Lec 6/7 - Bioethanol production from lignocellulose

Question 1

Give the advantages of using bioethanol compared to fossil fuels

Answer 1

• 65% less CO2 emissions
• 80% less NO emissions
• no C particulates
• C neutral
• beneficial to developing countries - lots of land mass/climate for growing these crops

Question 2

Give the disadvantages of using bioethanol compared to fossil fuels

Answer 2

• production = v energy intensive
• only 2/3rds amount of energy released per unit volume
• ethanol is soluble in water & hygroscopic therefore can become diluted on storage
• only 10-15% ethanol petrol mix

Question 3

Give the 3 sources of lignocellulose

Answer 3

• forestry waste (although not enough waste here to supply amount of bioethanol used as substitute for petrol because so much petrol currently used)
• plant crop waste
• energy crop plant grown on non-arable soil (eg switchgrass)

Question 4

Give the 3 steps that are required to convert lignocellulose to bioethanol

Answer 4

1) chemical pretreatment of lignocellulose @ high temps
2) enzyme hydrolysis of hemi/cellulose to c5/c6 sugars
3) fermentation -> bioethanol

Question 5

Give the methods that are followed in the chemical pretreatment step of lignocellulose (state the method which is most commonly used)

Answer 5

• conc acid
• dilute acid @ high temp (most favoured - but high temp :( therefore expensive)
• alkaline (NaOH) treatment
• ozonolysis

Question 6

In terms of enzyme hydrolysis what is the most expensive step In this process?

Answer 6

cost of the cellulase enzymes

Question 7

If we were to combine steps 1 and 2 what properties will the cellulases need to have for them to be effective?

Answer 7

withstand low pHs
withstand high temps
- both found in step 1

Question 8

Describe the components of lignocellulose

Answer 8

50 % cellulose
20 % hemicellulose
rest = non-fermentable lignin

Question 9

Describe the composition of hemicellulose

Answer 9

heteropolysaccharide of C5/C6 sugars
C6 -> glucose, galactose, mannose
C5 -> xylose, arabinose

Question 10

What is the problem of using wild type S. cerevisiae for fermentation?

Answer 10

cannot digest xylose (2nd most abundant molecule in hemicellulose)

Question 11

How is WT S. cerevisiae modified in order for digestion of xylose?

Answer 11

• genes from P. stipitis (xylose reductase - XLY1 and xylitol dehydrogenase - XYL2) expressed in S. cerevisiae
• also modified so it can take up xylose from the environment

Question 12

Describe the bacterial enzyme that converts xylose to xylulose that can also be inserted into S. cerevisiae

Answer 12

xylose isomerase

Question 13

Why, initially, were poor growth and fermentation rates seen when the genes were overexpressed in S. cerevisiae and how was this problem corrected?

Answer 13

imbalance of NAPH and NADH
NADPH dependent Glutamate dehydrogenase (GDH1) deleted and over expressed NADH dependent GDH
this restored the redox balance & stopped xylitol being excreted into the medium

Question 14

Is it possible to combine Steps 2 and 3?

if so how is it possible?

Answer 14

yes

use thermotolerant yeast strains (enzymatic hydrolysis requires high temps)

Question 15

Describe high substrate ethanol production

Answer 15

high substrate conc initially gives an increased efficiency lignocellulose process

Question 16

What are the advantages and disadvantages of high substrate ethanol production ?

Answer 16

+

• 10% yield of ethanol (normally 4-8%)
• reduced energy usage
• reduced water usage
• cost competitive
• high viscosity means that mixing is v hard
• build up of inhibitory products (from pretreatment). can be overcome by using a fed batch approach in step 3. adding the sugars in periodically reduces the build up of inhibitory products

Question 17

What is the general issue that affects all of the processes to do with producing ethanol from lignocellulose?

Answer 17

removal of non-fermentable lignin