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Background: Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. Results: We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium. Conclusions: This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future. [ABSTRACT FROM AUTHOR]

Background: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. Results: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol. Conclusions: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes. [ABSTRACT FROM AUTHOR]

Background: The replacement of fossil fuels and petrochemicals with sustainable alternatives is necessary to mitigate the effects of climate change and also to counteract diminishing fossil resources. Acetogenic microorganisms such as Clostridium spp. are promising sources of fuels and basic chemical precursors because they efficiently utilize CO and CO2 as carbon source. However the conversion into high titers of butanol and hexanol is challenging. Results: Using a metabolic engineering approach we transferred a 17.9-kb gene cluster via conjugation, containing 13 genes from C. kluyveri and C. acetobutylicum for butanol and hexanol biosynthesis, into C. ljungdahlii. Plasmid-based expression resulted in 1075 mg L−1 butanol and 133 mg L−1 hexanol from fructose in complex medium, and 174 mg L−1 butanol and 15 mg L−1 hexanol from gaseous substrate (20% CO2 and 80% H2) in minimal medium. Product formation was increased by the genomic integration of the heterologous gene cluster. We confirmed the expression of all 13 enzymes by targeted proteomics and identified potential rate-limiting steps. Then, we removed the first-round selection marker using CRISPR/Cas9 and integrated an additional 7.8 kb gene cluster comprising 6 genes from C. carboxidivorans. This led to a significant increase in the hexanol titer (251 mg L−1) at the expense of butanol (158 mg L−1), when grown on CO2 and H2 in serum bottles. Fermentation of this strain at 2-L scale produced 109 mg L−1 butanol and 393 mg L−1 hexanol. Conclusions: We thus confirmed the function of the butanol/hexanol biosynthesis genes and achieved hexanol biosynthesis in the syngas-fermenting species C. ljungdahlii for the first time, reaching the levels produced naturally by C. carboxidivorans. The genomic integration strain produced hexanol without selection and is therefore suitable for continuous fermentation processes. [ABSTRACT FROM AUTHOR]

Background: Clostridium carboxidivorans P7 is capable of producing ethanol and butanol from inexpensive and non-food feedstock, such as syngas. Achieving improved ethanol and butanol production in the strain for industrial application depends on the energetics and biomass, especially ATP availability. Results: This study found that exogenous addition of citrulline promoted accumulation of ATP, increased specific growth rate, and reduced the doubling time of C. carboxidivorans P7. In heterotrophic fermentation experiments, the addition of citrulline increased intracellular ATP by 3.39-fold, significantly enhancing the production of total alcohol (ethanol + butanol) by 20%. Moreover, in the syngas fermentation experiments, the addition of citrulline improved the level of intracellular ATP and the biomass by 80.5% and 31.6%, respectively, resulting in an 18.6% and 60.3% increase in ethanol and the alcohol/acid production ratio, respectively. Conclusions: This is the first report that citrulline could promote the growth of C. carboxidivorans P7 and increase the level of intracellular ATP, which is of great significance for the use of C. carboxidivorans P7 to synthesize biofuels. [ABSTRACT FROM AUTHOR]

Background: Ethanol production through fermentation of gas mixtures containing CO, CO2 and H2 has just started operating at commercial scale. However, quantitative schemes for understanding and predicting productivities, yields, mass transfer rates, gas flow profiles and detailed energy requirements have been lacking in literature; such are invaluable tools for process improvements and better systems design. The present study describes the construction of a hybrid model for simulating ethanol production inside a 700 m3 bubble column bioreactor fed with gas of two possible compositions, i.e., pure CO and a 3:1 mixture of H2 and CO2. Results: Estimations made using the thermodynamics-based black-box model of microbial reactions on substrate threshold concentrations, biomass yields, as well as CO and H2 maximum specific uptake rates agreed reasonably well with data and observations reported in literature. According to the bioreactor simulation, there is a strong dependency of process performance on mass transfer rates. When mass transfer coefficients were estimated using a model developed from oxygen transfer to water, ethanol productivity reached 5.1 g L−1 h−1; when the H2/CO2 mixture is fed to the bioreactor, productivity of CO fermentation was 19% lower. Gas utilization reached 23 and 17% for H2/CO2 and CO fermentations, respectively. If mass transfer coefficients were 100% higher than those estimated, ethanol productivity and gas utilization may reach 9.4 g L−1 h−1 and 38% when feeding the H2/CO2 mixture at the same process conditions. The largest energetic requirements for a complete manufacturing plant were identified for gas compression and ethanol distillation, being higher for CO fermentation due to the production of CO2. Conclusions: The thermodynamics-based black-box model of microbial reactions may be used to quantitatively assess and consolidate the diversity of reported data on CO, CO2 and H2 threshold concentrations, biomass yields, maximum substrate uptake rates, and half-saturation constants for CO and H2 for syngas fermentations by acetogenic bacteria. The maximization of ethanol productivity in the bioreactor may come with a cost: low gas utilization. Exploiting the model flexibility, multi-objective optimizations of bioreactor performance might reveal how process conditions and configurations could be adjusted to guide further process development. [ABSTRACT FROM AUTHOR]

Background: This study aimed at producing C6-C8 medium-chain carboxylates (MCCAs) directly from gaseous CO using mixed culture. The yield and C2-C8 product composition were investigated when CO was continuously fed with gradually increasing partial pressure. Results: The maximal concentrations of n-caproate, n-heptylate, and n-caprylate were 1.892, 1.635, and 1.033 mmol L-1, which were achieved at the maximal production rates of 0.276, 0.442, and 0.112 mmol L-1 day-1, respectively. Microbial analysis revealed that long-term acclimation and high CO partial pressure were important to establish a CO-tolerant and CO-utilizing chain-elongating microbiome, rich in Acinetobacter, Alcaligenes, and Rhodobacteraceae and capable of forming MCCAs solely from CO. Conclusions: These results demonstrated that carboxylate and syngas platform could be integrated in a shared growth vessel, and could be a promising one-step technique to convert gaseous syngas to preferable liquid biochemicals, thereby avoiding the necessity to coordinate syngas fermentation to short-chain carboxylates and short-tomedium-chain elongation. Thus, this method could provide an alternative solution for the utilization of waste-derived syngas and expand the resource of promising biofuels. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures. Results: Here, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n-caproic acid, which were up to 40 mM day-1 (4.64 g L-1 day-1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n-caproic acid production. In addition, n-caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions. Conclusions: Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5. [ABSTRACT FROM AUTHOR]