1. No category

Selective production of acetone during continuous
synthesis gas fermentation by engineered biocatalyst
Clostridium sp. MAceT113
Katelyn McKindles Bio 381
November 29th, 2012
Experimental Aims
• To engineer an acetogen biocatalyst capable
of fermenting synthesis gas blend to acetone
as the only liquid carbonaceous product
Acetone
• Acetone is used in the industry as an organic solvent
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Currently produced from propylene and benzene mixture resulting from
petroleum cracking and refining processes
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Thinning fiberglass resin
Metal degreaser
Paint component
Organic reaction solvent (Jones Oxidation)
Acne treatments
Nail polish remover
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
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.
Biomass Gasification
Clostridium
• Clostridium species are Grampositive, rod-shaped, sporeformers. 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)
Basic Clostridium Metabolism
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
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 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 hydroxymethylglutarylCoA 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
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 grow recombinants:
• Choloramphenicol
• Erythromycin
Syngas Fermentation Broth/Agar
Chemical
g/L
Chemical
g/L
Indicator
NaCl
0.8
KAl(SO4)2*12H2O
0.0004
Resazurin
NH4Cl
1.0
H3BO3
0.0001
0.2ml
KCl
0.1
Na2MoO4*2H2O
0.0002
MgCl2*6H2O
0.2
Na2WO4*2H2O
0.0002
CaCl2*2H2O
0.03
Cysteine-HCL*H2O
0.25
KH2PO4
0.1
Na2S*9H2O
0.25
NaHCO3
1.0
Nicotinic Acid
2.5E-4
Nitrilotriacetic Acid
0.02
Cyancobalamin
2.5E-4
MnSO4*2H2O
0.01
p-Aminobenzoic acid
2.5E-4
(NH4)2Fe (SO4)2*6H2O 0.008
D-Ca-Pantothenate
2.5E-4
CoCl2*6H2O
0.004
Thiamine-HCl
2.5E-4
ZnCl2
0.000
3
Riboflavin
2.5E-4
NiCl2*6H2O
2.5E-4
Lipoic acid
0.0003
Na2SeO4
0.000
3
Folic acid
0.0001
Continuous bioreactor
•Vertical bioreactor BioFlo 2000
inoculated with 250ml of overnight
seed cultures with OD600 3.65±0.15
No liquid
Flow
• Bioreactors were run with no liquid
flow and syngas flow at 25ml min-1
until optimal OD is reached
(6.60±0.15)
Liquid
Flow
•Flow gradually increased from 0 to
2ml min-1 to maintain OD for 25 days
•Waste 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
Results
• RT-PCR reveals the inactivation of pta and aldh
in Clostridium sp. MAceT113 (lanes 3 and 5)
1 23 45
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.
Products (gas and liquid)
mM
Single stage continuous syngas
fermentation using strain
Clostridium sp. MT896 producing
acetate and ethanol
M
Single stage continuous syngas
fermentation using strain
Clostridium sp. MAceT113
producing acetone.
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
ALDH
PTA- Phosphotransacetylase
ALDH- acetaldehyde dehydrogenase
R10- thiolase
CoAT- acetoacetyl-CoA transferase
R16- acetoacetate decarboxylase
2.3.1.9- Acetyl-CoA cacetyltransferase
2.3.3.10- HMG-CoA
synthase
4.1.3.4- HMG-CoA lyase
4.1.1.4- Acetoacetate
decarboxylase
Found in
Eukaryote cells
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
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
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 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.
http://www.kegg.jp/keggbin/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