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

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

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

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

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.

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.

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

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

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

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

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.

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.

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.

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.

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

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

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.

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.

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