https://bio.as.uky.edu/sites/default/files/Electrobiocommodities%202013%20copy.pdf

Electrobiocommodities: powering microbial production of fuels and commodity chemicals from carbon dioxide with electricity

Acetogenic bacteria employing the Wood–Ljungdahl pathway can be used as biocatalysts in syngas fermentation for the production of biofuels such as ethanol or butanol as well as biocommodities such as acetate, lactate, butyrate, 2,3 butanediol, and acetone. The potential of such processes can be projected by the global syngas output, which was 70,817 megawatts thermal in 2010 and is expected to increase up to 72% in 2016. To date, different acetogens are used as commercial production strains for industrial syngas fermentations in pilot or demonstration plants (Coskata, INEOS Bio, LanzaTech) and first commercial units are expected to launch operation in the near future (INEOS Bio, LanzaTech). Considerations on potential yields are quite promising for fermentative production. New methods for metabolic engineering were established to construct novel recombinant acetogenic biocatalysts. Synthetic biology will certainly play a major role in constructing strains for commercial operations. This way, a cheap and a...

Bacterial synthesis gas (syngas) fermentation

https://orbit.dtu.dk/files/85977334/1_s2.0_S0958166913007167_main.pdf

Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms

Synthesis gas, a mixture of CO, H2, and CO2, is a promising renewable feedstock for bio-based production of organic chemicals. Production of medium-chain fatty acids can be performed via chain elongation, utilizing acetate and ethanol as main substrates. Acetate and ethanol are main products of syngas fermentation by acetogens. Therefore, syngas can be indirectly used as a substrate for the chain elongation process. Here, we report the establishment of a synthetic co-culture consisting of Clostridium autoethanogenum and Clostridium kluyveri. Together, these bacteria are capable of converting CO and syngas to a mixture of C4 and C6 fatty acids and their respective alcohols. The co-culture is able to grow using solely CO or syngas as a substrate, and presence of acetate significantly stimulated production rates. The co-culture produced butyrate and caproate at a rate of 8.5 ± 1.1 and 2.5 ± 0.63 mmol/l/day, respectively. Butanol and hexanol were produced at a rate of 3.5 ± 0.69 and 2.0 ± 0.46 mmol/l/day, respectively. The pH was found to be a major factor during cultivation, influencing the growth performance of the separate strains and caproate toxicity. This co-culture poses an alternative way to produce medium-chain fatty acids and higher alcohols from carbon monoxide or syngas and the process can be regarded as an integration of syngas fermentation and chain elongation in one growth vessel.

/pdf/production-of-medium-chain-fatty-acids-and-higher-alcohols-ntne2l0eff.pdf

Production of medium-chain fatty acids and higher alcohols by a synthetic co-culture grown on carbon monoxide or syngas

Reactor systems for syngas fermentation processes: A review

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60 % CO and 40 % H2 in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.

Selective n-butanol production by Clostridium sp. MTButOH1365 during continuous synthesis gas fermentation due to expression of synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase.

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p<0.005) and 321 mM ethanol (p<0.005) from synthesis gas (syngas) blend 60 % CO and 40 % H(2). Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610 mM (p<0.005). Inactivation of pta rendered only 502 mM of ethanol (p<0.005). The acetogen biocatalyst with eliminated aldh produced 450 mM acetate (p<0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.

Cre-lox66/lox71-based elimination of phosphotransacetylase or acetaldehyde dehydrogenase shifted carbon flux in acetogen rendering selective overproduction of ethanol or acetate.

Methanol-resistant mutant acetogen Clostridium sp MT1424 originally producing only 365 mM acetate from CO2/CO was engineered to eliminate acetate production and spore formation using Cre-lox66/lox71-system to power subsequent methanol production via expressing synthetic methanol dehydrogenase, formaldehyde dehydrogenase and formate dehydrogenase, three copies of each, assembled in cluster and integrated to chromosome using Tn7-based approach Production of 22 M methanol was steady (p < 0005) in single step fermentations of 20 % CO2 + 80 % H2 blend (v/v) 25 day runs each in five independent repeats If the integrated cluster comprised only three copies of formate dehydrogenase the respective recombinants produced 95 mM formate (p < 0005) under the same conditions For commercialization, the suggested source of inorganic carbon would be CO2 waste of IGCC power plant Hydrogen may be produced in situ via powered by solar panels electrolysis

Selective methanol or formate production during continuous CO2 fermentation by the acetogen biocatalysts engineered via integration of synthetic pathways using Tn7-tool

Acetogen strain Clostridum sp. MT653 produced acetate 273 mM (p < 0.005) and ethanol 250 mM (p < 0.005) from synthesis gas blend mixture of 64 % CO and 36 % H2. Clostridum sp. MT653 was metabolically engineered to the biocatalyst strain Clostridium sp. MTEtOH550. The biocatalyst increased ethanol yield to 590 mM with no acetate production during single-stage continuous syngas fermentation due to expression of synthetic adh cloned in a multi-copy number expression vector. The acetate production was eliminated by inactivation of the pta gene in Clostridium sp. MTEtOH550. Gene introduction and gene elimination were achieved only using Syngas Biofuels Energy, Inc. electroporation generator. The electrotransformation efficiencies were 8.0 ± 0.2 × 106 per microgram of transforming DNA of the expression vector at cell viability ~15 %. The frequency of suicidal vector integration to inactivate pta was ~10−5 per the number of recipient cells. This is the first report on elimination of acetate production and overexpression of synthetic adh gene to engineer acetogen biocatalyst for selective biofuel ethanol production during continuous syngas fermentation.

Elimination of Acetate Production to Improve Ethanol Yield During Continuous Synthesis Gas Fermentation by Engineered Biocatalyst Clostridium sp. MTEtOH550

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO2/H2 blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60 % CO and 40 % H2 (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO2/H2 blend was fed instead of syngas (p < 0.005). Mevalonate from CO2/H2 blend might serve as a commercial route to mitigate global warming in proportion to CO2 fermentation scale worldwide.

Michael Kiriukhin

Papers

Selective n-butanol production by Clostridium sp. MTButOH1365 during continuous synthesis gas fermentation due to expression of synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase.

Cre-lox66/lox71-based elimination of phosphotransacetylase or acetaldehyde dehydrogenase shifted carbon flux in acetogen rendering selective overproduction of ethanol or acetate.

Selective methanol or formate production during continuous CO2 fermentation by the acetogen biocatalysts engineered via integration of synthetic pathways using Tn7-tool

Elimination of Acetate Production to Improve Ethanol Yield During Continuous Synthesis Gas Fermentation by Engineered Biocatalyst Clostridium sp. MTEtOH550

Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO2/H2 blend