Sustainable processing of agricultural products: Ethanol production for fuel

Publiceret Juli 2007

Ethanol produced by fermentation of biomass may be used as extender or octane booster in motor fuel. The carbohydrate raw materials have to be hydrolysed to fermentable sugars. Enzymes are the catalytic tools in the production of these sugars for simultaneous saccharification and fermentation of starch containing raw materials. Yeast nutrients may be added or released from the grain by enzyme hydrolyses and the efficiency of the fermentation process may be improved using for example cell wall degrading carbohydrases or proteases.


Many traditional chemical processes based on acidic - or base catalysed reactions for processing of agricultural products have inherent drawbacks from a commercial and environmental point of view. Non-specific reactions may result in poor product yields. High temperatures and high pressures needed to drive reactions may lead to high costs and requirement of large volumes of cooling water downstream. Harsh and hazardous processes involving high temperatures, pressures, acidity or alkalinity need high capital investment, and specially designed equipment and control systems. Unwanted by-products may prove difficult or costly to dispose of. High chemical and energy consumption, and harmful by-products have a negative impact on the environment. The use of enzymes may virtually eliminate these drawbacks within the non-food as well as within the food area.

Fermentation processes for brewing, baking and the production of alcohol have been used in ancient China and Japan. The production of fermented alcoholic drinks from crops rich in starch has been practiced for centuries.

The enzymatic processes are related to fermentation. As such modern history of enzymes dates back to 1833 when the French chemists Payen and Persoz (1) described the isolation of an amylase complex from germinating barley and called it diastase. Like malt itself, this product converted gelatinized starch into sugars, primarily maltose.


In countries with surplus of agricultural capacity, ethanol produced from biomass may represent a sensible substitute, extender or octane booster for traditional motor fuel. While sugar-based raw materials such as cane juice or molasses can be fermented directly, this is not possible for starch-based raw materials. They have to be converted to fermentable sugars first. Although the equipment is different, the principle of using enzymes to produce fuel alcohol is similar to that for producing potable alcohol.

Ethanol was first used as a motor fuel in the late 19th century. In fact, the first automobiles developed in both the United States and France was designed to run on grain-based ethanol. However, petroleum quickly displaced ethanol as the fuel of choice because it was less expensive.

Grain-based ethanol had to be produced using malt or koji as the enzyme source. The grain-based ethanol industry did not become a viable source of fuel until industrial microbial enzymes became readily available like today.

The use of microbial enzymes for alcohol production from starch was first reviewed by Aschengreen (2) and various enzyme-based cooking processes were described in 1981 (3). A review of the production of ethanol from whole grain was made by Lyons in 1983 (4), and later by Lewis in 1996 (5). Fuel ethanol is recovered by distillation after anaerobic fermentation using yeast, primarily species of Saccharomyces cerevisia.

Raw materials such as corn (maize), wheat, barley, rye or sorghum need mechanical and enzymatic pre-treatment to release the starch in a free form and to make it suitable for hydrolysis to fermentable sugars, mainly glucose and maltose.

Over the last 8-10 years new enzymes systems have been developed for the bioethanol industry (6). Thanks to efficient enzyme systems dry-milling processes including continuous starch liquefaction followed by a so-called “very high gravity” fermentation (SSF) in which the saccharification is carried out simultaneous. To minimise investment and operating cost novel enzyme systems have been developed.

Different substrates for bioethanol production

Bioethanol can be produced from nearly any readily available crop. Wheat, barley, rye, triticale and sugar beet are used in Northern Europe. I western Canada wheat and barley are used and in eastern Canada and the U.S. feed-grade corn is used. The current technology for ethanol production from cereals is based on the so-called “dry milling processes”, and “wet milling processes”. New production units are mainly based on dry milling. Brazil as the world’s second largest producer of fuel alcohol uses sugar cane and molasses. Some compositional data for raw materials is shown in Table 1.


% content of dry matter






























Total dietary fibre






- of which soluble fibre






Table 1. Some general composition data for corn, barley, rye, wheat and oats (Data from several sources).

Cellulose is being intensively researched as a potential source of fuel alcohol. Using cellulose is more complicated than using grain because lignocelluloses are resistant to breakdown by enzymes before fermentation (7).

Current technology for ethanol production using Starch-based raw materials

Liquefaction actually comprises two steps: gelatinization and dextrinization. Gelatinization is accomplished by raising the temperature of the mash above that of the upper limit of the gelatinization temperature range. An overview of different starch raw materials for ethanol production is shown in Table 2.

Raw material

Typical starch content in % (as is)

Gelatinisation temperature, °C

Alcohol yield, litres per 100 kg

Protein content in %


54 - 65

53° - 63°

34 - 41

9 - 14


60 - 63

68° - 74°

38 - 40

9 - 10

Manioc/Tapioca – Meal

65 - 80

51° - 65°

40 - 50

0.5 - 2


55 - 62

55° - 70°

35 - 37

 8 - 16


55 - 65

70° - 78°

36 - 42

 8 - 10


63 - 69

55° - 70°

40 - 44

13 - 16


58 - 62

58° - 65°

36 - 39

10 - 14

Table 2. Overview of starch content, gelatinization temperature and expected yield of alcohol for various raw materials used for alcohol production.

The main process stages in alcohol production from starch-containing crops are summarized in Figure 1. First, the raw material is treated with viscosity reducing enzymes, then gelatinized with steam and liquefied with alpha-amylase to dissolve and dextrinize the starch carbohydrate. This treatment is referred to as “cooking”. Then, the resulting crude mash is saccharified with glucoamylase, and fermented with ordinary yeast. Finally, the fermented mash is separated by distillation into alcohol and stillage.

Alcohol production: Main process stages
Figure 1. Alcohol production: Main process stages (6) and (8).

The starch may be liquefied and pre-saccharified using first alpha-amylase and then glucoamylase. The resulting sugar is cooled and transferred to the fermentor where yeast is added. If the fermentation processes are performed continuously the fermentation time is around 24-30 hours. After fermentation, beer and yeast is separated. The beer stream is transferred to the distillation process where the ethanol is separated from the remaining “stillage.” The ethanol is concentrated using conventional distillation and dehydrated. The anhydrous ethanol is blended with denaturant, often gasoline, ready for shipment into the fuel market.

In the dry milling process hammer mills with screens grind the corn so that 60-90 % has a particle size of 250-350 ?M. The resulting meal is mixed with water to form a mash. Principally the process shown in figure 1 is used. In this process the pre-liquefaction consumes a minimum of steam for mash cooking. This may be obtained using a raw material slurring and a two-step liquefaction process as shown in figure 2. Alpha-amylase may be added during the pre-liquefaction at 70-90°C and again during the post liquefaction at ca. 85°C.

"Warm or Hot Slurry Pre-liquefaction Processes"
Figure 2. "Warm or Hot Slurry Pre-liquefaction Processes".

Lower ethanol production cost versus investment and operating cost – some suggestions and possibilities

The most economical effect is judged to be when the same plant volume is applied to treat more grain per hour. The intake of grain is increased without altering the investment. The effect of the enzyme treatment would here influence on the rest of the unit operations of the complete plant. Higher plant productivity vs. invested capital, results in lower production cost (8).

Viscosity reduction of the pre-slurry

Viscosity reduction is essential for alcohol processes when raw materials like wheat, barley and rye are used because of the importance of easy mash stirring, pumping and avoiding local overheating.

Problems can be encountered during both mashing and liquefaction due to high viscosity, which reduces the efficiency of heat exchangers, enzyme kinetics, and fermentation. Using pentosanases and beta-glucanases for example the products Viscozyme® Wheat, Viscozyme® Barley or Viscozyme® Rye developed by Novozymes A/S the pre-mash becomes a thin liquid within 30 minutes at the beginning of the process – the mixing of milled grain in the slurry tank.

Viscosity data is shown in Figure 3 for a process on wheat with jet cooking.

Effect on viscosity reduction during processing wheat to ethanol
Figure 3. Effect on viscosity reduction during processing wheat to ethanol. Viscozyme® Wheat was used up-front during mixing in whole ground wheat at 30-35% DS.

Reducing the viscosity of mashes and liquids in all stages of the process will facilitate use of higher content of dry solids, energy savings, and higher production capacity of alcohol in a given plant. Furthermore better pumping using smaller equipment, the avoidance of local overheating, more successful cleaning (CIP) and higher overall throughput of the plant are obtained. The overall result is a greater fermentation yield of ethanol.

The extraction/solubilisation of all viscous polysaccharides such as starch, celluloses, pentosans or beta-glucans during the process very much depends on the composition of the raw materials (table 1).


Measuring the viscosity with a Haake Viscotester VT-02 on a slurry of ground wheat treated with increasing dosages of Viscozyme Wheat is shown in Figure 4. The viscosity was measured at different time intervals. Without enzyme added we found a slight reduction of the viscosity due to enzyme activity in the wheat itself.

Viscosity measured at different time intervals after treatment of the wheat slurry with increasing dosages of Viscozyme Wheat.
Figure 4. Viscosity measured at different time intervals after treatment of the wheat slurry with increasing dosages of Viscozyme Wheat.

Starch conversion – Liquefaction

In the cooking stage the individual characteristics of different raw materials are significant. Because the dry-milling process is automated and highly controlled in a plant the liquefaction step is highlighted. Some of the concerns of the dry-milling industry for a liquefaction amylase include consistent conversion at decreased calcium ion levels and at lower pH values. Furthermore a rapid viscosity reduction in the mash, energy cost reductions, and efficient utilization of recycle streams is demanded.

The Liquefying Amylases

Liquefaction is easily accomplished at 35-38% solids when using Liquozyme® SC from Novozymes. However, above 38 % solids the slurry becomes increasingly viscous. Liquozyme SC is a liquid enzyme preparation containing a heat-stable alpha-amylase expressed in and produced by a genetically modified strain of a Bacillus microorganism. Liquozyme SC can operate at lower pH (pH=4.5) and at lower calcium levels than conventional thermostable alpha-amylases (6). This brings advantages to its application which all result in reduced operating cost. Liquozyme SC was introduced on the market in 1999 especially designed to decrease viscosity rapidly (Figure 5).

Pasting curves made on corn starch with enzymes present in the temperature range 50-95°C
Figure 5. Pasting curves made on corn starch with enzymes present in the temperature range 50-95°C.

Energy reduction

Typical values for energy consumption in the most demanding process stages are listed in Table 3. Substantial savings can be obtained by replacing traditional batch pressure-cooking by continuous processes. Additional savings can be obtained in the distillation and stillage evaporation stages - mainly by improving heat recovery. Recently, various engineering companies have quoted even lower energy consumption


Energy consumption MJ/litre ethanol




Stillage evaporation

Traditional batch




Modern continuous




Table 2. Process energy consumptions (9)

Simultaneous saccharification and fermentation (SSF)

Both continuous fermentation and batch fermentation are successfully utilised in the dry-milling processes. The advantages of continuous fermentation include the full utilisation of fermentation vessel capacities (no filling/draining/sanitisation), the ease of controlling continuous flows and the consistency of the products. The disadvantages are the susceptibility to infection from the whole grain and stillage recycle, and the disruption caused to production by the occasional sanitisation of the fermenters.

Ethanol production and fermentation efficiency may be quantified in the laboratory by measuring the CO2 production as weight decreases or by direct measurement of the ethanol using HPLC analysis. Based on the metabolic conversion rate, the amount of ethanol produced can be calculated from the CO2 produced.

Saccharifying amylases (glucoamylases) for ethanol production

Spirizyme® Fuel is used to saccharify whole-grain mashes for ethanol production. This glucoamylase is used in simultaneous saccharification and fermentation (SSF) as well as pre-fermentation saccharification processes. It is produced by submerged fermentation of a genetically modified microorganism.

It has higher activity and greater thermostability than traditional glucoamylases from Aspergillus niger. It allows saccharification systems to be operated up to 70°C. A greater flexibility in operating conditions is an advantage for an SSF process to follow.

Improved yeast efficiency provides increased ethanol yield

The bottleneck in an alcohol plant is often the fermentation tanks. The effect of addition of enzymes, which improve the nutritional status of the yeast, may result in a higher production capacity of the other unit operations. Improving the yeast nutrition by addition of enzymes has been shown to be able to secure that a higher intake of corn per hour in the plant can be made without extra investments in tanks, distillation towers etc. It is thus assumed that capacity increase based on corn up to 20-30 % may be implemented without extra investments that change the investments costs. Cereals, in particular maize (corn), tend to be low in soluble nitrogen compounds. This results in poor yeast growth and increased fermentation time, which can be overcome by adding ammonia, urea, or a protein-degrading enzyme, to the mash. A way to do this may be by reduction of yeast flocculation effects, by increase of the content of free amino acids and yeast nutritious compounds like minerals and vitamins (10).

World production of bioethanol for fuel

In the late 1970’s, the first major oil crisis occurred; the need for renewable liquid fuel such as ethanol was recognized.  In 2007, world production of bioethanol is 49.7 million m3 (11).


Bioethanol is today very important as extender or octane booster in motor fuel. The sustainable technology for production of ethanol from grain has improved considerably not at least as a result of the biotechnological results obtained in modern enzymology.


(1) Anselme Payen and Jean-François Persoz,  Annales de Chemie et de Physique, 2me. Série 55, 73-92 (1833).

(2) Aschengreen N.H. Microbial Enzymes for Alcohol production. Process Biochemistry, August 1969 3pp.(1969).

(3) Hagen, H. A. “Production of Ethanol from Starch-containing Crops - Various Cooking Procedures”. Paper given at a Meeting on bio-fuels in Bologna, June 1981. Available as Available as Article A-5762a GB from Novozymes A/S, (1981).

(4) Lyons, T. P Alcohol – Power/Fuel. In Industrial Enzymology. (Ed. Tony Godfrey & Jon Reichelt), Macmillan Publishers Ltd., England, (1983).

(5) Lewis, S. M. Fermentation alcohol. In Industrial Enzymology. (Ed. Tony Godfrey & Stuart West), Macmillan Publishers Ltd., England, (1996).

(6) Sejr Olsen, H. and Schäfer, T. Ethanol Produktion aus pflanzlicher Biomasse, in Antranikian: Angewandte Mikrobiologie (Chapter 19), Springer Verlag, Berlin Heidelberg (2006).

(7) Michael E. Himmel, Shi-You Ding, David K. Johnson, William S. Adney, Mark R. Nimlos, John W. Brady, Thomas D. Foust. Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production. Science 315, 804-807 (2007).

(8). Sejr Olsen, H. Using enzymes in ethanol production. Hand book available from Novozymes Customer center. Luna 2004-13388-02 (2005).

(9) H.A. Hagen and B. Helwiig Nielsen.: Ethanol from Starch-containing Crops. – Energy-saving Cooking Processes. Paper presented at the 5th Int. Fuel Alc. Symp. in Auckland, N.Z., May 1982. Available as Article A-5783a GB from Novozymes A/S.

(10) Devantier, R; Olsen, L; Pedersen, S; Olsson, L. Investigation of the mechanism behind the beneficial effect of protease addition to very high gravity ethanol fermentation of corn mash. Paper in Devantier, R. Saccharomyces cerevisiae in very high gravity ethanol fermentations using simultaneous saccharification and fermentation. Ph.D. Thesis BioCentrum-DTU, Technical University of Denmark Novozymes A/S, Bagsværd, Denmark (2005).

(11) F.O. Lichts. World Ethanol & Biofuels Report. Vol. 6 No. 4 page 63. October 23rd, 2007.