Design and application of microbial consortia for enhanced biomanufacturing

The global energy demand is experiencing rapid growth and fossil reserves cannot anymore meet this demand. Moreover, environmental issues such as global warming and glacier melting, underscore the need for alternative renewable energy sources. To address these challenges and align with the long-term goals of a sustainable circular economy, lignocellulosic biomass (LCB), an abundant and renewable feedstock, has garnered significant attention as resource that can fulfill energy needs while reducing dependence on fossil fuels and mitigating environmental concerns.
Establishing biorefineries for microbial fermentation of LCB for value-added products and energy production is a promising way to convert such materials efficiently and economically. To be used in fermentation processes, LCB requires a previous step of pretreatment to disassemble lignin, breakdown hemicellulose, diminish cellulose crystallinity and liberate monomeric sugars for fermentation. However, despite releasing sugars, pretreatment also generate distinct toxic compounds including furans, phenolic compounds, and weak acids, which inhibit the microbial metabolism during fermentation. Therefore, to have a successful fermentation, the concentration of inhibitory compounds needs to be reduced to a minimum that do not affect the microbial performance. Detoxification methods to reduce the concentration of inhibitors in hemicellulosic hydrolysates are currently challenged by low efficiency, prolonged operational time, high costs, undesired sugar loss, and environmental concerns. Alternatively, microbial detoxification with bacteria able to degrade specific inhibitors could be a valuable option to avoid such challenges.
In this thesis, the ability of six bacteria to consume inhibitory compounds present in brewer’s spent grain (BSG - a scarcely valorized and abundant by-product of beer industry) hemicellulosic hydrolysate was assessed. Two out of the six bacteria, namely P. putida and Rhodococcus sp., were further studied for their ability to metabolize some of the most frequent lignocellulose-derived inhibitors as sole carbon source. Then, BSG hemicellulosic hydrolysate was used to produce 2,3-butanediol (2,3-BDO) by Paenibacillus polymyxa. 2,3-BDO is a versatile platform chemical with diverse applications across industries, and with a current global market value of US$ 270.4 Mn (million), projected to grow 3.5% in the next 8 years. Presently, the industrial production of 2,3-BDO primarily relies on petroleum-derived hydrocarbons but high production yield has been shown by the bacterium Paenibacillus polymyxa. Different strategies have been studied to enhance the production of 2,3-BDO and the development of a more sustainable bioprocess for its production, including: 1) reducing by-products formation; 2) improving bacterial productivity by genetic engineering; 3) optimizing the fermentation parameters. Utilization of cheap and abundant agro-industrial biomasses remains the best alternative for the development of biomanufacturing in-line with the principle of circular economy. In this thesis, simultaneous saccharification and fermentation (SSF), and simultaneous saccharification and co-fermentation (SSCF) processes were used in monoculture of P. polymyxa and also in co-culture of this strain with an engineered P. putida (unable to use sugars), utilizing BSG as a feedstock. Co-culture was proposed for detoxification of lignocellulosic-derived inhibitory compounds. An enhanced production of 2,3-BDO (20.94 g/L) was achieved using SSCF, when the microbial detoxification of inhibitory compounds was operated. This approach represents a significant progress for the establishment of an efficient biomanufacturing of 2,3-BDO from lignocellulosic biomass. This study was concluded with a techno-economic assessment of the SSCF process using a co-culture system of P. polymyxa with Pseudomonas putida for 2,3-BDO production from BSG.