Unlocking the potential of one-carbon gases (CO2, CO) for concomitant bioproduction of ß-carotene and lipids.

This study investigates the use of a Yarrowia lipolytica strain for the bioconversion of syngas-derived acetic acid into ß-carotene and lipids. A two-stage process was employed, starting with the acetogenic fermentation of syngas by Clostridium aceticum, metabolising CO, CO2, H2, to produce acetic acid, which is then utilized by Y. lipolytica for simultaneous lipid and ß-carotene synthesis. The research demonstrates that acetic acid concentration plays a pivotal role in modulating lipid profiles and enhancing ß-carotene production, with increased acetic acid consumption leading to higher yields of these compounds. This approach showcases the potential of using one-carbon gases as substrates in bioprocesses for generating valuable bioproducts, providing a sustainable and cost-effective alternative to more conventional feedstocks and substrates, such as sugars.

Carbon dioxide as key player in chain elongation and growth of Clostridium kluyveri: Insights from batch and bioreactor studies.

Chain elongation technology allows medium-chain fatty acids (MCFAs) production as an alternative to fossil resources. Clostridium kluyveri generates n-caproate primarily from ethanol and acetate, presumably requiring CO2 for growth. Here, the impact of CO2 on C. kluyveri was explored. Bottle studies revealed the bacterium's adaptability to low CO2 levels, even in conditions with minimal dissolved NaHCO3 (0.0003 M) and unfavorable pH (below 6) under 1 bar CO2. Bioreactor investigations demonstrated a direct correlation between CO2 availability and bacterial growth. The highest n-caproate production (11.0 g/L) with 90.1 % selectivity was achieved in a bioreactor with continuous CO2 supply at 3 mL/min. Additional bottle experiments pressurized with 1 bar CO2 and varying ethanol:acetate ratios (1:1, 2:1, 4:1) also confirmed CO2 consumption by C. kluyveri. However, increasing the ethanol:acetate ratio did not enhance n-caproate selectivity, likely due to overly acidic pH conditions. These findings provide insights into chain-elongators responses under diverse conditions.

Effect of electron acceptors on product selectivity and carbon flux in carbon chain elongation with Megasphaera hexanoica.

Megasphaera hexanoica is a bacterial strain following the reverse ß-oxidation pathway to synthesize caproate (CA) using lactate (LA) as an electron donor (ED) and acetate (AA) or butyrate (BA) as electron acceptors (EA). Differences in the type and concentration of EA lead to distinctions in product distribution and energy bifurcation of carbon fluxes in ED pathways, thereby affecting CA production. In this study, the effect of various ratios of AA, BA, and AA+BA as EA on carbon flux and CA specific titer during the carbon chain elongation in M. hexanoica was explored. The results indicated that the maximum levels of CA were 18.81 mM and 31.48 mM when the molar ratios of LA/AA and LA/BA were 10:1 and 3:1, respectively. Meanwhile, when AA and BA were used as combined EA (LA, AA, and BA molar amounts of 100, 23, and 77 mM), a maximum CA production of 39.45 mM was obtained. Further analysis revealed that the combined EA exhibited a CA production carbon flux of 49 % (4.3 % and 19.5 % higher compared to AA or BA, respectively) and a CA production specific titer of 45.24 mol (80.89 % and 58.51 % higher compared to AA or BA, respectively), indicating that the effective carbon utilization rate and CA production efficiency were greatly improved. Finally, a scaled-up experiment was conducted in a 1.2 L (working volume) automated bioreactor, implying high biomass (optical density at 600 nm or OD600 = 1.809) and a slight decrease in CA production (28.45 mM). A decrease in H2 production (4.11 g/m3) and an increase in CO2 production (0.632 g/m3) demonstrated the appropriate metabolic adaptation of M. hexanoica to environmental changes such as stirring shear.

Effect of pH and medium composition on chain elongation with Megasphaera hexanoica producing C4-C8 fatty acids.

Introduction: Chain elongation technology, which involves fermentation with anaerobic bacteria, has gained attention for converting short and medium chain substrates into valuable and longer-chain products like medium chain fatty acids (MCFAs). In the recent past, the focus of studies with pure chain elongating cultures was on species of other genera, mainly Clostridium kluyveri. Recently, other chain elongators have been isolated that deserve further research, such as Megasphaera hexanoica. Methods: In this study, batch studies were performed in bottles with two different media to establish the optimal conditions for growth of M. hexanoica: (a) a medium rich in different sources of nitrogen and (b) a medium whose only source of nitrogen is yeast extract. Also, batch bioreactor studies at pH values of 5.8, 6.5 and 7.2 were set up to study the fermentation of lactate (i.e., electron donor) and acetate (i.e., electron acceptor) by M. hexanoica. Results and discussion: Batch bottle studies revealed the yeast extract (YE) containing medium as the most promising in terms of production/cost ratio, producing n-caproate rapidly up to 2.62 ± 0.24 g/L. Subsequent bioreactor experiments at pH 5.8, 6.5, and 7.2 confirmed consistent production profiles, yielding C4-C8 fatty acids. A fourth bioreactor experiment at pH 6.5 and doubling both lactate and acetate concentrations enhanced MCFA production, resulting in 3.7 g/L n-caproate and 1.5 g/L n-caprylate. H2 and CO2 production was observed in all fermentations, being especially high under the increased substrate conditions. Overall, this study provides insights into M. hexanoica's behavior in lactate-based chain elongation and highlights optimization potential for improved productivity.

Production of biofuels from C1 -gases with Clostridium and related bacteria-Recent advances.

Clostridium spp. are suitable for the bioconversion of C1 -gases (e.g., CO2 , CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C4 or C6 fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.

Efficient production of n-caproate from syngas by a co-culture of Clostridium aceticum and Clostridium kluyveri.

In recent years, the possibility of merging technologies for waste recovery such as those based on syngas fermentation and chain elongation has been studied for the production of medium chain fatty acids (MCFAs) and bioalcohols, in an attempt to integrate the concept of circular economy in the industry. Nevertheless, one of the main issues of this approach is the pH mismatch between acetogens and chain elongating microorganisms. This work reports, for the first time, the suitability of a co-culture of C. aceticum and C. kluyveri metabolizing syngas at near neutral pH in stirred tank bioreactors. For this purpose, bioreactor studies were carried out with continuous syngas supply. In the first experiment, maximum concentrations of n-butyrate and n-caproate of 7.0 and 8.2 g/L, respectively, were obtained. In the second experiment, considerable amounts of n-butanol were produced as a result of the reduction, by C. aceticum, of the carboxylates already formed in the broth. In both experiments, ethanol was used as an exogenous electron agent at some point. Finally, batch bottle assays were performed with a pure culture of C. aceticum grown on CO in presence of n-butyrate to assess and confirm its ability to produce n-butanol, reaching concentrations up to 951 mg/L, with a n-butyrate conversion efficiency of 96%, which had never been reported before in this species. Therefore, this work contributes to the state of the art, presenting a novel system for the bioproduction of MCFAs by combining syngas fermentation and chain elongation at near neutral pH, as opposed to the acidic pH range used in all previously reported literature.