1. ETHANOL PRODUCTION FROM LIGNOCELLULOSIC MATERIALS
Dr. TERESA FERNÁNDEZ ALDAMA
“SAMARA UNIVERSITY”

2. Molasses Inoculum propagation Fermentation Distillation Ethanol
That it is a Flow chart for production of ethanol from simple sugars. They have simple structure and microorganisms can convert them into ethanol directly.
Ethanol

3. Ethanol production from LCM
Lignocellulosic Materials ?
However, when we have more complex structures they need to be converted in simple sugars before fermentation. This conversion can occur by hydrolysis. But sometimes hydrolysis is not efficient due to the complex structure of LCM and is necessary to pretreat the material to increase sugar concentration for producing ethanol.
Ethanol

4. Flowchart for ethanol production from LCM
Lignocellulosic Materials
Pretreatment
Physical
Biological
Chemical
Physico-chemical
Hydrolysis
Fermentation
After pretreatment there is by products formation that can inhibit the fermentation process and just today we will study how to overcome the inhibition problems before fermentation of lignocellulosic hydrolysate.
Ethanol
Distillation

5. Outline
Composition of lignocellulosic hydrolysates. Formation of inhibitory. Inhibitory compounds.
Strategies to overcome the fermentation inhibitors.
To understand the methods about removing toxic compounds from the hydrolysates we will study the composition of lignocellulosic hydrolysates. Formation of inhibitory and inhibitory compounds. Finally we will see some strategies to overcome the fermentation inhibitors.

6. Objectives
To identify inhibitors formed during pretreatment of lignocellulosic materials
To explain how to overcome inhibition problems
When we finish this activity, you are be able to identify inhibitors formed during pretreatment of lignocellulosic materials and
To explain how to counteract inhibition problems.

7. Formation of inhibitors
Hemicellulose
Cellulose
Lignin
Extractives
Phenols
Glucose
Manose
Galactose
Xylose
Arabinose
Furfural
Hydroxymethyl furfural (HMF)
Acetic acid
We know what are the mayor components of lignocellulosic materials. They are composed by cellulose, hemicellulose, lignin and extractives. When we pretreat materials there are glucose from cellulose and hemicellulose. Manose, galactose, xylose and arabinose from hemicellulose. From lignin and extractives are produced phenolic and others aromatic compounds. During pretreatment of lignocellulosic materials and due to the conditions of process there are degradation of the components. Under strong pretreatment conditions such as long reaction time, high temperature and acid concentration we can find hydroximethylfurfural, furfural and levulinic acid, formic acid and acetic acid.
Levulinic acid
Formic acid

8. Formation of inhibitors
Hemicellulose
Cellulose
Lignin
Extractives
Phenols
Glucose
Manose
Galactose
Xylose
Arabinose
Furfural
Hydroxymethyl furfural (HMF)
Acetic acid
There are three different groups of inhibitory compounds. Furan derivatives (furfural and hydromethylfurfural), organic acids (levulinic, formic and acetic acids).
Levulinic acid
Formic acid

9. Inhibitory compounds Inhibitory effects
Furan derivatives: furfural and HMF
Organic compounds: levulinic, formic and acetic acids
Fenolic compounds
Inhibitory effects
The generation of by-products from pretreatment depends on feedstokcs and pretreatment methods. By-products formed affect Cell physiology (decrease viability) and there is a low ethanol yield and productivity. Fermentation of a mixture of hexose, pentose and inhibitors can introduce further problems in production of ethanol from lignocellulosic materials. This is the reason for Strategies to overcome inhibition problems.
☹ Cell physiology (decrease viability)
☹ Low ethanol yield and productivity

10. Strategies to overcome inhibition problems
Preventing formation of inhibitors
Reduction of amounts of formed inhibitors (Detoxification)
Adaptation of strains to inhibitors
Genetical transformation of microorganism to enhance its tolerance
Here you have some of these strategies.
Preventing formation of inhibitors
Reduction of amounts of formed inhibitors (Detoxification)
Adaptation of strains to inhibitors
Genetical transformation of microorganism to enhance its tolerance

11. Strategies to overcome inhibition problems
1. Preventing formation of inhibitors
To select less recalcitrant feedstock and to utilize mild pretreatment conditions
Simultaneous saccharification and fermentation (SSF)
Preventing formation of inhibitors. This strategy tries to reduce the formation of inhibitors during the hydrolysis. One possibility is To select less recalcitrant feedstock and to utilize mild pretreatment conditions. But it will be cause other problem. If you select mild conditions, may be you will obtain a few amount of sugars and it is not desirable from the economic point of view. Other possible solution is to use simultaneous saccharification and fermentation. With this strategy we hydrolyze cellulosic materials and at the same time fermentation process occurs and inhibition of by-products is reduced.

12. Strategies to overcome inhibition problems
2. Reduction of amounts of formed inhibitors (Detoxification)
Biological: envolve the use of microorganism that convert toxic compounds by changing their chemical structure.
Physical: promote the removal of toxic compounds from the medium without changing their chemical structures.
Chemical: based on the use of chemical additives (overliming, reducing agents, polymers) and ph modifications.
Detoxification method is one of the most powerful ways to overcome inhibition problems.
There are biological, physical and chemical detoxification. Biological: involve the use of microorganism that convert toxic compounds by changing their chemical structure. Physical: promote the removal of toxic compounds from the medium without changing their chemical structures. Chemical: based on the addition of chemical additves (overliming, reducing agents, polymers) and ph modifications.

13. Strategies to overcome inhibition problems
Detoxification methods:
☺Improve fermentability and enzymatic saccharification of cellulose.
☹ A separate process step is required. Then is possible to perform the treatment ¨in situ¨ (in the bioreactor) and there is no need for extra process step for detoxificaction.
Detoxification methods in general, improve fermentability and enzymatic saccharification of cellulose, but a separate process step is required. Then is possible to perform the treatment ¨in situ¨ (in the bioreactor) and there is no need for extra process step for detoxification.

14. Strategies to overcome inhibition problems
3. Adaptation of strains to inhibitors: ability of microorganism to grow in lignocellulosic hydrolysates.
They can increase the tolerance:
Agaisnt phenolic compounds, furaldehydes and aliphatic acids.
To high temperature.
Adaptation of strains to inhibitors: ability of microorganism to grow in lignocellulosic hydrolysates.
They can increase the tolerance:
Agaisnt phenolic compounds, furaldehydes and aliphatic acids and to high temperature.

15. Strategies to overcome inhibition problems
Adaptation of strains to inhibitors
Screening
0 %
25 %
50 %
75 %
100 %
Agar plates with hydrolysate
Liquid culture médium with Hidrolysates
There is an example of adaptation of strain to inhibitors. Adaptation starts with a screening of the cells that grow at different concentrations of the inhibitors in solid media, from 0 to 100 %. Then is selected the best cells growth and it is inoculated in liquid medium with different concentration of hydrolysate. Finally is taken strain from the best results. The result cells are adapted to grow at the given concentration of inhibitors.
50 %
65 %
80 %
90%
92,5 %
95 %
Resistant
strain

16. Adaptation of strains to inhibitors
30oC
32oC
34oC
36oC
38oC
40oC
YPD
YPD-H
Cepa original
Cepa termotolerante
Adaptation of strains to inhibitors
OriginalStrain
YPD
30 0C
32 0C
34 0C
36 0C
38 0C
400C
32 0C
34 0C
36 0C
38 0C
40 0C
There is an example of adaptation the cells to high temperature. From the original strain we inoculate at 30 0C. From there are taken cells and they will be inoculated at higher temperature, from there to the next plate and as a result we obtain cells capable to grow at 40 Celsius degree.
YPD-H
YPD-H
YPD-H
YPD-H
YPD-H
Thermotolerant strain

17. Strategies to overcome inhibition problems
4. Genetical transformation of microorganism to enhance its tolerance.
Recombinant xylose utilizing S. cerevisiae
Utilize xylose
Produce ethanol
Ethanol tolerance
Inhibitors tolerance
Last lecture we were talking about this aspects. From this strategy is possible for example to get recombinant yeast for xylose assimilation. You know that pentose is at high concentration in the hydrolysates. Then it is necessary to convert it into ethanol.

18. Conclusions
Fermentation of a mixture of hexose, pentose and inhibitors can introduce problems in the production of ethanol from lignocellulosic materials.
A suitable strategy, such as detoxification of lignocellulosic hydrolysates, adapted microorganisms to grow at hard conditions and employing a suitable natural or engineered microorganism that can convert both pentose and hexoses is necessary for the fermentation.
Fermentation of a mixture of hexose, pentose and inhibitors introduce problems in the production of ethanol from lignocellulosic materials.
A suitable strategy, such as detoxification of lignocellulosic hydrolysates, adapted microorganisms to grow at hard conditions and employing a suitable natural or engineered microorganism that can convert both pentose and hexoses is necessary for the fermentation.

19. THANK YOU FOR YOUR ATTENTION!