ETHANOL PRODUCTION FROM SUGAR CANE Good afternoon. Last time we studied general aspects of industrial microbiology, because it is important to understand biocatalytic processes. Today we will start to study a very known process. It is production of ethanol from sugar cane. Dr.C. Teresa Fernández Aldama “Samara University”

OBJECTIVE To give a general overview of the main features of the ethanol production from sugar cane. When we finish this lecture you should be able to give a general overview of the principal features of the ethanol production from sugar cane.

OUTLINE Alcohols. Ethanol. Characteristics. Ethanol uses. Main sources for ethanol production. Feedstocks used for ethanol production. Sugar cane as raw materials for ethanol production. Technological schemes for ethanol production from molasses. Now, let’s see which aspects we can study. We will speak about the alcohols, ethanol and it’s characteristics, also something about the uses of ethanol. There are two ways to obtain ethanol and we will study only this one which occur by fermentation. Later we will hear about feedstocks used in this production, specially in the sugar cane as an important raw material in this production and finally we will understand how occur the production of ethanol from sugar cane.

C, H, O R-OH ALCOHOLS Aliphatic Aromatic Primary Secondary Tertiary Alcohols, in general, are substances that contains carbon, hydrogen and oxygen. The functional group, which distinguishes these substances is hydroxide. R, as you know can be any carbon chain. These alcohols may be aliphatic or aromatic. It means with opened chain or associated with aromatic ring. Aliphatic alcohols can be primary, secondary and tertiary depending on the amount of hydrogen atoms which are replaced in the molecule. Primary Secondary Tertiary H R R R C OH R C OH R C OH H H R

CH3-CH-CH3 CH3-OH | OH CH3-CH2-OH CH3-CH2-CH2-OH CH3 CH3-C-OH Primary Secondary CH3-CH-CH3 | OH CH3-OH Ethanol CH3-CH2-OH Tertiary CH3-CH2-CH2-OH CH3 | CH3-C-OH Ethanol is a primary alcohol, because it has only replaced one hydrogen atom in the molecule.

CHARACTERISTICS Under ordinary conditions: Volatile Flammable Clear Colorless liquid Miscible in water and non-polar solvents Under ordinary conditions ethanol has the following characteristics. It is volatile because at normal temperatures it can pass to gas phase. Also can burn, it’s clear, no color and until four carbon atoms molecule it is soluble in water and non-polar solvents. For molecules which have more carbon atoms, predominates polar part of molecule and it’s soluble in polar solvents. (Hasta 12 átomos de carbono los alcoholes son líquidos y hasta cuatro átomos son solubles en agua)

Ethanol uses Now let’s see uses of ethanol. As you can see the major part is used as fuel. The industrial use may be for pharmaceutical, chemicals and cosmetics. The rest is for beverages.

Ethanol uses Here is represented behavior of different uses of the ethanol during last years. You can observe that the increasing of uses is, more and less, the same for ethanol which is used in the industry and for beverages. And for using ethanol as fuel the increasing is higher, year by year. We will study the reason later, even we will study the production of ethanol as fuel. Fuel Industrial Beverages Fuel Industrial Beverages

Main sources for ethanol production Catalytic hydration of ethylene in vapor phase Fermentation There are two ways for obtention of etanol: catalytic hydration of ethilen in vapor phase or by fermentation.

Main sources for ethanol production Synthesis, as you see is not used so much. The major part of production of ethanol is from fermentation.

Main sources for ethanol production The alcoholic fermentation was the first biotechnological process known by human society C6H12O6 + enzymes  2 CH3CH2OH + 2 CO2 The sugars (sucrose, glucose, fructose) are transformed into ethanol by enzymes contained in an ethanologenic microorganism (yeast Saccharomyces cerevisiae). The alcoholic fermentation is the first biotechnology process, known by human society. It is the reaction that occurs. We obtain ethanol and carbon dioxide from sugar (glucose) and enzymes. The enzymes come from yeast. In the industry of ethanol various yeasts can be used including species of Saccharomyces, Kleyveromyces and Candida, but most ethanol is produced by Saccharomyces.

Feedstocks used for ethanol production -Monosaccharides - Disaccharides Sugarcane juice Beet molasses Sugarcane molasses Simple sugars We have been seen that during ethanol fermentation is necessary some saccharide compound and a microorganism such as yeast, specially in this process we use Saccharomyces. Here you have simple sugars. They can be monosaccharides, they are only composed by one kind sugar and disaccharides, composed by two sugars. We can find these simple sugars in sugarcane juice, beet molasses and sugarcane molasses. The main advantage with these materials is that sugars are readily fermentable. It means that is not necessary to do hydrolysis. Later we will study this process: hydrolysis as an important step in the bioethanol production. They are mainly used in tropic climate countries. Their share on the global ethanol production is about 46 %.

Feedstocks used for ethanol production -Starch - Lignocellulose Grains (corn, wheat, rice) Tubers (cassava, potato) Polysaccharides Agricultural residues Forest wastes Municipal solid wastes They are promising materials for ethanol production without affecting food sector !! On the contrary of the simple sugars, which are able to be used directly in the fermentation process, polysaccharides are substances with bigger molecules, then it’s not possible to use them directly in the fermentation. They need to be hydrolysed. Between these substances we can find starch and lignocellulose. Starch includes corn, wheat and rice; and lignocellulose includes agricultural residues and forest and municipal solid wastes. They are promising materials for ethanol production without affecting the food sector Later we will study the methods which can be used for hydrolysis, before ethanol production.

Sugarcane as raw material for ethanol production Today we are going to study ethanol production from sugar cane, it’s real name is Saccharum officinarum. First, let’s see in which countries you can find it. As you see it is possible to find sugar cane in America, Europe, Asia, Africa…The major producers are Brazil, India, China, Pakistan, México, Thailand and Cuba. Saccharum officinarum

The lower part of the stem CANE It is possible to harvest sugar cane automatically with machine or man can cut it with his hands, but now the major places do it automatically. During the harvest time we get two things: cane, which goes directly to process and straw, it is residue and it can be later used in production of other kind ethanol that we are going to study. The lower part of the stem

Industrial use of sugar cane Clarification Evaporation Grinding Juice Crystallization Bagasse + Straw Centrifugation Sugar cane is processed and as result there are two main by products: bagasse and straw as a residue and juice. Then the juice is submitted to different steps: clarification, evaporation, crystallization and centrifugation. As a result there is another by product: molasses and final product: sugar. Molasses Sugar

Industrial use of sugar cane Juice Molasses Bagasse + Straw ? FUEL From these by products we can obtain ethanol for different uses as we saw at the beginning of the lecture. But here we only show the use of ethanol for beverages. The processes which we use are good established, it means that we produce ethanol from molasses or juice by fermentation from these raw material. As you see the first material: juice, is used in Brazil and the second one: molasses, are used in Cuba. For instance for preparing Havana Club! From the third by product we also can produce ethanol as fuel. It can be used in mixes with gasoline. This process is not good established like this from sugar cane. We will study it later.

The lower part of the stem After grinding (extraction of the juice), there are bagasse and straw.

Sugarcane juice This is the sugarcane juice. People can drink it with ice!

Sugarcane molasses The quality of molasses depends on the maturity of the sugar cane, the amount of sugar extracted, and the method of extraction. Molasses contain significant amounts of vitamins and minerals. Can be used in the manufacture of animal feed, and for other industrial uses. Compared to beet molasses, cane molasses have a higher content of vitamins and minerals. And they are molasses.

Technological scheme of ethanol production from molasses Now let’s see the technological scheme of ethanol production from molasses. At the beginning preparation of substrate is considered. It involves dissolution, clarification and pasteurization of molasses. The pH is adjusted according to requirement of used microorganisms. On the other side, it is necessary to prepare the inoculum for fermentation and after yeast removal allows distillation and dehydration. Now we will see each step separately. SUBSTRATE PREPARATION INOCULUM PREPARATION AND FERMENTATION YEAST REMOVAL DISTILLATION DEHYDRATION

Molasses Inoculum propagation Fermentation Distillation Ethanol Molasses should be diluted in adequate concentration (180 g/L) to be assimilated by yeast. Inoculum propagation is very important before starting fermentation. The aim is to obtain enough amount of cell biomass for inoculating the fermentors. Distillation Ethanol

A pure culture of yeast (S. cerevisiae) is propagated. Yeast pure culture Nutrients Air Developed yeast culture (inoculum) Diluted molasses Inoculum propagation A pure culture of yeast (S. cerevisiae) is propagated. The aim is to obtain enough amount of cell biomass for inoculating the fermentors. A yeast pure culture (Saccharomyces cerevisiae) is propagated. The yeasts are facultative anaerobic microorganisms. They can grow in aerobic and anaerobic conditions. In the presence of oxygen, and at low sugar concentration, all the carbon sources are used for energy production and cell reproduction. There is an intensive biomass growth. It is the exponential phase as we saw las t activity, do you remember?? Because of that is necessary full aeration, acidic pH, adequate sugar concentrations and nutrients. Also sterile conditions are necessary to ensure the presence only of desired microorganisms or to avoid contamination.

Yeast pure culture Nutrients Air Developed yeast culture (inoculum) Diluted molasses Inoculum propagation The yeasts are facultative anaerobic microorganisms. They can grow in aerobic and anaerobic conditions. In the presence of oxygen, and at low sugar concentration, all the carbon sources are used for energy production and cell reproduction.

Sugar concentration around 120 g/L Yeast pure culture Nutrients Air Developed yeast culture (inoculum) Diluted molasses Inoculum propagation Full aeration Acidic pH Sugar concentration around 120 g/L Sterile conditions in the first steps Nutrients: urea, (NH4)2SO4 , NH4H2PO4, (NH4)3PO4

Molasses Inoculum propagation Fermentation Distillation Ethanol Fermentation is biocatalytic process, which occurs anaerobically. The sugar concentration (as total soluble solids) is adjusted to 150-200 g/L (15-20o Brix) Distillation Ethanol

Alcoholic fermentation C6H12O6 + enzymes  2 CH3CH2OH + 2 CO2 GLUCOSE ETHANOL Anaerobic conditions. Acidic pH Sugar concentration around 180 g/L Non sterile conditions Nutrients: urea, (NH4)2SO4, NH4H2PO4, (NH4)3PO4 Intense ethanol formation (9-12%)-Higher ethanol concentration inhibits fermentation; Low biomass growth. In this step yeast convert sugars into ethanol, carbon dioxide and energy. Here sugar concentration is higher than during inoculum preparation. The necessary enzymes for degrading glucose to ethanol come from yeast Saccharomyces cerevisiae. This microorganisms grow in anaerobic conditions and acidic pH. In this step is not necessary sterile conditions, but nutrients are added. The sugar concentration (as total soluble solids) is adjusted to 150-200 g/L (15-20o Brix) and fermented by yeast.

Saccharomyces cerevisiae GLUCOSE ETHANOL Acetaldehyde By-products It is the biochemical path way to produce etanol by the cells of yeast, but we will not study how it occur. Acetaldehyde By-products

PROCESSES FOR ETHANOL PRODUCTION Batch processes Fed-batch (semicontinuous processes) Continuous processes

Molasses Inoculum propagation Fermentation Distillation The yeast cells are separated by centrifugation; The supernatant is pumped to the separation system. When the fermentation is finished, the yeast cells are separated by centrifugation. They are recycled to fermentation in order to reach high cell concentration during cultivation. The supernatant (wine, must) is pumped to the separation system. Distillation Ethanol

Wine Distillation Ethanol Stillage (vinasse) The separation is based on the difference in boiling points (ethanol 78oC, water 100oC); We can obtain ethanol by heating a fermented liquid at a high temperature that volatilizes most of the alcohol. The alcohol is then condensed and collected. The distillation is based on the difference in boiling points of components of liquid. Ethanol is more volatile than water and it can be separated before.

Distillation Ethanol separation is carried out in distillation columns

Final ethanol concentration: 96% Distillation columns are made up of a series of plates. The feeding stream enters the column, and flows over the plates. As liquid goes down, vapor comes in contact with it many times. Evaporation of ethanol occurs, and the ethanol-enriched vapor goes up to the top of the column. Distillation columns are made up of a series of plates. The feeding stream enters the column, and flows over the plates. As liquid goes down, vapor comes in contact with it many times. Evaporation of ethanol occurs, and the ethanol-enriched vapor goes up to the top of the column. Final ethanol concentration: 96% Final ethanol concentration: 96%

CONCLUSIONS Ethanol is a very important raw material It can be used in: Alcoholic beverages Chemical, pharmaceuticals and cosmetic products Automotive fuel 2. The major part of etanol is produced by fermentation. 3. Sugar cane is one of the most used raw material for etanol production. Today we studied the main steps for production of etanol. We saw alcohols, its characteristics. The most important materials for etanol production and also we could understand the steps in which alcoholic fermentation runs. First we need to

CONCLUSIONS 4. During fermentation is very important to control environmental parameters: Preparation of the inoculum (aeration, nutrients, ph, sterile conditions, sugar concentration) Fermentation (anaerobic conditions, acidic ph, sugar concentration, nun sterile conditions, nutrients) 5. Distillation (Boiling temperature: 78 0C)

THANK YOU FOR YOUR ATTENTION!