Labor division in engineered cross-kingdom consortia: Consolidated bioprocessing of lignocellulosic biomass to carboxylic acids

Lignocellulose is a renewable source of fixed carbon and a promising substrate for the production of chemicals and fuels in second generation biorefineries. In this thesis, we engineered artificial microbial consortia for consolidated bioprocessing (CBP) of lignocellulose to carboxylic acids and ethanol. Inspired by natural ecosystems following the principle of labor division, we distributed the required metabolic capabilities among different specialized microorganisms. As a vast majority of product-forming microorganisms is non-cellulolytic the fungus Trichoderma reesei was employed to produce cellulolytic enzymes.

For the production of lactic acid from steam-pretreated beech wood, the aerobic fungus T. reesei was co-cultivated with the non-cellulolytic facultative anaerobic bacterium Lactobacillus pentosus. The known instability problem of such a cooperator-cheater community was overcome by enforced niche differentiation. To this end, a novel stirred tank bioreactor was engineered that was equipped with a continuously aerated, oxygen permeable membrane that locally provided oxygen for the aerobic fungus in an otherwise anaerobic bioreactor. The success of this strategy was shown by the formation of a fungal biofilm on the membrane's surface and a lactic acid concentration of 19.8 gL-1 (85.2 % of theoretical maximum).

For the production of various carboxylic acids, obligate and strict anaerobic bacteria were added to the community of T. reesei and L. pentosus. The lactic acid bacterium metabolically funneled the heterogeneous lignocellulosic carbohydrates to lactate as central intermediate in synthetic food chains and with that, compensated for missing metabolic capabilities of the anaerobic secondary fermenters. The feasibility and modularity of the lactate platform was shown by the direct production of acetic, propionic, butyric, valeric and hexanoic acid from beech wood. Integration of the obligate anaerobes Clostridium tyrobutyricum or Veillonella criceti resulted in 196.5 kg butyric acid or 113.6 kg propionic and 133.3 kg acetic acid, respectively, per ton beech wood. For the production of mixtures of volatile fatty acids (VFAs) with up to six carbon atoms from beech wood the non-cellulolytic strict anaerobe Megasphaera elsdenii was integrated into the lactate platform and 220 kg total VFAs per ton beech wood were produced. By adding C. tyrobutyricum or V criceti to a three-member microbial community it was shown that the ratio of odd- and even-numbered VFAs can be tuned through intra-consortium competition.

For the production of ethanol from lignocellulose, T. reesei and Saccharomyces cerevisiae were co-cultivated. They formed a two-layered biofilm on the aerated membrane. The in situ degradation of ethanol was dedicated to aerobic metabolism and strategies to tune the oxygen supply were provided.

An advanced bioreactor was engineered and applied that provided multiple niches for the co-cultivation of microorganisms with highly diverse requirements for abiotic parameters (e.g. oxygen, light) to widen the product range. The demonstrated co-cultivation of T. reesei and the microalgae Chlamydomonas reinhardtii in a two-layered biofilm is an important milestone towards the direct production of lipids from lignocellulose.

The results of this thesis illustrate the potential of community-based CBP of renewable lignocellulosic biomass to fuels and chemicals in second generation biorefineries in tomorrow's bioeconomy.