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Producing shorterchain fatty acids with Acinetobacter
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Producing shorter-chain fatty acids with Acinetobacter. Ralph Seelke & Dan Levings ... Supernatant from cells was then analyzed by HPLC ... – PowerPoint PPT presentation
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Title: Producing shorterchain fatty acids with Acinetobacter
1
Producing shorter-chain fatty acids with
Acinetobacter
- Ralph Seelke Dan Levings
- Lake Superior Research Institute
- University of Wisconsin-Superior
2
Overview
- Discussion of the problem
- Capabilities of Acinetobacter calcoaceticus HO 1N
- Path to successful use of Acinetobacter in
biofuel production - Progress to date
- Future plans
3
The problem with producing a bio-jet fuel
- Jets fly where it is cold
- Many fuels solidify in the cold, including
biodiesel - Freezing point of fuel is related to carbon chain
length- longer chains solidify at higher
temperatures than shorter chains - Diesel C14-C20
- JP-8 C10-C14
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Potential Solutions for Bio-Jet Fuel
- Bio-diesel is made from fatty acids esterified to
methanol - Since fatty acid chain length is the problem, we
can - Shorten the chain length chemically
- Shorten the chain length enzymatically
5
Acinetobacter
- A very large genus, with multiple species
- Gram-negative, coccobaccillus
- Grows off hydrocarbons
- Makes WAX!
- Reported to have the unusual ability to split
hydrocarbons
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What would have to happen to make this a reality?
- The enzyme(s) involved would need to be stable,
produced in large quantities, and able to be
immobilized on columns
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- Steps towards commercial utilization of
Acinetobacter in shortening fatty acid chains
Identify chain shortening activity (CSA) in vitro
and/or in vivo
Find CSA in related, sequenced Acinetobacter
strains
Sequence HO1N
Clone CSA into E. coli Purify and produce CSA
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Identifying Chain-Shortening Activity
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Results of HPLC Analysis
- Cultures were grown in Soybean oil or Hexadecane
(C16) - Cultures were concentrated and then exposed to
either Soybean oil or C16. - Supernatant from cells was then analyzed by HPLC
- Examined for product with retention similar to
Azaleic acid (nonanedioic acid)
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Growth Soy Exposed - Soy
Growth C16 Exposed - Soy
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Cell-Free Extract activity
- Several attempts to show metabolic activity of
cell-free crude extracts - NAD linked oxidation of alcohols
- Published methods have NOT proven successful
- Testing sonication as a means of cell disruption.
- Particulate material from sonicated cells shows
weak alcohol dehydrogenase activity
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- Increased Absorbance at 340 nm indicates
reduction of NAD NADH - Unlike published reports, activity is low and
remains with particulate fraction.
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Next Steps
- Demonstrate CSA in vitro
- Bioinformatic analysis to search for gene(s)
close to genes involved in alkane utilization. - Cloning of candidate genes and testing for
effects on E. coli ability to utilize alkanes and
show CSA
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- Steps towards commercial utilization of
Acinetobacter in shortening fatty acid chains
Identify chain shortening activity (CSA) in vitro
and/or in vivo
Find CSA in related, sequenced Acinetobacter
strains
Sequence HO1N
Clone CSA into E. coli Purify and produce CSA
17
Conclusions
- The carbon-splitting activity of Acinetobacter
has high potential value - Many obstacles to be overcome before commercial
application would be feasible - High-risk, but high-payoff, research endeavor
18
Thanks to
- UW-Superior Bio-fuels project
- Tom Markee
- Eta Obeya
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