Butanol as a major product during ethanol and acetate chain elongation

Abstract

Chain elongation is a relevant bioprocess in support of a circular economy as it can use a variety of organic feedstocks for production of valuable short and medium chain carboxylates, such as butyrate (C4), caproate (C6), and caprylate (C8). Alcohols, including the biofuel, butanol (C4), can also be generated in chain elongation but the bioreactor conditions that favor butanol production are mainly unknown. In this study we investigated production of butanol (and its precursor butyrate) during ethanol and acetate chain elongation. We used semi-batch bioreactors (0.16 L serum bottles) fed with a range of ethanol concentrations (100–800 mM C), a constant concentration of acetate (50 mM C), and an initial total gas pressure of ∼112 kPa. We showed that the butanol concentration was positively correlated with the ethanol concentration provided (up to 400 mM C ethanol) and to chain elongation activity, which produced H2 and further increased the total gas pressure. In bioreactors fed with 400 mM C ethanol and 50 mM C acetate, a concentration of 114.96 ± 9.26 mM C butanol (∼2.13 g L−1) was achieved after five semi-batch cycles at a total pressure of ∼170 kPa and H2 partial pressure of ∼67 kPa. Bioreactors with 400 mM C ethanol and 50 mM C acetate also yielded a butanol to butyrate molar ratio of 1:1. At the beginning of cycle 8, the total gas pressure was intentionally decreased to ∼112 kPa to test the dependency of butanol production on total pressure and H2 partial pressure. The reduction in total pressure decreased the molar ratio of butanol to butyrate to 1:2 and jolted H2 production out of an apparent stall. Clostridium kluyveri (previously shown to produce butyrate and butanol) and Alistipes (previously linked with butyrate production) were abundant amplicon sequence variants in the bioreactors during the experimental phases, suggesting the microbiome was resilient against changes in bioreactor conditions. The results from this study clearly demonstrate the potential of ethanol and acetate-based chain elongation to yield butanol as a major product. This study also supports the dependency of butanol production on limiting acetate and on high total gas and H2 partial pressures.

High Efficacy Two-Stage Metal Treatment Incorporating Basic Oxygen Furnace Slag and Microbiological Sulfate Reduction

“…The sequences were truncated to 250 base pairs for quality control and then denoised using the DADA2 pipeline . Taxonomy was assigned to the amplicon sequence variants (ASVs) by referencing the SILVA database (v.138) − as described in previous studies. ,− Bray–Curtis diversity analyses, Faith’s phylogenetic diversity indices, and Pielou’s evenness indices were generated via the q2-diversity plugin core-metrics-phylogenetic method. For these analyses, the sampling depth was 3872, which is the lowest sequence number in the samples from this study.…”

CO2 supply is a powerful tool to control homoacetogenesis, chain elongation and solventogenesis in ethanol and carboxylate fed reactor microbiomes

“…Solventogenesis is commonly observed in microbial chain elongation bioprocess development, leading to the production of not only carboxylates but also alcohols beyond ethanol, including branched alcohols ( de Leeuw et al, 2021 ; Robles et al, 2023 ). Various pathways are now considered for alcohol formation in the development of chain elongation bioprocesses, including: 1) hydrogenotrophic carboxylate reduction (e.g., butyrate reduction to butanol with hydrogen) ( Steinbusch et al, 2008 ): 2) carboxyl-hydroxyl exchanging, which couples hydrogenogenic ethanol oxidation with hydrogenotrophic carboxylate reduction (K. D. De Leeuw et al, 2021 )); 3) carbon monoxide-driven carboxylate reduction ( Diender et al, 2016 ; Richter et al, 2016 ); 4) bioelectrochemical carboxylate reduction using electrons or hydrogen from a cathode ( Sharma et al, 2013 ); or 5) alcohol production as an apparent result of the ethanol-based chain elongation process itself, such as propanol, butanol, and hexanol, as demonstrated with strains from Clostridium kluyveri ( Kenealy and Waselefsky, 1985 ; Candry et al, 2020 ).…”

“…The steering of open culture chain elongation processes involves the meticulous control of various parameters, including pH, temperature, substrate (electron donor and acceptor) species and concentrations, N 2 and CO 2 gas supply, H 2 partial pressure, and hydraulic retention time (HRT) ( Contreras-Dávila et al, 2021a ; Contreras-Dávila et al, 2021b ; De Groof et al, 2020 ; K. D; de Leeuw et al, 2020 ; Grootscholten et al, 2013 ; Robles et al, 2023 ; Roghair, Hoogstad, et al, 2018a ; Shrestha et al, 2023 ). CO 2 gas supply stands out as a particularly crucial parameter, given its dual role as growth nutrient for chain elongating organisms and as an electron acceptor for various other microbes ( Tomlinson, 1954 ; Tomlinson and Barker, 1954 ).…”

Converting food waste into high-value medium chain fatty acids and long chain alcohols via chain elongation with an internally produced electron donor

“…24 Also, as yeast inoculation can affect substrate accessibility for microorganisms, 16 the presence of yeast is likely to influence butanol formation. Moreover, since greater ethanol availability was recently found to be conducive for butanol formation 25,26 and substrate hydrolysis can be influenced by ethanol availability, 14 the higher in situ ethanol yield resulting from the yeast inoculation may also stimulate the butanol biosynthesis from the FW-fed fermentation system. To further back up the assumptions proposed herewith and increase the yield of the valuable end-products, it is necessary to analyse the conversions among various metabolites in this novel fermentation system with yeast inoculation and the underlying mechanism for synthesising these end-products.…”