Lecture 8 - The future of biofuels - fantasy or reality?

Question 1

What are the political drivers of biofuels in the UK?

Answer 1

• the 2005 RTFO (renewable transport fuels obligation) requires that a proportion (5%) of all transport fuel sold in the UK should come from renewable sources
  
  • 1m tonnes a year, 1300 million litres
  • Enforced in 2008
  • Likely through blends and flexible fuel vehicles
  • ideally 2nd generation biofuels
• The Renewable Fuel Standard to get 5% of all fuel as biofuel by 2012
  
  • US department of agriculature established Biomass Crop ASsistance program
  • reduce the risk for farmers growing biofuel crops

Question 2

What is the status of making biofuels in the UK?

Answer 2

2 of the largest bioethanol plants in europe are Ensus (400m litres) and Vivergo (420m litres)

• use wheat as the feedstock
• if working at capacity they would have a major effect on the demand for wheat in the UK (as both are first generation biofuel plants)
• Together use 2 million tonnes of wheat
• DEFRA total wheat demand: 7.5m tonnes with total production ~11.5-12 million tonnes
• pressures on food security
• at mercy of the weather - need to import

Ensus are a UK boifuels company base in Teesside

• Use 1st generation process using whole wheat, produce animal feed as main waste product
• Rising wheat price (valuable feed crop) the process becomes uneconomical (low value product)
• shut down 4 times since opening in 2010
• has a knock on impact on the farmers whose wheat they were going to buy
• price of oil also has an effect, need to be at $70-80 dollars a barrel

Ensus bought by CropEnergies AG (german producer of bioethanol)

• £50m into the site
• employ 100 people and support many other job in the wider supply chain: farmers, hauliers, people emplyed in the engineering support and storage sectors

Question 3

How does producing biofuels have an impact on the demand for wheat?

Answer 3

• 2 of the largest bioethanol plants in europe are Ensus (400m litres) and Vivergo (420m litres)
• use wheat as the feedstock
• if working at capacity they would have a major effect on the demand for wheat in the UK (as both are first generation biofuel plants)
• Together use 2 million tonnes of wheat
• DEFRA total wheat demand: 7.5m tonnes with total production ~11.5-12 million tonnes
• pressures on food security
• at mercy of the weather - need to import

Question 4

Give an example of the harsh reality of making biofuels

Answer 4

• Ensus are a UK boifuels company base in Teesside
• Use 1st generation process using whole wheat and also get value from the animal feed that they prouce as their main waste product
• as soon as wheat prices rise (valuable feed crop) the process becomes uneconomical (low value product)
• shut down 4 times since opening in 2010
• has a knock on impact on the farmers whose wheat they were going to buy
• proce of oil also has an effect, need to be at $70-80 dollars a barrel

Question 5

Give an example of how biofuel production has extreme sensitivity to economic fluxuation

Answer 5

• Ensus bought by CropEnergies AG (german producer of bioethanol)
• £50m into the site
• employ 100 people and support many other job in the wider supply chain
  
  • farmers
  • hauliers
  • people emplyed in the engineering support and storage sectors

Question 6

How have the US attempted to link biofuels and crops?

Answer 6

• The Renewable Fuel Standard to get 5% of all fuel as biofuel by 2012
• US department of agriculature established Biomass Crop ASsistance program
  
  • reduce the risk for farmers growing biofuel crops
• $300million Highlands ethanol project in florida lead by BP as the first major cellulosic bioethanol plant in USA
  
  • capacity of 35 m gallons per year
  • Lykes providing the feedstock from 20 000 farmable acres near the site

Question 7

Why did BP pull out of cellulosic bioethanol?

Answer 7

• tight margins
• high feedstock prices - economically viable cellulosic feedstocks
• overcapacity
• depressed sugar and power prices

Question 8

What is the trend of biofuel production between 100 and 2010?

Answer 8

Up!

Total increased from ~20 billion litres to ~100 billion litres in 10 years

Question 9

What is DuPonts involvment in biofuels?

Answer 9

• opening 30m gallon cellulosic ethanol plant in Nevade, LA, 2015
• corn stover as feedstock (bits of corn left over after sweetcorn ears)
• using unique strain of Zymomonas to do the fermentation
• Bacterial commercial process
• Oil has to be around $70-80 a barrel to be competitive

Question 10

What is project liberty?

Answer 10

• Lignocellulosic ethanol plant in emmetsburg, Iowa
• partners with DSM (enzyme company)

Question 11

What is the future for biofuels?

Answer 11

• despite BPs withdrawal from the bioethanol market there are still many other operators starting cellulosic bioethanol plants in 2014
• hugh amounts of investment coming from government to promote research into second generation biofuels e.g. BSBEC
• UK companies pushing biobutanol, algal biodiesel and syngas
• new feedstocks like municipal solid waste are also attractive as are much cheaper and sustainable
  
  • especially with growing population
  • use anaerobic fermentation - potential for increased optimisation and engineering
• Very few going to make money under current market conditions (aside from biogas)

Question 12

Who generate petrol as a biofuel?

Answer 12

Yong Jun Choi and Sang Yup Lee

Question 13

What are short chain alkanes? (petrol)

Answer 13

• `fully saturated (no double bonds) hydrocarbons which form open chains
• General formular is C_nH_2n+2
• The petrol in car is a mixture of SCAs
• combusted in car engine by reacting with oxygen to form CO₂ and H₂O
• the liberation of the gas and heat forces the piston back down the cylinder
• e.g butane C4H10
• Nonane C9H20
• Dodecane C12H26

Question 14

What was the ain of the paper?

Answer 14

To engineer pathways into E.coli to produce:

• Fatty esters
• Fatty alchohols
• Short chain carbons to amek alkanes, with acyl-ACP as starting point

Engineer fadE out to get more flux to the production of these moecules

Question 15

What stages were there to consider in engineering a new SCA pathway to make alkanes, moving from acyl-ACP as the starting point?

Answer 15

5 stages

1. removing fadE
2. engineering thioesterases
3. removing transcriptional control
4. enhancing fatty acyl-CoA synthase
5. converting fatty acyl-CoAs to SCAs

Question 16

How and why did the authors want to remove fadE?

Answer 16

• As well as making free fatty acids E.coli will catabolise them using the beta-oxidation pathway
• In the ideal pathway free FFAs are intermediates so don’t want them to be removed by a competeing pathway
• removed flux through the beta-oxidation pathway by deleting the acyl-CoA dehydrogenase gene fad1 (GAS1 strain)
• Used the recombineering method

Question 17

What was the recombineering method used to remove fadE?

Answer 17

1. made a marked mutation where the fadE gene replaced b an antibiotic resistance gene
2. antibiotic resistnace gene is flanked with sites for a specific recombinase
3. the gene for the recombinase is transformed on a helper plasmid and catalyses the removal of the antibiotic resistance gene
4. This leaves a clean deletion of the original gene on the chromosome (unmarked)

Question 18

How did the authors engineer thioesterases?

Answer 18

• Wanted thioesterase enzymes that created a large amount of FFAs
• Created a fadD deletion strain (in the same way as the fadE mutant)
• Expressed genes for three different enzymes in trans
• TesA was the best
  
  • periplasmic enzyme as the signal peptide was removed to keep it in the cytoplasm
  • knew a point mutation L109P made it better at generating short chain FFAs
  • this was seen in the profile of FFAs that accumulate in the fadD deletion strain

Question 19

How did the authors remove transcriptional control?

Answer 19

• fadR is a transcription factor that regulates the beta-oxidation genes and expression of fabAB
• removal of fadR led to a change in the activity of the fatty acid elongastion cycle - resulting in a profile of FFAs shifted towards shorter chains and the removal of any unsaturated FFAs
• Moved the fadR deletion into GAS1 to create GAS2

Question 20

How did the authors enhance fatty acyl-CoA synthetase?

Answer 20

• got right FFas at high levels
• needed to direct the flux of the FFAs to make alkanes
• put fadD under the control of the strong trc promoter on the chromosome
• when this is added to the GAS2 they have final background strain GAS3 (mutant: fadE, fadR; additional fadD::P_trc)
• also put in on a plasmid
  *

Question 21

How did the authors concert fatty actl-CoAs to SCAs?

Answer 21

• to complete route to SCAs, introduce two new enzyme into E.coli
• clostridium acetobutylicum acr gene
  
  • encodes a fatty acyl-CoA reductase for the reduction of fatty acyl-CoAs to fatty aldehydes
• The arabidopsis taliana CER1 gene
  
  • encoding a fatty aldehyde decarboxylase
  • decarbonylation of fatty aldehydes to corresponding hydrocardon
• both were introduced on plasmids drivedn from either Ptac or Ptrc
• Cer1 codon optimised for E.coli using a synthetic gene

Question 22

What was the result of the genetic engineering by the authors to make petrol?

Answer 22

• main FFA was C10
• Cer1 enzyme removes a carbon
• length of the final SCA - 9 carbons (nonane)
• overall yeild 400mg/L using batch fed fermentation
• CER1 more active at 30 degrees
• Yeild increased to 580 mg/L
• Though CER1 was active against long chain fatty acids (above 30Cs)
• still potential to manipulate further

Question 23

What are the global trends of biofuel production?

Answer 23

$300million Highlands ethanol project in florida lead by BP as the first major cellulosic bioethanol plant in USA

• capacity of 35 m gallons per year
• Lykes providing the feedstock from 20 000 farmable acres near the site

2013 BP pulled out

• tight margins
• high feedstock prices - economically viable cellulosic feedstocks
• overcapacity
• depressed sugar and power prices

Global trend is up - Total increased from ~20 billion litres to ~100 billion litres in 10 years

Sucess stories:

• Dupont opening 30m gallon cellulosic ethanol plant in Nevade, LA, 2015
  
  • corn stover as feedstock (bits of corn left over after sweetcorn ears)
  • using unique strain of Zymomonas to do the fermentation
  • Bacterial commercial process
  • Oil has to be around $70-80 a barrel to be competitive
• POET-DSM, “Project liberty”
  
  • Lignocellulosic ethanol plant in emmetsburg, Iowa
  • partners with DSM (enzyme company)