Towards continuous industrial bioprocessing with solventogenic and acetogenic clostridia: challenges, progress and perspectives.

The sustainable production of solvents from above ground carbon is highly desired. Several clostridia naturally produce solvents and use a variety of renewable and waste-derived substrates such as lignocellulosic biomass and gas mixtures containing H2/CO2 or CO. To enable economically viable production of solvents and biofuels such as ethanol and butanol, the high productivity of continuous bioprocesses is needed. While the first industrial-scale gas fermentation facility operates continuously, the acetone-butanol-ethanol (ABE) fermentation is traditionally operated in batch mode. This review highlights the benefits of continuous bioprocessing for solvent production and underlines the progress made towards its establishment. Based on metabolic capabilities of solvent producing clostridia, we discuss recent advances in systems-level understanding and genome engineering. On the process side, we focus on innovative fermentation methods and integrated product recovery to overcome the limitations of the classical one-stage chemostat and give an overview of the current industrial bioproduction of solvents.

Genetic Cell-Surface Modification for Optimized Foam Fractionation.

Rhamnolipids are among the glycolipids that have been investigated intensively in the last decades, mostly produced by the facultative pathogen Pseudomonas aeruginosa using plant oils as carbon source and antifoam agent. Simplification of downstream processing is envisaged using hydrophilic carbon sources, such as glucose, employing recombinant non-pathogenic Pseudomonas putida KT2440 for rhamnolipid or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., rhamnolipid precursors) production. However, during scale-up of the cultivation from shake flask to bioreactor, excessive foam formation hinders the use of standard fermentation protocols. In this study, the foam was guided from the reactor to a foam fractionation column to separate biosurfactants from medium and bacterial cells. Applying this integrated unit operation, the space-time yield (STY) for rhamnolipid synthesis could be increased by a factor of 2.8 (STY = 0.17 gRL/L·h) compared to the production in shake flasks. The accumulation of bacteria at the gas-liquid interface of the foam resulted in removal of whole-cell biocatalyst from the reactor with the strong consequence of reduced rhamnolipid production. To diminish the accumulation of bacteria at the gas-liquid interface, we deleted genes encoding cell-surface structures, focusing on hydrophobic proteins present on P. putida KT2440. Strains lacking, e.g., the flagellum, fimbriae, exopolysaccharides, and specific surface proteins, were tested for cell surface hydrophobicity and foam adsorption. Without flagellum or the large adhesion protein F (LapF), foam enrichment of these modified P. putida KT2440 was reduced by 23 and 51%, respectively. In a bioreactor cultivation of the non-motile strain with integrated rhamnolipid production genes, biomass enrichment in the foam was reduced by 46% compared to the reference strain. The intensification of rhamnolipid production from hydrophilic carbon sources presented here is an example for integrated strain and process engineering. This approach will become routine in the development of whole-cell catalysts for the envisaged bioeconomy. The results are discussed in the context of the importance of interacting strain and process engineering early in the development of bioprocesses.

Uncoupling Foam Fractionation and Foam Adsorption for Enhanced Biosurfactant Synthesis and Recovery.

The production of biosurfactants is often hampered by excessive foaming in the bioreactor, impacting system scale-up and downstream processing. Foam fractionation was proposed to tackle this challenge by combining in situ product removal with a pre-purification step. In previous studies, foam fractionation was coupled to bioreactor operation, hence it was operated at suboptimal parameters. Here, we use an external fractionation column to decouple biosurfactant production from foam fractionation, enabling continuous surfactant separation, which is especially suited for system scale-up. As a subsequent product recovery step, continuous foam adsorption was integrated into the process. The configuration is evaluated for rhamnolipid (RL) or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA, i.e., RL precursor) production by recombinant non-pathogenic Pseudomonas putida KT2440. Surfactant concentrations of 7.5 gRL/L and 2.0 gHAA/L were obtained in the fractionated foam. 4.7 g RLs and 2.8 g HAAs could be separated in the 2-stage recovery process within 36 h from a 2 L culture volume. With a culture volume scale-up to 9 L, 16 g RLs were adsorbed, and the space-time yield (STY) increased by 31% to 0.21 gRL/L·h. We demonstrate a well-performing process design for biosurfactant production and recovery as a contribution to a vital bioeconomy.

Perspectives for the microbial production of methyl propionate integrated with product recovery.

A new approach was studied for bio-based production of methyl propionate, a precursor of methyl methacrylate. Recombinant E. coli cells were used to perform a cascade reaction in which 2-butanol is reduced to butanone using alcohol dehydrogenase, and butanone is oxidized to methyl propionate and ethyl acetate using a Baeyer-Villiger monooxygenase (BVMO). Product was removed by in situ stripping. The conversion was in line with a model comprising product formation and stripping kinetics. The maximum conversion rates were 1.14 g-butanone/(L h), 0.11 g-ethyl acetate/(L h), and 0.09 g-methyl propionate/(L h). The enzyme regioselectivity towards methyl propionate was 43% of total ester. Starting from biomass-based production of 2-butanol, full-scale ester production with conventional product purification was calculated to be competitive with petrochemical production if the monooxygenase activity and regioselectivity are enhanced, and the costs of bio-based 2-butanol are minimized.

Prospects and challenges for the recovery of 2-butanol produced by vacuum fermentation - a techno-economic analysis.

The conceptual design of a bio-based process for 2-butanol production is presented for the first time. Considering a hypothetical efficient producing strain, a vacuum fermentation is proposed to alleviate product toxicity, but the main challenge is the energy-efficient product recovery from the vapor. Three downstream scenarios were examined for this purpose: 1) multi-stage vapor recompression; 2) temperature swing adsorption; and 3) vapor absorption. The processes were simulated using Aspen Plus, considering a production capacity of 101 kton/yr. Process optimization was performed targeting the minimum selling price of 2-butanol. The feasibility of the different configurations was analyzed based on the global energy requirements and capital expenditure. The use of integrated adsorption and absorption minimized the energy duty required for azeotrope purification, which represents 11% of the total operational expenditure in Scenario 1. The minimum selling price of 2-butanol as commodity chemical was estimated as 1.05 $/kg, 1.21 $/kg, and 1.03 $/kg regarding the fermentation integrated with downstream scenarios 1), 2), and 3), respectively. Significant savings in 2-butanol production could be achieved in the suggested integrated configurations if more efficient microbial strains were engineered, and more selective adsorption and absorption materials were found for product recovery.