Continuous Production of Ethanol, 1-Butanol and 1-Hexanol from CO with a Synthetic Co-Culture of Clostridia Applying a Cascade of Stirred-Tank Bioreactors.

Syngas fermentation with clostridial co-cultures is promising for the conversion of CO to alcohols. A CO sensitivity study with Clostridium kluyveri monocultures in batch operated stirred-tank bioreactors revealed total growth inhibition of C. kluyveri already at 100 mbar CO, but stable biomass concentrations and ongoing chain elongation at 800 mbar CO. On/off-gassing with CO indicated a reversible inhibition of C. kluyveri. A continuous supply of sulfide led to increased autotrophic growth and ethanol formation by Clostridium carboxidivorans even at unfavorable low CO concentrations. Based on these results, a continuously operated cascade of two stirred-tank reactors was established with a synthetic co-culture of both Clostridia. An amount of 100 mbar CO and additional sulfide supply enabled growth and chain elongation in the first bioreactor, whereas 800 mbar CO resulted in an efficient reduction of organic acids and de-novo synthesis of C2-C6 alcohols in the second reactor. High alcohol/acid ratios of 4.5-9.1 (w/w) were achieved in the steady state of the cascade process, and the space-time yields of the alcohols produced were improved by factors of 1.9-5.3 compared to a batch process. Further improvement of continuous production of medium chain alcohols from CO may be possible by applying less CO-sensitive chain-elongating bacteria in co-cultures.

Novel synthetic co-culture of Acetobacterium woodii and Clostridium drakei using CO2 and in situ generated H2 for the production of caproic acid via lactic acid.

Acetobacterium woodii is known to produce mainly acetate from CO2 and H2, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A. woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO2 and H2 via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5 g L-1 were achieved with the AiO-electrode, whereas 8.1 g L-1 lactate were produced with direct H2 sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO2.

Artificial microbial consortia for bioproduction processes.

The application of artificial microbial consortia for biotechnological production processes is an emerging field in research as it offers great potential for the improvement of established as well as the development of novel processes. In this review, we summarize recent highlights in the usage of various microbial consortia for the production of, for example, platform chemicals, biofuels, or pharmaceutical compounds. It aims to demonstrate the great potential of co-cultures by employing different organisms and interaction mechanisms and exploiting their respective advantages. Bacteria and yeasts often offer a broad spectrum of possible products, fungi enable the utilization of complex lignocellulosic substrates via enzyme secretion and hydrolysis, and microalgae can feature their abilities to fixate CO2 through photosynthesis for other organisms as well as to form lipids as potential fuelstocks. However, the complexity of interactions between microbes require methods for observing population dynamics within the process and modern approaches such as modeling or automation for process development. After shortly discussing these interaction mechanisms, we aim to present a broad variety of successfully established co-culture processes to display the potential of artificial microbial consortia for the production of biotechnological products.

Synthetic co-culture of autotrophic Clostridium carboxidivorans and chain elongating Clostridium kluyveri monitored by flow cytometry.

Syngas fermentation with acetogens is known to produce mainly acetate and ethanol efficiently. Co-cultures with chain elongating bacteria making use of these products are a promising approach to produce longer-chain alcohols. Synthetic co-cultures with identical initial cell concentrations of Clostridium carboxidivorans and Clostridium kluyveri were studied in batch-operated stirred-tank bioreactors with continuous CO/CO2 -gassing and monitoring of the cell counts of both clostridia by flow cytometry after fluorescence in situ hybridization (FISH-FC). At 800 mbar CO, chain elongation activity was observed at pH 6.0, although growth of C. kluyveri was restricted. Organic acids produced by C. kluyveri were reduced by C. carboxidivorans to the corresponding alcohols butanol and hexanol. This resulted in a threefold increase in final butanol concentration and enabled hexanol production compared with a mono-culture of C. carboxidivorans. At 100 mbar CO, growth of C. kluyveri was improved; however, the capacity of C. carboxidivorans to form alcohols was reduced. Because of the accumulation of organic acids, a constant decay of C. carboxidivorans was observed. The measurement of individual cell concentrations in co-culture established in this study may serve as an effective tool for knowledge-based identification of optimum process conditions for enhanced formation of longer-chain alcohols by clostridial co-cultures.

Monitoring co-cultures of Clostridium carboxidivorans and Clostridium kluyveri by fluorescence in situ hybridization with specific 23S rRNA oligonucleotide probes.

The development of co-cultures of clostridial strains which combine different physiological traits represents a promising strategy to achieve the environmentally friendly production of biofuels and chemicals. For the optimization of such co-cultures it is essential to monitor their composition and stability throughout fermentation. FISH is a quick and sensitive method for the specific labeling and quantification of cells within microbial communities. This technique is neither limited by the anaerobic fermenter environment nor by the need of prior genetic modification of strains. In this study, two specific 23S rRNA oligonucleotide probes, ClosKluy and ClosCarb, were designed for the monitoring of C. kluyveri and C. carboxidivorans, respectively. After the optimization of hybridization conditions for both probes, which was achieved at 30% (v/v) formamide, a high specificity was observed with epifluorescence microscopy using cells from different pure reference strains. The discriminating properties of the ClosKluy and ClosCarb probes was verified with samples from heterotrophic co-cultures in anaerobic flasks as well as autotrophic stirred-tank bioreactor co-cultures of C. kluyveri and C. carboxidivorans. Besides being suited to monitor defined co-cultures of these two species, the new specific FISH oligonucleotide probes for C. kluyveri and C. carboxidivorans additionally have potential to be applied in environmental studies.