Katelyn McKindles Bio 381 November 29 th , 2012

1. Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113 Katelyn McKindles Bio 381 November 29th, 2012

2. Experimental Aims • To engineer an acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product

3. Acetone • Acetone is used in the industry as an organic solvent • Thinning fiberglass resin • Metal degreaser • Paint component • Organic reaction solvent (Jones Oxidation) • Acne treatments • Nail polish remover • Currently produced from propylene and benzene mixture resulting from petroleum cracking and refining processes • cracking is the process whereby complex organic molecules such as kerogens or heavy hydrocarbons are broken down into simpler molecules such as light hydrocarbons, by the breaking of carbon-carbon bonds in the precursors. • Expensive process- ~$1000/ton

4. Synthesis Gas Fermentation The structural materials that plants produce to form the cell walls, leaves, stems, stalks, and woody portions of biomass are composed mainly of three biobased chemicals called cellulose, hemicellulose, and lignin. Together, they are called lignocellulose, a composite material of rigid cellulose fibers embedded in a cross-linked matrix of lignin and hemicellulose that bind the fibers. Lignocellulose material is by necessity resistant to physical, chemical, and biological attack, but it is of interest to biorefining because the cellulose and hemicellulose can be broken down through a process called hydrolysis to produce fermentable, simple sugars. Lignocellulosic biomass is often a waste material of the food processing and forest products industries that may be locally, readily available at low cost. Biomass gasification is a high-temperature process (600 to 1000oC) to decompose the complex hydrocarbons of biomass into simpler gaseous molecules, primarily hydrogen, carbon monoxide, and carbon dioxide.

5. Biomass Gasification

6. Clostridium species are Gram-positive, rod-shaped, spore-formers. These generally obligate anaerobes are ubiquitous saprophytes or part of our normal flora. • Clostridia employ butyric fermentation pathways to generate energy and, as a result, often produce a foul odor. • Novel metabolic pathways: Wood-Ljungdahl pathway, Solventgenesis, ABE pathway • Experiment utilized Clostridium sp. MT896 as it had a tolerence of acetone at 2.5mol l-1 • Mutant can only produce Ethanol and acetate (no butanol pathway) Clostridium

7. Basic Clostridium Metabolism

8. Acetone-Butanol-Ethanol Process • Among the saccharolytic butyric acid-producing clostridia, there are a number of species capable of producing significant amounts of neutral solvents during the later stages of batch fermentation. • As the culture enters the stationary growth phase, the metabolism of the cells undergo a shift to solvent production • Research including this pathway fell off after World War II, when feed stocks such as maize and molasses were in high demand • Byproducts tend to be highly toxic, produced in low concentrations • Butanol is toxic at low concentrations, limiting the level of solvent in the final fermentation broth around 2% • Acetone sensitivity is about 86mmol l-1

10. Gene Inactivation • Inactivation of phosphotransacetylase which prevents production and accumulation of acetic acid (leads to growth inhibition) and inactivation of acetaldehyde dehydrogenase to prevent acetaldehyde production • Genes inactivated through the introduction of synthesized suicidal vectors (1) pMTerm(B)pta23 and (2) pMTcat_aldh13 • (1) Uses Erythromycin resistance gene erm(B) from Moorellathermoacetica for screening. Contains genes for thiolase (thio) and hydroxymethyglutaryl-CoA synthase (hmgCoAs), as well as a fragment of pta from Clostridium ljungdahlii • (2) Uses Chloramphenicol acetyltransferase (resistance) gene cat from M. thermoacetica for screening. Contains genes for hydroxymethylglutaryl-CoA lyase (hmgCoAl) and acetoacetate decarboxylase (adc), as well as a fragment of aldh from Cl. Ljungdahlii • pUC19 used as DNA backbone. From E. coli JM109

11. Experiment Growth Conditions • Anaerobic Chamber or anaerobic Vacu-Quick Jars • Syngas (60% CO, 40% H2) • Cell exposure to nonsyngas conditions caused sporulation • Incubated at 36°C • Antibiotics used to growrecombinants: • Choloramphenicol • Erythromycin

12. Syngas Fermentation Broth/Agar

13. Continuous bioreactor

14. Results • RT-PCR reveals the inactivation of pta and aldh in Clostridium sp. MAceT113 (lanes 3 and 5) 1 2 3 4 5 Expression of thio, pta, thio + hmgCoAs and aldh in Clostridium sp. MAceT113 (rtPCR). Lines: 1 – 1 kb Ladder; rtPCR products for mRNA: thio (2), pta (3), thio + hmgCoAs (4) and aldh (5). Single plasmid recombinants were detected at 7.2±0.11*10-6 per non-transformed cell, while double plasmid recombinants were detected at 8.3±0.02*10-7 per single plasmid recombinant.

15. Products (gas and liquid) mM Single stage continuous syngas fermentation using strain Clostridium sp. MAceT113 producing acetone. Single stage continuous syngas fermentation usingstrainClostridium sp. MT896 producing acetate and ethanol M

16. Discussion • The use of syngas to produce acetone does not depend on food or petroleum market • Acetone yield 20x of that achieved during conventional ABE fermentation • Continuous bioreactor reduces cost due to intercyclic maintenance • The addition of a solar panel to the system would enhance hydrogen production for the process needs that would enhance energy recovery when coupled with water electrolysis

17. X X ALDH PTA- Phosphotransacetylase ALDH- acetaldehyde dehydrogenase R10- thiolase CoAT- acetoacetyl-CoA transferase R16- acetoacetate decarboxylase

18. 2.3.1.9- Acetyl-CoA c-acetyltransferase2.3.3.10- HMG-CoA synthase4.1.3.4- HMG-CoA lyase4.1.1.4- Acetoacetate decarboxylase Found in Eukaryote cells

19. Cl. sp. MAceT113 • Lost the ability to produce ethanol and acetate due to gene inactivation. • Because the new genes added were eukaryotic, the prokaryotic cell had no regulation system in place to prevent high productions of acetone through the ketone synthesis pathway • Elevated levels of production likely due to the expression of multiple copies of the synthetic constructs stabilized by integration into multiple sites of the target genes

20. Limitations and Problems • The bioreactors were kept at 1.8L of culture at all times. The paper did not discuss the possible issues that would occur when scaling up for industrial use. Ideally, the project wanted to have zero free carbon (CO2 production as a byproduct), which would be harder at a larger scale as larger bioreactor tanks would have more headspace • The original Cl. sp. MT896 was UV mutated from Cl. sp. MT962, but screening processes and species descriptions are never given. It is never specified exactly how the UV mutant is different than the parent species

21. References • “Fermentation of Lignocellulose Biomass,” Wisconsin biorefining development initiative. www.wisbiorefine.org • Jones and Woods (1986) Acetone-Butanol Fermentation Revisited. Microbiological Reviews. 50.4:484-524 • Tyurin, Kiriukhin, Berzin(2012) Electrofusion of cells of AcetogenClostridium sp. MT 351 with erm(B) or cat in the chromosome. Journal of Biotech Research. 4:1-12 • “Synthesis and Degradation of Ketone Bodies,” Kegg Pathway. http://www.kegg.jp/kegg-bin/highlight_pathway?scale=1.0&map=map00072&keyword=ketone • “Clostridium,” http://www.cehs.siu.edu/fix/medmicro/clost.htm • Kopke, Held, Hujer et al. (2010) Clostridium ljungdahlii represents a microbial production platform based on syngas. PNAS 107.29:13087-13092 • Berzin, Kiriukhin, Tyurin (2012) Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113. Letters in Applied Microbiology. 55:149-154 • Rubio, Sierra, Guerrero (2011) Gasification from waste organic material. Ing. Investig. 31:17-25