General SeminarTopic- Microdiesel, an extension to the Biodiesel

research

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).

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)

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.

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.

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

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…

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..

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.

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

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

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.

Aerated Fed-Batch fermentation using pMicrodiesel

• Glucose was added at regular intervals at around neutral pH.

• Yield- 1.28 g/l after 72 hrs.

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.

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.