loss of gene iolR leads to an accumulation of the sugar acid D-xylonate. Aiming for its production, strain C. glutamicum ΔiolR was constructed and the native enzyme IolG was revealed as xylose dehydrogenase. Batch and fed-batch processes with defined medium were used to quantify performance indicators, demonstrating that D-xylonic acid can be produced at a high rate with maximum yield. The applicability in a biorefinery was shown by establishing a novel one-pot sequential hydrolysis and fermentation process using bagasse, a waste product of sugarcane processing