micro diesel ppt
TRANSCRIPT
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General SeminarTopic- Microdiesel, an extension to the Biodiesel
research
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A word on Biodiesel
• An alternative source, substitute for petroleum based diesel fuel.
• Production: Transesterification to produce FAME or FAEE
Trans-esterification of oil to biodiesel. R1-3 are hydrocarbon groups (Chisti Yusuf, 2007).
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Biodiesel- Pros and Cons
Advantages• Maintains a balanced CO2
cycle
• Reduced emissions during combustion(CO,S, etc.)
• High flash point, low flammability, so safe
• Completely biodegradable
• Good lubrication properties
Limitations• Sufficient vegetable oil feed
stocks reqd (rapeseed, soyabean, palm oil), so production limited
• V.O mainly consist of TAGs, can’t be used directly, Viscosity , Filter plugging.
• FAME (source, toxic) / FAEE (cost)
• Extensive Acreage (only 1300 l ha-1 from rapeseed)
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Consequence of disadvantages
• Biodiesel based on oilseed crops will probably not be able to substitute more than 5–15 % of petroleum based diesel in the future. (Rainer Kalscheuer , Torsten Stölting andAlexander Steinbüchel, 2006)
• What is reqd??
-To develop processes enabling biodiesel production from bulk plant materials like sugars, starch etc.
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The Next Level : Microdiesel
• Engineering microbes to produce biodiesel:microdiesel.
• The concept is to genetically and metabolically engineer microbes to direct them towards the production of biodiesel from bulk and
easily available materials.
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E.Coli engineered for fuel production(Rainer Kalscheuer, Torsten Stölting and Alexander
Steinbüchel,2006)
• The authors established biosynthesis of biodiesel-adequate FAEEs, referred to as Microdiesel, in metabolically engineered Escherichia coli
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The Basic Concept employed:
• The unspecific acyltransferase (wax ester synthase/acyl-coenzyme A
: diacylglycerol acyltransferase (WS/DGAT; the atfA gene
product)simultaneously synthesizes wax esters and TAGs by
utilizing long-chain fatty alcohols or diacylglycerols and fatty acid
coenzyme A thioesters (acyl-CoA) as substrates (Kalscheuer &
Steinbüchel, 2003) in its natural host Acinetobacter baylyi strain
ADP1.
• Exhibits very low substrate specificity.
Cntd…
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Cntd…
• So, it should be possible to produce fatty acid ethyl esters (FAEE) or FAME for use as biodiesel in recombinant bacteria such as E.coli or Bacillus sp or fungi class Actinomycetes..
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What was done?• Created a recombinant E.coli havingfollowing plasmids were used
along with the inserts: pLOI297- genes pdc and adhB from Z.mobilis pBBR1MCS-2 : : atfA harbouring the WS/DGAT gene from A. baylyi
strain ADP1
• Result: FAEE synthesis was achieved along with other products like ethyl
palmitoleate, ethyl palmitate etc.
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Pathway of FAEE biosynthesis inrecombinant E. coli. FAEE formation wasachieved by coexpression of the ethanolicenzymes pyruvate decarboxylase (Pdc) andalcohol dehydrogenase (AdhB) from Z.mobilis and the unspecific acyltransferaseWS/DGAT from A. baylyi strain ADP1
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Yield in Batch fermentation
• Aerobic-0.26 g/l (also saturation after 18 hrs)• Anaerobic-0.05 g/l• O2 restrained conditions- 0.16 g/l
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Improvement: the very desired
• pMicrodiesel
3.2 kbp BamHI fragment of pLOI297, which comprised the Z.
mobilis genes pdc and adhB and the upstream lacZ promoter
region, was cloned into BamHI-linearized pKS : : atfA, a derivative of
the highcopy-number plasmid pBluescript KS2 (Kalscheuer
&Steinbu¨chel, 2003), yielding pMicrodiesel.
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Aerated Fed-Batch fermentation using pMicrodiesel
• Glucose was added at regular intervals at around neutral pH.
• Yield- 1.28 g/l after 72 hrs.
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Microbial production of fatty-acid-derived fuels and chemicals from plant biomass
(Eric J. Steen, Yisheng Kang et.al.,2010)
• Engineering ofEscherichia coli to produce structurally tailored fatty
esters (biodiesel), fatty alcohols, and waxes directly from simple
sugars. Furthermore, we show engineering of the biodiesel-
producing cells to express hemicellulases, a step towards producing
these compounds directly from hemicellulose, a major component
of plant derived biomass.
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Future Perspectives:• It is still a challenge to establish microdiesel production solely from
bulk plant materials
• Using actinomycetes (lipid accumulating bacteria)(they are capable of synthesizing high amounts of FA under growth
limiting conditions from glucose, accumulate them as TAGs) Solution: expression of ethanol producing genes in these aerobic
bacteria may be a promising.
• Low reaction rate of WS/DGAT with ethanol than long chain fatty acids.
Solution: search for homologues with more specificity for ethanol.
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