<i>Moorella</i> Strains for the Production of Biochemicals from Syngas

In the process of sugar fermentation, a significant portion of lignocellulosic biomass is left unused. An alternative is the gasification into syngas, a carbon-rich gas mixture. Syngas serves as energy and carbon source for acetogenic bacteria, which can then produce biochemicals. In the syngas fermentation process even the recalcitrant lignin portion can be fully converted into higher value compounds. Still the cost-effectiveness of this process requires better understanding of the metabolism and modification of the acetogenic strains. In my PhD project I am laying the basis for production of higher value biochemicals (acetone) from syngas using Moorella strains as cell factories. Moorella has outstanding abilities that make it especially suitable for the syngas fermentation process (thermophily, carbon source utilization). Firstly, the project focuses on understanding the primary metabolism in acetogenic bacteria. The main research aspect is to determine acceptance of, and the exact growth rates on different carbon sources (C1, C6, gaseous substrates) in different Moorella strains. Genome analysis on pathway level is performed to link the genotype to the phenotype. Differential expression analysis between heterotrophic and autotrophic growth (RNA-seq) serves to elucidate the regulatory mechanisms underlying carbon source utilization. In the second part of my project I am developing tools for genetic manipulation of Moorella strains. For example, a pyrF deletion strains, which allows heterologous gene expression was constructed. These tools developed in my project will be applied to engineer bacterial cell factories for production of higher value biochemicals like acetone

Production of 1,3-PDO and butanol by a mutant strain of Clostridium pasteurianum with increased tolerance towards crude glycerol

The production of biodiesel results in a concomitant production of crude glycerol (10% w/w). Clostridium pasteurianum can utilize glycerol as sole carbon source and converts it into 1,3-propanediol, ethanol, butanol, and CO(2). Reduced growth and productivities on crude glycerol as compared to technical grade glycerol have previously been observed. In this study, we applied random mutagenesis mediated by ethane methyl sulfonate (EMS) to develop a mutant strain of C. pasteurianum tolerating high concentrations of crude glycerol. At an initial crude glycerol concentration of 25 g/l the amount of dry cell mass produced by the mutant strain was six times higher than the amount produced by the wild type. Growth of the mutant strain was even detected at an initial crude glycerol concentration of 105 g/l. A pH controlled reactor with in situ removal of butanol by gas-stripping was used to evaluate the performance of the mutant strain. Utilizing stored crude glycerol, the mutant strain showed significantly increased rates compared to the wild type. A maximum glycerol utilization rate of 7.59 g/l/h was observed along with productivities of 1.80 g/l/h and 1.21 g/l/h of butanol and 1,3-PDO, respectively. These rates are higher than what previously has been published for C. pasteurianum growing on technical grade glycerol in fed batch reactors. In addition, high yields of the main products (butanol and 1,3-PDO) were detected and these two products were efficiently separated in two steams using gas-stripping

Exploiting the potential of gas fermentation

The use of gas fermentation for production of chemicals and fuels with lower environmental impact is a technology that is gaining increasing attention. Over 38 Gt of CO2 is annually being emitted from industrial processes, thereby contributing significantly to the concentration of greenhouse gases in the atmosphere. Together with the gasification of biomass and different waste streams, these gases have the potential for being utilized for production of chemicals through fermentation processes. Acetogens are among the most studied organisms capable of utilizing waste gases. Although engineering of heterologous production of higher value compounds has been successful for a number of acetogens, the processes are challenging due to the redox balance and the lack of efficient engineering tools. In this review, we address the availability of different gaseous feedstock and gasification processes, and we focus on the advantages of alternative fermentation scenarios, including thermophilic production strains, multi-stage fermentations, mixed cultures, as well as mixotrophy. Such processes have the potential to significantly broaden the product portfolio, increase the product concentrations and yields, while enabling the exploitation of alternative and mixed feedstocks. The reviewed processes also have the potential to address challenges associated with product inhibition and may contribute to reducing the costs of downstream processing. Given the widespread availability of gases, such processes will likely significantly impact the transition towards a more sustainable society

Increasing growth of a co2 fixing thermophile bacterium

Provided are methods for increasing the growth of Moorella species bacteria, genetically modified bacteria derived from such methods, and use of such bacteria for metabolizing a carbon-containing substrate, optionally in the production of a biochemical.</p

Probing efficient microbial CO2 utilisation through metabolic and process modelling

Abstract Acetogenic gas fermentation is increasingly studied as a promising technology to upcycle carbon‐rich waste gasses. Currently the product range is limited, and production yields, rates and titres for a number of interesting products do not allow for economically viable processes. By pairing process modelling and host‐agnostic metabolic modelling, we compare fermentation conditions and various products to optimise the processes. The models were then used in a simulation of an industrial‐scale bubble column reactor. We find that increased temperatures favour gas transfer rates, particularly for the valuable and limiting H2, while furthermore predicting an optimal feed composition of 9:1 mol H2 to mol CO2. Metabolically, the increased non‐growth associated maintenance requirements of thermophiles favours the formation of catabolic products. To assess the expansion of the product portfolio beyond acetate, both a product volatility analysis and a metabolic pathway model were implemented. In‐situ recovery of volatile products is shown to be within range for acetone but challenging due to the extensive evaporation of water, while the direct production of more valuable compounds by acetogens is metabolically unfavourable compared to acetate and ethanol. We discuss alternative approaches to overcome these challenges to utilise acetogenic CO2 fixation to produce a wider range of carbon negative chemicals

Thermodynamics and economic feasibility of acetone production from syngas using the thermophilic production host Moorella thermoacetica

Background: Syngas fermentation is a promising option for the production of biocommodities due to its abundance and compatibility with anaerobic fermentation. Using thermophilic production strains in a syngas fermentation process allows recovery of products with low boiling point from the off-gas via condensation. Results: In this study we analyzed the production of acetone from syngas with the hypothetical production host derived from Moorella thermoacetica in a bubble column reactor at 60°C with respect to thermodynamic and economic feasibility. We determined the cost of syngas production from basic oxygen furnace (BOF) process gas, from natural gas, and from corn stover and identified BOF gas as an economically interesting source for syngas. Taking gas-liquid mass transfer limitations into account, we applied a thermodynamics approach to derive the CO to acetone conversion rate under the process conditions. We estimated variable costs of production of 389 $/t acetone for a representative production scenario from BOF gas with costs for syngas as the main contributor. In comparison, the variable costs of production from natural gas- and corn stover-derived syngas were determined to be higher due to the higher feedstock costs (1724 and 2878$/t acetone, respectively). Conclusion: We applied an approach of combining thermodynamic and economic assessment to analyze a hypothetical bioprocess in which the volatile product acetone is produced from syngas with a thermophilic microorganism. Our model allowed us to identify process metrics and quantify the variable production costs for different scenarios. Economical production of bulk chemicals is challenging, making rigorous thermodynamic/economic modeling critical before undertaking an experimental program and as an ongoing guide during the program. We intend this study to give an incentive to apply the demonstrated approach to other bioproduction processes.BT/Bioprocess Engineerin