Presentation on theme: "Katelyn McKindles Bio 381 November 29th, 2012"— Presentation transcript:
1 Katelyn McKindles Bio 381 November 29th, 2012 Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113Katelyn McKindles Bio 381 November 29th, 2012
2 Experimental AimsTo 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 resinMetal degreaserPaint componentOrganic reaction solvent (Jones Oxidation)Acne treatmentsNail polish removerCurrently produced from propylene and benzene mixture resulting from petroleum cracking and refining processescracking 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.
6 Clostridium species are Gram- positive, rod-shaped, spore- formers 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 pathwayExperiment utilized Clostridium sp. MT896 as it had a tolerence of acetone at 2.5mol l-1Mutant can only produce Ethanol and acetate (no butanol pathway)Clostridium
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 productionResearch including this pathway fell off after World War II, when feed stocks such as maize and molasses were in high demandByproducts tend to be highly toxic, produced in low concentrationsButanol 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 InactivationInactivation of phosphotransacetylase which prevents production and accumulation of acetic acid (leads to growth inhibition) and inactivation of acetaldehyde dehydrogenase to prevent acetaldehyde productionGenes inactivated through the introduction of synthesized suicidal vectors (1) pMTerm(B)pta23 and (2) pMTcat_aldh13(1) Uses Erythromycin resistance gene erm(B) from Moorella thermoacetica 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. LjungdahliipUC19 used as DNA backbone. From E. coli JM109
11 Experiment Growth Conditions Anaerobic Chamber or anaerobic Vacu-Quick JarsSyngas (60% CO, 40% H2)Cell exposure to nonsyngas conditions caused sporulationIncubated at 36°CAntibiotics used to grow recombinants:CholoramphenicolErythromycin
13 Continuous bioreactor Vertical bioreactor BioFlo 2000 inoculated with 250ml of overnight seed cultures with OD ±0.15No liquid FlowBioreactors were run with no liquid flow and syngas flow at 25ml min-1 until optimal OD is reached (6.60±0.15)Liquid FlowFlow gradually increased from 0 to 2ml min-1 to maintain OD for 25 daysWaste is gravity collected and headspace is tested every 15 min for CO, CO2, H2; Fluid HPLC tested; fresh cells collected for DNA extraction and electrotransformation
14 ResultsRT-PCR reveals the inactivation of pta and aldh in Clostridium sp. MAceT113 (lanes 3 and 5)Expression of thio, pta, thio + hmgCoAs and aldh in Clostridiumsp. MAceT113 (rtPCR). Lines: 1 – 1 kb Ladder; rtPCR productsfor 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) mMSingle stage continuous syngas fermentation using strain Clostridium sp. MT896 producing acetate and ethanolMSingle stage continuous syngas fermentation using strain Clostridium sp. MAceT113 producing acetone.
16 DiscussionThe use of syngas to produce acetone does not depend on food or petroleum marketAcetone yield 20x of that achieved during conventional ABE fermentationContinuous bioreactor reduces cost due to intercyclic maintenanceThe 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 PTA- Phosphotransacetylase ALDH- acetaldehyde dehydrogenase R10- thiolaseCoAT- acetoacetyl-CoA transferaseR16- acetoacetate decarboxylase
18 Found in Eukaryote cells Acetyl-CoA c-acetyltransferase HMG-CoA synthase HMG-CoA lyase Acetoacetate decarboxylaseFound in Eukaryote cells
19 Cl. sp. MAceT113Lost 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 pathwayElevated 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 headspaceThe 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.Jones and Woods (1986) Acetone-Butanol Fermentation Revisited. Microbiological Reviews. 50.4:Tyurin, Kiriukhin, Berzin (2012) Electrofusion of cells of Acetogen Clostridium 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.“Clostridium,”Kopke, Held, Hujer et al. (2010) Clostridium ljungdahlii represents a microbial production platform based on syngas. PNAS :Berzin, Kiriukhin, Tyurin (2012) Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113. Letters in Applied Microbiology. 55:Rubio, Sierra, Guerrero (2011) Gasification from waste organic material. Ing. Investig. 31:17-25