Etanol

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Naim Kosaric1, ZdravkoDuvnjak2, Farkas3,
Adalbert Hermann Sahm4, Print this page
Stephanie Bringer-Meyer5, Otto Goebel6, Dieter Mayer7 SEARCH THIS TITLE
1The University of Western Ontario, London, Ontario N6A 5B9,
Canada
2University of Ottawa, Ottawa, Ontario K1N 9B4, Canada
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3Consulting Chemist, Delray Beach, Florida 33483, United States
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4Institut fr Biotechnologie, Forschungszentrum Jlich GmbH,
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Jlich, Federal Republic of Germany
5Institut fr Biotechnologie, Forschungszentrum Jlich GmbH,
Jlich, Federal Republic of Germany
6CORA Engineering, Chur, Switzerland
7Hoechst Aktiengesellschaft, Frankfurt, Federal Republic of
Germany
Copyright 2002 by Wiley-VCH Verlag GmbH & Co. KGaA. All rights
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DOI: 10.1002/14356007.a09_587
Article Online Posting Date: June 15, 2001
Abstract | Full Text: HTML
Abstract
The article contains sections titled:
1. Introduction
2. Physical Properties
3. Chemical Properties
4. Synthesis
4.1. Direct Catalytic Hydration of Ethylene
4.1.1. Chemistry
4.1.2. Catalysts
4.1.3. Production Process
4.2. Indirect Hydration of Ethylene
4.2.1. Chemistry
4.3. Other Methods
4.3.1. Homologation of Methanol
4.3.2. Carbonylation of Methanol and Methyl Acetate
4.3.3. Conversion of Synthesis Gas
4.3.3.1. Synthesis by Heterogeneous Catalysis
4.3.3.2. Synthesis by Homogeneous Catalysis
5. Fermentation
5.1. Production by Yeast
5.1.1. Nutrients
5.1.2. Fermentation Pathways
5.1.3. Fermentation Variables
5.1.4. Direct Fermentation
5.2. Production by Bacteria
5.2.1. Ethanol Tolerance
5.2.2. Fermentation of Glucose by Z. mobilis
5.2.3. Fermentation of Other Sugars by Recombinant Z. mobilis Strains
5.2.4. Ethanol Production by Recombinant Enterobacteria
5.2.5. Comparison of Recombinant Bacterial Strains
5.3. Fermentation Modes
page 1 of 57
5.3.1. Batch Processes
5.3.2. Continuous Processes
5.3.3. Other Processes
5.4. Raw Materials and Processes
5.4.1. Readily Fermentable Carbohydrates
5.4.2. Starch
5.4.3. Lignocellulosic Materials
5.4.4. Waste Materials and Residues
6. Recovery and Purification
6.1. Distillation
6.1.1. Distillation of Azeotropic Ethanol
6.1.2. Dehydration by Azeotropic Distillation
6.1.3. Motor Fuel Ethanol
6.1.4. Reduction of Energy Costs
6.2. Nondistillative Methods
6.2.1. Solvent Extraction
6.2.2. Carbon Dioxide Extraction
6.2.3. Adsorptive Dehydration
6.2.4. Membrane Technology
6.2.5. Yarn-Filled Column
6.3. Storage and Transportation
7. Comparison of Process Economics for Synthetic and Fermentation Ethanol
7.1. Summary of Cost Analysis for Synthetic and Fermentation Ethanol
7.2. Production Costs of Synthetic Ethanol
7.3. Production Costs of Fermentation Ethanol
8. Analysis
9. Uses
10. Economic Aspects
10.1. Worldwide Production of Synthetic and Fermentation Ethanol
10.2. Major Producers of Fermentation Ethanol from Regenerable Resources
11. Toxicology
[Top of Page]
1. Introduction
Naim Kosaric and Zdravko Duvnjak
Ethanol [64-17-5] or ethyl alcohol (CH3CH2OH), Mr 46.7, is also referred to as alcohol spirit, spirit of wine, grain alcohol,
absolute alcohol, and ethyl hydrate. Depending on its water content, preparation, and final use, several ethanol products
exist on the market. The 99 % alcohol (often referred to as absolute alcohol) is used extensively for tinctures and
pharmaceutical preparations, as a solvent and preservative, as an antiseptic, and in perfume. Ethanol is an important
functional component of alcoholic beverages, which are produced by fermentation of fermentable carbohydrates. The
fermentation broth itself may constitute (after processing and aging) a beverage, e.g., in the case of beer or wine, or the
alcohol can be concentrated from the broth to produce high-alcohol-containing spirits. If the alcohol is used for purposes
other than as a beverage, it is denatured by the addition of substances such as methanol, pyridine, formaldehyde, or
sublimate. The denatured alcohol is then used by industry and commerce, principally as a solvent, as a raw material for
manufacturing chemicals, or as a fuel.
Chemically synthesized ethanol is usually derived from petroleum sources by the hydration of ethylene (see Chap.
Synthesis) and has found wide application as industrial alcohol. Various routes for the production of ethanol are depicted in
Figure 1.
Figure 1. Industrial sources of ethanol
History. Ethanol can be considered to be one of the oldest human food products. Fermented beer was consumed in
Babylon, and wine was produced as early as 3000 B.C. The distillation process probably orginated in the 10 14th centuries.
At this time, the spiritual effect of ethanol was recognized; thus, the name spiritus was given to alcoholic drinks. The first
wine distillates were used for medicinal rather than beverage purposes.
page 2 of 57
Until the 17th century, alcoholic fermentation was considered to be a spoilage process whereby the yeast produced was
eliminated. The nature of fermentation was initially clarified in the 19th century with the discovery of the microscope, which
showed that yeast cells were living organisms. However, recognition of the fact that these living organisms are responsible
for the fermentation process took about 150 years.
In the 19th century, two theories were developed to explain the mechanism of fermentation: the mechanistic and the vital
processes. LOUIS PASTEUR (1822 1895) promoted the vital theory, which stated that living organisms were responsible for
the conversion of sugar to alcohol. The mechanistic theory was supported by JUSTUS FREIHERR VON LIEBIG (1803 1873)
and by FRIEDRICH WHLER (1800 1882). A convincing proof of the mechanistic mechanism, by which physicochemical
processes lead to chemical conversion of sugar to ethanol, came from EDWARD BUCHNER (1860 1917), who demonstrated
that alcoholic fermentation is related not to the living cell but to a substance in the fermentation broth, which was later
identified as an enzyme. As is now known, enzymes are ultimately responsible for the complex conversion of carbohydrates
to ethanol.
[Top of Page]
2. Physical Properties
Ethanol in its pure form (absolute alcohol) is a colorless liquid. It is miscible in all proportions with water and also with ether,
acetone, benzene, and some other organic solvents. Anhydrous alcohol is hygroscopic; at a water uptake of 0.3 0.4 %, a
certain stability does occur. Various physical properties of anhydrous ethanol are as follows:
bp 78.39 C
fp 114.15 C
n20 1.36048
0.79356
0.78942
0.79425
0.79044
Surface tension at 20 C 22.03 mN/m
Cp (16 21 C) 2.415 J g1 K1
Heat of fusion 4.64 kJ/mol
Heat of evaporation
At 70 C 855.66 kJ/kg
At 80 C 900.83 kJ/kg
At 100 C 799.05 kJ/kg

Heat of combustion
(at constant volume) 1370.82 kJ/mol
Thermal conductivity at 20 C 18 W m1 K1
Dynamic viscosity 1.19 mPa s
Volumetric expansion coefficient 1.1103 K1 *
Heat of mixing 30 wt % ethanol
and 70 wt % water at 17.33 C 39.32 J/g
Flash point (in a closed vessel) 13 C
Autoignition temperature 425 C
Explosion limit (amount of
ethanol in a mixture with air)
Lower, 3.5 vol % 67 g/m3
Upper, 15 vol % 290 g/m3
Maximum explosion pressure 736 kN/m2
Specific conductivity 1351011 1 cm1
Dilution number at 20 C
(diethyl ether = 1) 8.2
Diffusion coefficient for vapors
at 20 C and 101.3 kPa 0.12 cm1
Heating value
Upper 29 895 kJ/kg
page 3 of 57
Lower 29 964 kJ/kg
* In practice, the volume increase for 1000 L is taken as 1 L/K.
Tables 1 and 2 show the freezing and flash points of ethanol water mixtures. Table 3 shows the vapor liquid equilibria of
ethanol water mixtures; the azeotropic mixture contains 95.57 wt % ethanol and 4.43 wt % water. Therefore, the highest
concentration of ethanol, obtained by distillation from an ethanol water mixture, is 95.57 wt %. Azeotropic distillation, with
the help of a tertiary solvent (e.g., benzene), must be introduced to produce absolute (anhydrous) ethanol.
Table 1. Freezing points of ethanol water mixtures
Ethanol, fp, C Ethanol, fp, C

vol % vol %
50 36.9 30 15.3
45 28.1 25 11.3
40 24 20 7.6
38 22.3 15 5.1
32 16.8 10 3
Table 2. Flash points of ethanol water mixtures
Ethanol, Flash Ethanol, Flash

wt % point, C wt % point, C
100 13 50 24.5
94.5 16 40 26.5
80 19.5 30 30
70 21.5 10 46
60 22.6 5.5 56
Table 3. Vapor liquid equilibria and boiling points of the ethanol water system at 101.3 kPa
Ethanol in liquid, Ethanol in vapor, bp, C

wt % wt %
0 0.00 100.00
1 6.5 98.90
3 20.5 96.75
5 38.0 94.95
10 52.0 91.45
15 59.5 88.95
20 64.8 87.15
25 68.6 85.75
30 71.4 84.65
35 73.3 83.75
40 74.7 83.10
45 75.9 82.45
50 77.1 81.90
55 78.2 81.45
60 79.4 81.00
page 4 of 57
65 80.7 80.60
70 82.2 80.20
75 83.9 79.80
80 85.9 79.35
85 88.3 78.95
90 91.3 78.50
95 95.04 78.15
97 96.86 78.20
99 98.93 78.25
100 100.00 78.30
When ethanol is mixed with water, the volume contracts slightly, as shown in Table 4. For example, when 52 volumes of
absolute ethanol and 48 volumes of water are mixed, 96.3 volumes of diluted ethanol result.
Table 4. Volume contraction for 1 mol of ethanol water mixture at 15C
x *C2H5OH V **
1.00000 0.79354 0.0000

0.97483 0.79666 0.1063
0.92674 0.80270 0.2903
0.88143 0.80851 0.4429
0.81817 0.81688 0.6250
0.74155 0.82756 0.8028
0.65639 0.84032 0.9495
0.56708 0.85510 1.0540
0.47734 0.87186 1.1123
0.35078 0.89980 1.1116
0.22077 0.93482 0.9695
0.15550 0.95355 0.7864
0.07909 0.97311 0.4030
0.05987 0.97790 0.2869
0.03726 0.98435 0.1586
0.01605 0.99196 0.0582
0.00792 0.99542 0.0267
0.00000 0.99913 0.0000
* Mole fraction of ethanol.

** Change in volume.
[Top of Page]
3. Chemical Properties
The chemical properties of ethanol are dominated by the functional OH group, which can undergo many industrially
important chemical reactions, e.g., dehydration, halogenation, ester formation, and oxidation ( Alcohols, Aliphatic).
Because ethanol can be produced efficiently not only by chemical synthesis from petroleum and coal-based feedstock, but
also by fermentation of abundantly available organic materials, its commercial role as a raw material for various chemicals is
of increasing importance.
Conversion of ethanol to alkochemicals is an entirely new approach to producing familiar petrochemicals (Fig. 2). Some of
these routes are already being used industrially in large alcohol-producing countries.
Figure 2. Chemicals produced from ethanol
page 5 of 57
[Top of Page]
4. Synthesis
Adalbert Farkas
The formation of ethanol from ethylene [74-85-1] by reaction with sulfuric acid was demonstrated over a hundred years ago
[1], [2]. Economic production of synthetic ethanol on an industrial scale was first accomplished in 1930 by the Carbide and
Chemical Corporation (now Union Carbide). The process used involved absorption of ethylene in sulfuric acid and
subsequent hydrolytic cleavage of the resulting ethyl sulfates. This indirect process has been gradually superseded by direct
catalytic hydration of ethylene, introduced by the Shell Chemical Company in 1948 [3].
More recent routes for ethanol synthesis depend on coal-based starting materials such as methanol [67-56-1] or synthesis
gas (a mixture of carbon monoxide and hydrogen). They include homologation and carbonylation processes and the direct
conversion of synthesis gas to ethanol.
4.1. Direct Catalytic Hydration of Ethylene

4.1.1. Chemistry
In the temperature range at which industrial catalysts operate, the hydration of ethylene to ethanol is a reversible reaction
controlled by the equilibrium
(1)
Calculation of the equilibrium is described in [4-6]. The kinetics of Reaction (1) have been studied on a phosphoric acid
silica gel catalyst [7], [8] and on a blue tungsten oxide silica gel catalyst (energy of activation = 125 kJ/mol) [9].
A nomogram for the correlation between ethylene conversion in this reaction and several process variables (pressure,
temperature, and the water ethylene molar ratio) has been developed [10]. Under usual reactor conditions (approximately
equimolar ethylene water feed, 250 300 C, 5 8 MPa), the equilibrium conversion of ethylene is 7 22 %.
A lower temperature favors higher ethylene conversion, but Reaction (1) is then accompanied by reversible formation of
diethyl ether [60-29-7] according to Reaction (2):
(2)
At high pressures, ethylene polymerizes to yield butenes and higher olefins, which are converted to higher alcohols by
hydration.
The mechanism of ethylene hydration is assumed to consist of four steps [11].
1. Formation of a -complex by the addition of a proton to the ethylene molecule:
2. Conversion of the -complex to a carbocation:
3. Addition of water to the carbocation:
4. Removal of a proton from the protonated ethanol:
page 6 of 57
4.1.2. Catalysts
The technical and patent literature describes numerous catalysts for ethylene hydration (see Heterogeneous Catalysis
and Solid Catalysts), [12]. Most of them are acidic because the reaction involves carbocations. However, only phosphoric
acid catalysts supported by diatomaceous earth (kieselguhr, Celite) [13-15], montmorillonite [16], bentonite [17], silica gel
[18-21], or opoka (Volga sandstone) [22] are of industrial importance.
Improved mechanical properties are claimed for catalysts on a diatomaceous earth support with added silica gel [23] or
bentonite [24]. Catalyst performance depends on the porosity of the support [21] which can be increased by incorporation of
combustible material [24].
Under operating conditions, the support must be resistant to phosphoric acid but able to retain it in large amounts; it must
also have adequate mechanical strength and a sufficiently long life and, ultimately, must provide an active, selective catalyst
for the hydration [13]. The support is impregnated with 50 77 % phosphoric acid but is first treated with hydrochloric or
sulfuric acid to remove most of its iron and aluminum. Otherwise, these materials are entrained on contact with phosphoric
acid and tend to clog the heat exchangers. Heating the supported catalyst to 700 1100 C has been recommended
because even though this treatment impairs catalyst activity and selectivity, it ensures that the phosphoric acid binds firmly to
the support [25-27].
The following compounds have also been suggested as catalysts for ethylene hydration: acidic oxides (such as tungsten
trioxide) on silica [11], silicotungstic acid [28], [29], silicoborotungstic acid [30], or phosphoric acid on silica alumina [31];
high-silica HZSM-5, dealuminated Y [32], HVSN-5 [33], or H-form [34] zeolites, as well as zeolites containing metal ions [35];
and salts such as boron phosphate [36-40], zirconium phosphate [41], other phosphates [40], cobalt sulfate [42], or
magnesium sulfate [43].

Ethanol production based on the catalytic hydration of ethylene is shown schematically in Figure 3. Ethylene and deionized
water (molar ratio range 1 : 0.3 1 : 0.8) are heated to 250 300 C at 6 8 MPa by passage through a heat exchanger and
a superheater. Since hydration is exothermic, the gaseous reaction products leave the reactor at a temperature 10 20 C
higher than when they entered and thus are used as a source of heat in the heat exchanger. Some phosphoric acid is
entrained by the gas stream and is neutralized by injecting a dilute solution of sodium hydroxide.
Figure 3. Synthesis of ethanol by direct hydration
a) Circulation compressor; b) Heat exchanger; c) Superheater; d) Reactor; e) Washer; f) Crude ethanol tank; g) Extractive
distillation column; h) Concentration column; i) Dehydration; j) Concentration column
After condensation and separation of the liquid reaction products, the gas is freed from residual ethanol by water washing
and then recompressed and recycled to the reactor. The phosphoric acid lost by entrainment and evaporation is replaced
continuously or periodically by spraying it on the catalyst bed. Crude product collects in the sump of the washer and contains
ca. 10 25 wt % ethanol. It is decompressed to recover the dissolved ethylene, which is recycled. The ethanol is then
purified, ideally by extractive distillation followed by rectification, to obtain a 95 vol % ethanol water azeotrope. The
azeotrope can be dehydrated by azeotropic distillation to give anhydrous ethanol. For further details of distillation see
Chapter Recovery and Purification.
Prior to distillation, the crude ethanol is often catalytically hydrogenated to convert acetaldehyde [75-07-0] and higher
aldehyde impurities to the corresponding alcohols. Acetaldehyde is a highly undesirable impurity because it tends to form
crotonaldehyde [4170-30-3].
Approximately 2 % diethyl ether is formed as a byproduct; it can be easily purified to a marketable product from the light ends
fraction ( Ethers, Aliphatic Diethyl Ether) or recycled to the reactor where it is quantitatively converted to ethanol.
Ethylene is available in high purity (>99.9 vol %); therefore, only small amounts need to be vented even if an ethylene level of
85 % must be maintained in the reactor stream.
Cylindrical steel containers with a diameter up to 4 m and an internal volume of up to 150 m3 are used as reactors. They are
lined with copper or carbon bricks for protection against attack by phosphoric acid. The heat exchangers and the connecting
piping exposed to phosphoric acid are made of, or lined with, copper or copper alloys. The remaining synthesis and
distillation equipment is made of steel. Waste gas does not present any problems. Vented ethylene is either returned to the
ethylene plant or burned. The synthesis step does not produce any liquid waste. Wastewater from distillation does not
contain any organic compounds, and its low phosphate content (0.3 kg of Na2HPO4 per cubic meter) permits ready disposal.
Optimal reaction conditions depend on the correlation of activity and selectivity of the catalyst with temperature, pressure,
residence time, and molar ratio of water to ethylene. These relationships have been studied extensively [44-48]. A rate
equation for the decrease in catalyst activity with time has been derived and used to develop a mathematical model for
computing optimum reaction parameters [49], [50].
page 7 of 57
4.2. Indirect Hydration of Ethylene
The indirect hydration, esterification hydrolysis, or sulfuric acid process is based on the absorption of a large volume of
ethylene in concentrated sulfuric acid; ethanol and some diethyl ether are formed when the sulfuric acid solution is diluted
with water.
4.2.1. Chemistry
Ethylene is absorbed in two steps:
(7)
(8)
Kinetic studies have shown that this reaction also proceeds via the mechanism in Reactions (3) (6) [51] but is complicated
by the fact that only part of the ethylene is physically dissolved in sulfuric acid.
Hydrolysis involves three steps:
(9)
(10)
(11)
The products are ethanol and 5 10 % diethyl ether. The ether yield can be controlled by varying the reaction conditions,
especially the ratio of ethylene to sulfuric acid, and the hydrolysis conditions ( Ethers, Aliphatic Diethyl Ether).
Transesterification. The ethyl sulfates formed in Reactions (7) and (8) can also be transesterified with acetic acid [64-19-7]
at 104 C to yield ethyl acetate [141-78-6], which is then recovered by distillation and hydrolyzed to ethanol and acetic acid
[52]. This process avoids problems associated with reconcentration of sulfuric acid.

Figure 4 shows the flow scheme for the production of ethanol by the sulfuric acid process. The feed gas must contain a
minimum of 35 vol % ethylene and only inert gases such as methane and ethane. Higher homologues of ethylene cause
resin formation and, therefore, must be removed from the feed.
Figure 4. Synthesis of ethanol by the sulfuric acid method
a) Wash column for residual gas; b) Absorber; c) Hydrolyzer; d) Stripping column; e) Wash column for crude ethanol; f)
Crude product tank; g) Diethyl ether distillation column; h) Ethanol distillation column
The absorption of ethylene increases almost linearly with pressure. Operating pressure is generally 1 3.5 MPa, the higher
pressure being used when the ethylene level in the feed is low. Each mole of sulfuric acid absorbs up to 1.4 mol of ethylene.
The absorption is carried out with 94 98 wt % sulfuric acid in wash towers at 65 85 C. Temperatures above 90 C lead to
formation of resins and oils and, therefore, must be avoided. The resulting liquid is agitated for several hours under pressure
to complete the reaction.
Hydrolysis is usually performed in two stages. First, diethyl sulfate is hydrolyzed at a low temperature (70 C) in the presence
of less than the equivalent amount of water. Then more water is added gradually, and the temperature is raised to ca. 100
C. Hydrolysis is complete within 1 h, and the sulfuric acid is then diluted to 40 55 wt %.
The ethanol formed is recovered, together with the diethyl ether byproduct, in a stripping column. The product mixture is then
washed with sodium hydroxide solution to neutralize any acid, and the diethyl ether is removed in the ether distillation
column. The ethanol is purified by distillation and concentrated to give a 95 vol % ethanol water azeotrope. Anhydrous
ethanol can be obtained as described in Chapter Recovery and Purification.
Reconcentration of the dilute sulfuric acid is the most expensive part of this process. It is encumbered by high energy
consumption, corrosion problems, and oxidation of the organic compounds remaining in the sulfuric acid. Although tar formed
in the absorption is separated as completely as possible, the sulfuric acid loss amounts to more than 5 % based on the
ethanol formed.
To avoid these disadvantages, an operating pressure below 0.5 MPa has been recommended [53]; less than 0.5 mol of
ethylene is absorbed per mole of sulfuric acid, which contains up to 7 % silver sulfate to accelerate the reaction. The sulfuric
acid can then be reconcentrated by simple flash evaporation.
The following construction materials are used in this process: steel for absorption and distillation; lead and acid-resistant
masonry for hydrolysis; silumin (an aluminum alloy), tantalum, cast iron, and lead for reconcentration of sulfuric acid.
4.3. Other Methods

Efforts have been made to develop methods for synthesizing ethanol that are not based on raw materials derived from
petroleum and are likely to be of industrial importance in the future. Methanol [67-56-1] and synthesis gas, which are
available from coal, can be used as starting materials.
page 8 of 57
4.3.1. Homologation of Methanol
The homologation (hydrocarbonylation) of alcohols discovered in 1949 [54], [55] allows the chain length of an alcohol to be
increased by one CH2 group by reaction with synthesis gas (CO + H2). The reaction occurs in the presence of a cobalt
carbonyl catalyst at elevated temperature and pressure:
When homologation was originally applied to methanol, the ethanol yield was relatively low and the reaction product
contained higher alcohols, formate and acetate esters, and other oxygenated products. The complexity of the mixture formed
was a result of further homologation of ethanol and other alcohols and of additional carbonylation reactions.
The selectivity for ethanol has been improved to up to 89 % by reaction in tetrahydrofuran [109-99-9] solution [56] or by
addition of iodine or iodide together with organic phosphines [57-61]. High ethanol selectivities have also been claimed for
combinations of cobalt catalysts with copper, iron, nickel, ruthenium, or rhodium compounds [62-70]. An alumina-supported
rhodium iron catalyst has been described as well [71]. Results of these studies are summarized in Table 5.
Table 5. Homologation of methanol to ethanol a, b
Catalyst Additive Ethanol selectivity, % Reference
Co(OAc)3 tetrahydrofuran 72 [56]

Bu3PCo(CO)3 I2 65 [57]
Co(OAc)3 I2, Bu3P 70 [58]
Co(OAc)3 I2, Ph3P 80 [59]
Co(OAc)3 I2, [Ph2P(CH2 )3 ]2 65 [60]
CoI3 89 [61]
Co2(CO)8, RuCl3 I2 79 [62]
Bu3PCo(CO)3, Ru(acac)3 I2 72 [63]
Co(acac)3, Ru(acac)3 I2, (Ph2PCH2 )2 82 c [64]
CoI3, FeI3, RuCl3 I2, MeOEt, MeOAc 86 [65]
CoI3, RhI3 N-methyl-2-pyrrolidone 62 [66]
Co20B10RhH6 78 [67]
Co2(CO)8, NiCl2 triphenylamine 82 [68]
CoS, Cu Bu3P, N-methyl-2-pyrrolidone 96 [69]
Co(acac)3, W(CO)6 HI, (Ph2PCH2 )2 89 c [70]
Co(acac)3, Fe(acac)3 HI, (Ph2PCH2 )2 83 d [70]
Rh Fe/ alumina 37 87 e [71]
a Except where otherwise specified, homologation was performed at 150 250 C and 20 35 MPa.
b Abbreviations: OAc = acetate; acac = acetylacetonate; Bu = butyl; Et = ethyl; Me = methyl; Ph = phenyl.
c Ethanol content of product.
d Selectivity for ethanol and acetaldehyde.
e Yield of ethanol and methyl acetate; conditions for homologation as described in [71].
4.3.2. Carbonylation of Methanol and Methyl Acetate

The first step of another method for converting methanol to ethanol is carbonylation of methanol to acetic acid [64-19-7] [72].
(13)
This reaction is the basis of a commercial process developed by Monsanto for synthesis of acetic acid [73] by using a
rhodium carbonyl iodine complex as catalyst [74] ( Acetic Acid Production).
Acetic acid can be hydrogenated directly to ethanol, but this requires expensive highpressure equipment, and the process is
corrosive [72]. According to a Davy McKee patent, carbonylation is carried out in a methanolic solution containing rhodium
trichloride [10049-07-7], methyl iodide [74-88-4], and acetic acid at 175 C and 7 MPa [75]. The acetic acid formed is
esterified with ethanol in the presence of sulfuric acid to yield ethyl acetate [141-78-6]. Ethyl acetate is then converted to
ethanol by hydrogenolysis in a tubular reactor at 200 C over a copper zinc oxide catalyst. Thus, the overall reaction
sequence is:
(13)
page 9 of 57
(14)
(15)
The net reaction is:
(16)
HALCON has shown that carbonyls of non-noble metals, such as molybdenum, tungsten, and chromium, catalyze the
carbonylation step [76], [77] and has developed an improved esterification procedure that is used commercially [72]. In a
modification of this process, methyl acetate [79-20-9] rather than methanol is carbonylated [76], [78]:
(17)
Reaction of the resulting acetic anhydride [108-24-7] with ethanol and methanol then yields ethyl and methyl acetates:
(18)
After separation of the acetates, the ethyl acetate is hydrogenolyzed to ethanol (Reaction 15) and the methyl acetate is
carbonylated (Reaction 17). The net reaction is given by Reaction (16).
Methyl acetate is carbonylated on a commercial scale in Eastman Kodak's acetic anhydride plant. Various techniques are
available for both the esterification (Reaction 18) and the hydrogenolysis (Reaction 15) steps [72].
4.3.3. Conversion of Synthesis Gas

Soon after methanol had been synthesized from carbon monoxide and hydrogen ( Methanol), and the Fischer Tropsch
synthesis had been discovered ( Coal Liquefaction), a search started for related routes to ethanol and higher alcohols,
based on synthesis gas and the use of heterogeneous [79] or homogeneous [80] catalysis.
Highly specific methods for producing ethanol from synthesis gas are not yet available, but some promising leads have been
found.
4.3.3.1. Synthesis by Heterogeneous Catalysis

Attempts to improve the conversion of synthesis gas to ethanol have been based on modifying industrial catalysts containing
oxides of zinc, chromium, or copper that are normally used for the synthesis of methanol. Early work showed that the
incorporation of alkali [81] and cobalt [82] improved yields of ethanol and higher alcohols.
The Institut Franais du Ptrole [83] has developed complex catalysts containing copper, cobalt, chromium, potassium,
lanthanum, and other elements for conversion of synthesis gas to alcohols to be blended with gasoline. The product contains
50 70 % methanol, 16 23 % ethanol, 8 14 % propanol, and some higher alcohols. Institut patents report on two
catalysts that can convert synthesis gas to an alcohol mixture containing 39 % ethanol, 24 % methanol, and 36 %
propanols + butanols at 250 C and 6 MPa [84], [85].
Use of a Cu Ni Ba catalyst is reported to give a product containing 46 % ethanol, 38 % methanol and 9 % propanol [86].
A Cu Zn Ni Na catalyst showed a 55 % selectivity for alcohols, but the alcohol fraction contained only 29 % ethanol
[87]. Iron and potassium have also been added to copper zinc catalysts normally used for methanol synthesis; their effects
on ethanol synthesis and the reaction mechanisms involved have been discussed [88], [89].
On certain supports, platinum-group metal catalysts prepared from carbonyl clusters convert synthesis gas to methanol
ethanol mixtures at 200 240 C and atmospheric pressure [90]. Thus, a supported rhodium lanthanum trioxide catalyst
gives ethanol as the predominant product, with a total alcohol selectivity up to 80 %. More recent studies using supported
rhodium catalysts promoted with cerium or europium report a similarly high selectivity for ethanol [91], [92].
The only catalysts derived from rhodium carbonyl clusters that produce ethanol as the major product are those which contain
added cobalt [93]. A number of formulations containing rhodium and a variety of cocatalysts and promoters are claimed by
the patent literature to be selective catalysts for the synthesis of ethanol, e.g., [94-96], acetaldehyde, and acetic acid, e.g.,
[97]. According to a Hoechst patent, a composition of cobalt, rhenium, gold, and barium oxides on a silica support produces
ethanol from synthesis gas with 60 % selectivity [98].
4.3.3.2. Synthesis by Homogeneous Catalysis

The homogeneous catalytic conversion of synthesis gas to ethanol and ethylene glycol [107-21-1] has been reviewed with
particular reference to the reaction mechanism, the nature of the active catalyst species, and their interaction with solvents,
cocatalysts, and other additives [80].
Ruthenium is the only group 8 metal to show a high selectivity for the conversion of synthesis gas to ethanol when used in
conjunction with organic phosphorus compounds. Synthesis gas can be converted to ethanol with a selectivity >50 % by
using an iodine-promoted ruthenium carbonyl catalyst dissolved in tripropylphosphine oxide [99], [100]. Lower ethanol
selectivities (20 30 %) have been reported for ruthenium catalysts in a melt of tetrabutylphosphonium bromide [101-103] or
alkyltriphenylphosphonium bromides [104].
A combination of ruthenium and cobalt carbonyls dissolved in toluene is reported to convert synthesis gas to a mixture of
ethanol, methanol, and acetic acid [105]. Ethanol is also reported to be the major product in a conversion catalyzed by
rhodium carbonyl, potassium chloroplatinate, and stannous chloride [106].
page 10 of 57
[Top of Page]
5. Fermentation
Naim Kosaric, Zdravko Duvnjak, Hermann Sahm, Stephanie Bringer-Meyer
5.1. Production by Yeast

Yeasts are unicellular, uninucleate fungi that can reproduce by budding, fission, or both. They have been used for centuries
to make alcoholic beverages.
Yeasts are the most commonly used organisms in the industrial production of ethanol. Some widely used, high-productivity
strains are Saccharomyces cerevisiae, S. uvarum (formerly S. carlsbergensis), and Candida utilis. Saccharomyces
anamensis and Schizosaccharomyces pombe are also used in some instances. Kluyveromyces species, which ferment
lactose, are good producers of ethanol from whey.
Ethanol production by yeast is characterized by high selectivity, low accumulation of byproducts, high ethanol yield, high
fermentation rate, good tolerance toward both increased ethanol and substrate concentrations, and lower pH value. Viability
and genetic stability of yeast cells under process conditions and at high temperature are also desirable. Although finding a
strain that has all these characteristics is difficult, some yeast strains can fulfill them to a great extent [107], [108].
5.1.1. Nutrients
Yeasts require the following for growth: carbon, nitrogen, phosphorus, sulfur, oxygen, hydrogen, minor quantities of
potassium, magnesium, calcium, trace minerals, and some organic growth factors (vitamins, nucleic acids, and amino acids).
Various carbon compounds can serve as a carbon source for yeast (Table 6). Nitrogen can be supplied as ammonia,
ammonium salts, urea, or amino acids. Orthophosphate salts and phosphoric acid are good sources of phosphorus. Sulfur,
potassium, magnesium, and calcium can be supplied in the form of their salts. Table 7 shows the amount of these elements
and trace minerals required by yeasts.
Table 6. Ability of Saccharomyces and Kluyveromyces species to ferment sugars [109]
Carbon Type of Sugar Basic unit Yeast e

number basic
of basic subunit S. S. uvarum K.

subunit cerevisiae fragilis
(carlsbergensis)
glucose glucose + + +
maltose glucose + +
maltotitrirose glucose + +
aldose cellobiose glucose
sugars trehalose glucose +/ +/
galactose galactose + + +
mannose mannose + + +
lactose glucose,
galactose +
C6 sugars melibiose glucose,
galactose +
ketose fructose fructose + + +
sugars sorbose sorbose
aldoses sucrose glucose, + + +
fructose
and raffinose glucose,
ketoses fructose,
galactose +/ + +/
deoxy rhamnose 6-
deoxymannose
sugars deoxyribose 2-deoxyribose +/ +/ +/
C5 sugars aldose arabinose arabinose
sugars xylose xylose
* + Indicates good ability to ferment sugars; cannot ferment sugars; +/ low ability to ferment sugars.
Table 7. Growth optimum and calculated inlet concentrations of inorganic ions required for optimum growth of
page 11 of 57
yeast in a CSTRa [110]
Ion Growth Cellular concentration, CSTR inlet concentration for a cell

optimum, mmol/100 g dry mass concentration of 10 g/L, mmol/L
mmol/L
B+ 0.0004 0.001 0.5103 0.0004 0.001

Ca2+ 0.5 5.0 b 0 1.5 0.5 5.0
Co2+ 0.0001 0.001c (0.03 1)103 0.0001 0.001
Cu2+ 0.001 0.01c 0.008 0.20 0.002 0.03
Fe2+ 0.001 0.01 0.036 0.18 0.005 0.02
K+ 2.0 10.0 b 8 56 3 15
Mg2+ 2.0 4.0 4 17 26
Mn2+ 0.002 0.01 b 0.007 0.055 0.003 0.01
Mo2+ 0.001 0.01 (0.04 0.08)103 0.001 0.01
Ni+ 0.001 0.05 (0.03 2)103 0.001 0.05
Zn2+ 0.005 0.015b, c 0.08 0.3 0.006 0.04
Cl 1 11 140 2 15
I 0.001 0.04 1103 0.001
1 0.6 15.0 2 10
H 24 40 65 6 10
2
1 700 100
a CSTR = continuous stirred tank reactor.

b Optimum concentration may be higher under conditions where specific inhibitory ions are present or any ion is
present at an inhibitory level.
c These ions are most likely to be deficient in a complex industrial medium.
Yeast extract is a good source of trace minerals and organic growth factors. Many raw materials used for industrial ethanol
production supply all the nutrients necessary for yeast growth; additional nutrient supplements are required in some cases.
5.1.2. Fermentation Pathways

Hexoses. Yeasts are able to metabolize various carbon compounds. Metabolic pathways differ under aerobic and under
anaerobic conditions. Conversion of glucose by Saccharomyces cerevisiae under both conditions is shown in Figure 5 [111].
Figure 5. Simplified chart of anaerobic and aerobic catabolism of Saccharomyces cerevisiae [111]
ADP = adenosine diphosphate; ATP = adenosine triphosphate; TCA = tricarboxylic acid (citric acid).
Under anaerobic conditions, the yeast produces ethanol, according to the Gay Lussac equation:
Therefore, from each gram of glucose consumed, 0.51 g of ethanol can be produced. However, some of the carbon source is
used for biomass generation so that the actual ethanol yield is ca. 90 95 % theoretical. In addition to ethanol, carbon
dioxide, and biomass, 2 mol of adenosine triphosphate (ATP) are produced per mole of glucose. A lower than theoretical
yield of ethanol is also caused by the formation of small amounts of byproducts, such as glycerol and succinate, at the
expense of the carbon source. If the carbon source is not used for the production of biomass and byproducts, the ethanol
yield based on sugar would increase by 1.6 and 2.7 %, respectively [112]. In ethanol production, some higher alcohols (fusel
oils) are formed partly from the carbon source and partly from deamination and subsequent conversion of certain amino
acids.
Under aerobic conditions glucose is converted to carbon dioxide and water according to the equation:
In addition, biomass and energy are also generated.
The sequence of intermediates and reactions in the transformation of glucose (or a polysaccharide) to ethanol and CO2 is
page 12 of 57
shown in Figure 6. The Embden Meyerhof Parnas or glycolytic pathway represents the major route of glucose catabolism
in most cells that convert hexoses to pyruvate. This pathway, which must provide all the intermediates and most of the
energy required for cell growth, is also used in microbial fermentation of glucose to ethanol, lactate, glycerol, glycols, and a
variety of other products.
Figure 6. Embden Meyerhof Parnas scheme of glycolysis [113]
Glycolysis: glucose + 2 P 2 lactate + 2 ATP (classical mammalian muscle or brain)

i + 2 ADP
glucose + Pi -glycerol phosphate + pyruvate (insect flight muscle,
striated muscle)
Fermentation: glucose + 2 P 2 ethanol + 2CO2 + 2 ATP + 2 H2O (first form)
i + 2 ADP
glucose + glycerol + acetaldehyde HSO3 + CO2(second form, no
net ATP)
2 glucose 2 glycerol + ethanol + acetate + 2 CO2 (third form, no net
ATP)
glucose + (Pi ) -glycerol phosphate + acetaldehyde + CO2
glycerol + Pi (third form, no net ATP)
Enzymes, characteristic inhibitors, and standard free energy changes for each of the reactions are listed in Table 8. The
reactions do not require oxygen and provide the cellular energy supply under completely anaerobic conditions.
Table 8. Reactions and thermodynamics of glycolysis and alcoholic fermentation [113]
Reaction Equation a Name of enzyme Characteristic G0 b,

number inhibitor kJ/mol
1 Glucose + ATP glucose 6-P + ADP hexokinase 14.32
cosubstrate: Mg2+ glucokinase

1a Glucose 6-P + H2O glucose + Pi glucose 6-phosphatase glucose, orinase 16.83
cosubstrate: Mg2+ nonspecific

phosphatases
2 Glycogen + n Pi n glucose 1-P -1,4-glucan 3.06
phosphorylase
3 Glucose 1-P glucose 6-P phosphoglucomutase F, 7.29
organophosphorus
cosubstrate: glucose 1,6-diP inhibitors
4 Glucose 6-P fructose 6-P phosphoglucose 2-deoxyglucose 2.09
(glucose
phosphate) isomerase 6-phosphate
5 Fructose 6-P + ATP phosphofructokinase ATP, citrate 14.24
fructose-1,6-diP + ADP + H+
cosubstrate: Mg2+,
activator: (ADP, AMP)K+
5a Fructose 1,6-diP + H2O Mg2+ fructose diphosphatase AMP, fructose 1,6- 16.75
diphosphate,
fructose
6-P + Pi nonspecific Zn2+, Fe2+
phosphatases
cosubstrate: Mg2+,
page 13 of 57
6 Fructose 1,6-diP dihydroxyacetone P (fructose phosphate) chelating agents 23.99
+ glyceraldehyde 3-P aldolase (microbial
enzymes only)
7 Dihydroxyacetone P glyceraldehyde 3- triose phosphate 7.66
P isomerase
8 2(Glyceraldehyde 3-P glyceraldehyde ICH2COR 2(6.28)
phosphate
+ Pi + NAD+ 1,3- dehydrogenase; triose D-threose
diphosphoglycerate
+ NADH + H+ phosphate 2,4-diphosphate
dehydrogenase
9 2(1,3-Diphosphoglycerate + ADP + phosphoglycerate 2(
kinase 28.39)
H+ Mg2+ 3
phosphoglycerate + ATP)
10 2(3-Phosphoglycerate phosphoglyceromutase 2(4.43)
2-phosphoglycerate
cosubstrate: glycerate 2,3-diP
11 2(2-Phosphoglycerate enolase Ca2+ 2(1.84)
(phosphopyruvate
phosphoenolpyruvate) hydratase) F plus Pi
cosubstrate: Mg2+ or Mn2+

12 2(Phosphoenolpyruvate + ADP pyruvate kinase Ca2+ vs. Mg2+ 2(
23.95)
+ H+ pyruvate + ATP) Na+ vs. K+
cosubstrate: Mg2+
activator: K+(Rb+, Cs+)
13 2(Pyruvate + NADH + H+ lactate lactate dehydrogenase oxamate 2(
25.12)
+ NAD+ )
14 2(Pyruvate + H+ acetaldehyde + CO2) pyruvate (de) 2(
carboxylase 19.76)
15 2(Acetaldehyde + NADH + H + alcohol dehydrogenase 2(
21.56)
ethanol + NAD+ )
Sums: (glucose)n + H2O 2 lactate + 2 H+ + (glucose)n1 219.40
(glucose)n + 3 Pi + 3 ADP 2 lactate + 3 ATP + (glucose)n1 glycolysis

(muscle) 114.38 c
glucose 2 lactate + 2 H+ 198.45
glucose + 2 Pi + 2 ADP 2 lactate + 2 ATP glycolysis or lactate fermentation
124.52c
glucose 2 ethanol + CO2 234.88
glucose + 2 Pi + 2 ADP 2 ethanol + 2 CO2 + 2 ATP alcoholic
fermentation 156.92 c
a P = phosphate.
b G0 values refer to pH 7.0 with all other reactants including H2O at unit activity; the free energy of formation of glucose in
aqueous solution equals 910.88 kJ/mol, its G0 of combustion to CO2 + H2O is 2872 kJ/mol, and G0 for (glucose)n + H2O
(glucose)n1 equals 21.06 kJ/mol.
c From this table.
As shown in Table 8 and Figure 6, conversion of D-glucose to ethanol starts with the formation of D-glucose 6-phosphate
(Robinson ester) (Reaction 1). This reaction is catalyzed by hexokinase (ATP : D-hexose 6-phosphotransferase). The
enzyme uses ATP as a phosphate donor and requires a divalent cation (usually Mg2+, but Mn2+ or frequently Ca2+ and Co2+
can replace the cofactor). This enzyme is relatively nonspecific and can catalyze the phosphorylation of a variety of hexoses
of appropriate configuration including D-fructose, D-mannose, 2-deoxy-D-glucose, and D-glucosamine. The equilibrium of the
reaction lies so far in the direction of product formation as to render the reaction virtually irreversible thermodynamically (K
eq 6.5103 at pH 7.4 and 25 C) and of no practical importance in gluconeogenesis.
page 14 of 57
The conversion of starch and other starchlike glucose-containing polysaccharides is catalyzed by -1,4-glucan
phosphorylases ( -1,4-glucose : orthophosphate glycosyltransferases), which are widely distributed in a variety of
organisms (Fig. 6, Reaction 2). This phosphorolysis commences at the free, nonreducing end of an amylose chain and
removes one glucose unit at a time, yielding a total of (n + 1) glucose 1-phosphate molecules until the reducing end is
reached. If the substrate is amylopectin, phosphorolysis continues only until the branch points are reached and the product is
a limit dextrin. The 1,6-bond at the branch may be cleaved by an amylo-1,6-glucosidase, yielding free glucose, after which
phosphorylase is able to act again until the next branch is reached, and so on.
Phosphoglucomutase ( -D-glucose 1,6-diphosphate : -D-glucose 1-phosphate phosphotransferase) is responsible for the

interconversion of D-glucose 1-phosphate (G1P) (Cori ester) and D-glucose 6-phosphate (G6P) (Fig. 6, Reaction 3). The
enzyme is specific for -D-pyranose phosphate and the Keq is 19 at pH 7 and 25 C for cG6P/cG1P. The reaction requires
Mg2+ and is inhibited by fluoride.
The interconversion of D-glucose 6-phosphate and D-fructose 6-phosphate (Neuberg ester) is catalyzed by
phosphoglucoisomerase (D-glucose 6-phosphate ketol-isomerase) (Fig. 6, Reaction 4). The enzyme is quite specific. It is
competitively and specifically inhibited by 2-deoxy-D-glucose 6-phosphate, which is readily formed from the corresponding
free deoxysugar in the presence of hexokinase.
Phosphofructokinase (ATP : D-fructose 6-phosphate 1-phosphotransferase) is a highly specific enzyme that controls the
formation of D-fructose 1,6-diphosphate (Harden Young ester) from fructose 6-phosphate. It requires ATP and Mg2+. The
enzyme can use phosphate from uridine triphosphate or from inosine triphosphate instead of from ATP. Its activity can be
inhibited by high ATP concentrations, which occurs when lactate and pyruvate are oxidized aerobically to CO2 via the Krebs
cycle. In that case, glycolysis is blocked and glucose synthesis is favored. At very low ATP concentration, glycolysis is
required for energy generation, and carbohydrate synthesis is shut off.
Fructose 1,6-diphosphatases (D-fructose 1,6-diphosphate 1-phosphohydrolases) are required for hydrolytic conversion of the
diphosphate to the 6-monophosphate (Fig. 6, Reaction 5 a). The enzymes are specific and can be inhibited by high
concentrations of their substrate and also by adenosine monophosphate (AMP).
The conversion of fructose 1,6-diphosphate to two molecules of triosephosphate [3-phosphoglyceraldehyde (Ficher Bauer
ester) and phosphodihydroxyacetone] (Fig. 6, Reaction 6) is carried out by aldolase (D-fructose 1,6-diphosphate : D-
glyceraldehyde 3-phosphate-lyase). These two triosephosphate molecules are in equilibrium, supported by triosephosphate
isomerase (D-glyceraldehyde 3-phosphate ketol-isomerase) (Fig. 6, Reaction 7). Only 3-phosphoglyceraldehyde undergoes
further conversion to ethanol. In the course of cleavage of the hexose, triose products must be removed because the
combined equilibrium lies in favor of hexose. The enzyme from yeast contains Zn2+ and, after removal or complexation of the
metal, is activated equally well by Zn2+, Fe2+, and Co2+. The activity of the enzyme is enhanced about 35 times in the
presence of K+ ions [112].
Glyceraldehyde 3-phosphate dehydrogenase [D-glyceraldehyde 3-phosphate: nicotinamide adenine dinucleotide (NAD)

oxidoreductase (phosphorylating) ] catalyzes Reaction 8. Free sulfhydryl groups are required for its activity. The presence of
NAD+ and Pi (inorganic phosphate) is necessary. In the reaction, NAD+ is reduced to NADH, which is then reoxidized in the
reduction of acetaldehyde to ethanol or pyruvate to lactate. As a dehydrogenase, the enzyme is specific for D-glyceraldehyde
3-phosphate, the free dephosphorylated aldehyde, or acetaldehyde, and is strongly inhibited by D-triose 2,4-diphosphate.
The enzyme phosphoglycerate kinase (ATP : 3-phospho-D-glycerate 1-phosphotransferase) catalyzes the conversion of 1,3-
diphosphoglycerate to 3-phosphoglycerate, with the generation of 1 mol of ATP (Fig. 6, Reaction 9). It requires a divalent
metal cofactor (Mg2+, Mn2+, or Co2+).
3-Phosphoglycerate, which is formed in Reaction (9) (Fig. 6), is isomerized to 2-phospho-D-glycerate by

phosphoglyceromutase (2,3-diphospho-D-glycerate : 2-phospho-D-glycerate phosphotransferase) (Fig. 6, Reaction 10). The
diester 2,3-diphosphoglycerate and Mg2+ are obligatory cofactors for the reaction.
The conversion of 2-phosphoglycerate to phosphoenolpyruvate (Fig. 6, Reaction 11) can be considered to be a dehydration
reaction that is catalyzed by enolase (phosphopyruvate hydratase). This enzyme is widely distributed in living organisms.
Divalent cations (Mn2+, Mg2+, Zn2+, or Cd2+) that can be antagonized by Ca2+ or Sr2+ are essential. Fluoride ion is an
effective inhibitor, especially in the presence of Pi.
Pyruvate kinase (ATP : pyruvate phosphotransferase) catalyzes Reaction (12), (Fig. 6). For this reaction, Mg2+ or Mn2+ is
required; Co2+ ions are competitive antagonists. Some enzymes of this type require K+, Rb+, or Cs+ for full activity. The ions
can be antagonized by Na+ or Li+. The reaction equilibrium is very favorable for the formation of pyruvate, and the turnover
number is 61013 compared to 121013 for the opposite direction [113].
To produce ethanol from pyruvate, yeasts that operate anaerobically irreversibly decarboxylate pyruvate with the aid of
pyruvate decarboxylase (2-oxo acid carboxylase) to yield acetaldehyde (Fig. 6, Reaction 14). Alcohol dehydrogenase
(alcohol : NAD oxidoreductase) then catalyzes the reduction of acetaldehyde to ethanol (Fig. 6, Reaction 15). The enzyme
responsible for the decarboxylation can satisfy its metal requirements by using either certain divalent ions (Mg2+, Mn2+, Co
2+, Ca2+, Cd2+, or Zn2+ ) or trivalent ions (Al3+ or Fe3+ ). Alcohol dehydrogenase from yeast contains Zn2+. The enzyme has
four catalytic sites and a molecular mass of 151 000. Both enzymes have been isolated from yeast and produced in
crystalline form.
page 15 of 57
Pentoses. Yeasts metabolize aldopentoses by an oxidation reduction reaction. They reduce D-xylose to xylitol, which is
subsequently oxidized to D-xylulose (Fig. 7). The reduction is catalyzed by aldose reductase (alditol : NADP 1-
oxidoreductase) [115]. The enzyme has a specificity for NADPH (reduced nicotinamide adenine dinucleotide phosphate).
Figure 7. D-Xylose and L-arabinose metabolism in yeast [114]
1) Aldose reductase; 2) D-Xylulose reductase; 3) D-Xylulokinase; 4) Transaldolase and transketolase; 5) D-Xylose isomerase
D-Xylulose reductase (xylitol : NAD 2-oxidoreductase) is involved in the oxidation of xylitol to D-xylulose. The reaction is
readily reversible, and NADH oxidation occurs with some ketoses including D-xylulose, D-fructose, and D-ribulose.
D-Xylulokinase catalyzes the phosphorylation of D-xylulose to D-xylulose 5-phosphate. The enzyme is found in bacteria [116],
[117]. The presence of D-xylulokinase in yeast is indicated by the ability of many yeasts to use D-xylulose under both aerobic
and anaerobic conditions [115], [118]. D-Xylulose 5-phosphate can be converted to pyruvate via both the pentose
phosphate and the Embden Meyerhof Parnas pathways.
Yeasts also metabolize L-arabinose by oxidation reduction. The aldose is reduced to L-arabitol, which is converted to D-
xylulose 5-phosphate, the key intermediate of the pentose phosphate pathway (Fig. 7) [114].
In the metabolism of D-xylose, the actual conversion of the sugar occurs after a lag period of adaption for the production of D-
xylose-metabolizing enzymes [119], [120].
Some yeasts, such as Candida utilis and Rhodotorula gracilis, have inducible D-xylose isomerase in addition to the initial
oxidation reduction reaction and can isomerize D-xylose directly to D-xylulose [119], [121].
Ethanol can be produced readily from D-xylulose in high yield by yeasts that normally produce ethanol from glucose [118],
[122].
About half of all yeasts can assimilate D-xylose under aerobic conditions [123]. Yeasts that could consume xylose or other
pentoses under anaerobic conditions were unknown. But anaerobic conversion of D-xylose to ethanol has been
demonstrated in the yeasts Pachysolen tannophilus [124] and Pichia stipitis [125]. Many yeasts can convert D-xylulose
(which is a keto isomer of D-xylose) to ethanol under anaerobic conditions. The growth of yeasts on D-xylose under
anaerobiosis has not been reported.
Certain yeasts can produce a high yield of ethanol directly from D-xylose (Candida sp. XF 217, Pichia stipitis), while others
(Saccharomyces cerevisiae, Schizosaccharomyces pombe) can do so after D-xylose is isomerized to xylulose by a bacterial
isomerase. Table 9 shows some advantages and disadvantages of these yeasts in the production of ethanol from xylose and
xylulose.
Table 9. Pentose (xylose and xylulose) fermenting yeasts *
Yeast Process Advantage Disadvantage
Candida sp. XF 217 batch yield (0.42 g per gram of low ethanol
xylose), low xylitol tolerance
formation
Kluyveromyces batch cellobiose also xylitol formation, low

cellobiovorus ethanol tolerance
Pachysolen tannophilus batch none xylitol formation, low

ethanol tolerance
Pichia stipitis batch yield (0.42 g per gram of low ethanol

xylose) low xylitol tolerance
formation
Saccharomyces simultaneous high ethanol tolerance, isomerization, cost

cerevisiae isomerization and fast, yield (0.45 g per (xylitol formation)
xylulose fermentation gram of xylulose)
Schizosaccharomyces simultaneous high ethanol tolerance, isomerization, cost

pombe isomerization and fast, yield (0.48 g per
xylulose fermentation gram of xylulose)
page 16 of 57
* Reprinted with permission from [126].
5.1.3. Fermentation Variables

Effect of Substrate Concentration. The carbon substrate concentration has a significant effect on ethanol production.
Previously, batch fermentation was carried out with an initial concentration of fermentable substrate of 14 18 wt %, which
resulted in a final ethanol concentration of ca. 6 9 % (wt/vol) [127]. Increasing sugar concentration was an advantage
because it led to higher final ethanol concentration and, subsequently, to reduction of distillation costs. At the same time, the
growth of osmosensitive contaminants was suppressed. However, at sugar concentrations >14 %, plasmolysis of yeast cells
begins. In addition, the initial rate of fermentation starts to decline before the ethanol concentration reaches a significant
value [109].
Under anaerobic conditions and at low-to-moderate glucose concentrations, the rate of ethanol production may be
represented by a Monod-type relationship [111]:
where V is the ethanol production rate, g h1; S is the glucose substrate concentration, g/L; and Ks is a constant with a
low value of 0.35 g/L.
This relationship shows that under anaerobic conditions, maximum ethanol production per cell is achieved when sugar
concentration is considerably > 3.5 g/L and <150 g/L. Inhibition of the ethanol production rate above 150 g/L of glucose is
significant. If the inhibition caused by ethanol is considered, it appears that it limits the concentration of fermentable sugars.
Effect of Oxygen. Although biomass is produced under aerobic conditions and anaerobic conditions favor production of
ethanol, oxygen was found to be essential for good fermentation. In addition to being a terminal acceptor of electrons from
the respiratory chain, it also acts as a yeast growth factor and is involved in the synthesis of unsaturated fatty acids and
ergosterol, which stimulate growth of yeast under anaerobic conditions and increase viability of cells [109], [128].
Oxygen is especially necessary when batch fermentation is carried out at high sugar levels requiring prolonged growth of
yeast, or in continuous processes, because the yeast is unable to grow for more than four to five generations under fully
anaerobic conditions. When oxygen disappears from the broth, the yeast continues to grow anaerobically. Cell division leads
to a redistribution of the sterols and unsaturated fatty acids that have accumulated during aeration. After several generations
the sterol and unsaturated fatty acid level is too low to enable normal functioning of the membrane causing a change in the
physiology of the yeast [129], [130].
The necessary level of oxygen depends on the strain of yeast. Usually an oxygen tension of 6.7 13.3 Pa is required in the
broth. If the concentration exceeds this value, cell growth is enhanced at the expense of ethanol production by promoting the
Pasteur effect, i.e., the complete oxidation of glucose to CO2 and H2O via the tricarboxylic acid (TCA) cycle and the
respiratory chain. In some yeasts, cell mass productivity increases drastically with a corresponding decrease in ethanol
production at an oxygen tension >0.23 kPa [131]. This process is controlled by the concentration of fermentable sugar in the
growth medium. If the concentration is high and conditions are aerobic, the Pasteur effect is likely to be diminished and
glucose will be degraded by the aerobic pathway. This phenomenon, known as aerobic fermentation, results from the
operation of the so-called reverse Pasteur or Crabtree effect, which represses both the synthesis and the activity of
respiratory enzymes at glucose or sucrose concentrations greater than 0.02 0.1 % [109], [132].
Air sparging through the growth medium can be omitted if certain supplements, such as oleic acid, linolenic acid, tween 80,
or ergosterol are present in the medium [129], [133]. Cultures enriched with these supplements can attain a high ethanol
concentration (15.5 wt %) with a high substrate conversion efficiency (95 %) under anaerobic conditions.
Various sterols and fatty acids increase the viability of resting cells and prolong their fermentative activity [133], [134].
Incorporation of these compounds into the cell membrane appears to increase its permeability to ethanol and permits a high
rate of ethanol transport out of the cells.
Effect of Ethanol. Ethanol is toxic to yeast. The general effect is most noticeable on the cell membrane; the major toxic
effect has been postulated as membrane damage or a change in membrane properties [135]. Ethanol inhibits both growth
and ethanol production in a noncompetitive manner [136]. When ethanol is present in concentrations of up to 2 %, the
observed inhibition is almost negligible for most yeasts. With a higher concentration, the effect of ethanol becomes more
evident (Figs. 8 and 9).
Figure 8. Comparison of the effect of various ethanol inhibition functions [137]
= specific growth rate; = maximum specific growth rate; Source of ethanol: added, ------ autogenous
a) Continuous [138]; b) Batch [139]; c) Continuous [140]; d) Continuous [136]; e) Batch [141]; f) Batch [140]; g) Continuous
[142]; h) Batch [143]
Figure 9. Effect of ethanol concentration on ethanol productivity [136]
page 17 of 57
a) [144]; b) [136]; c) [138]
Concentrations of ethanol > 110 g/L stop both growth and ethanol production in most yeast strains. However, with most
tolerant yeasts, ethanol production (not growth) is possible even in the presence of up to 20 % ethanol [109].
As shown in Figure 8, the effect of externally added ethanol on cell growth is much lower than the effect of autogenous
ethanol. The effect of ethanol on cell viability increases at higher temperature [145] or if the cells are exposed to heat stress
for a short period of time before coming into contact with ethanol [146].
Prolonged, continuous exposure of yeast to high ethanol concentration results in a loss of viability so that recycled cells must
be continually replenished or discarded completely at intervals [147].
Effect of pH. The concentration of hydrogen ions in a fermentation broth affects yeast growth, ethanol production rate,
byproduct formation, and bacterial contamination control.
Usually, industrial ethanol fermentation by yeast has an initial pH value of ca. 4 6, depending on the buffering capacity of
the medium. In lightly buffered media, the initial pH is ca. 5.5 6 and in more highly buffered media, ca. 4.5 4.7 [127],
[148]. If the pH value is <5 during fermentation, bacterial growth is severely repressed; the pH values for growth of most
strains of Saccharomyces cerevisiae are 2.4 8.6, with an optimum of 4.5. Yeast sugar fermentation rates are relatively
insensitive to pH values between 3.5 and 6 [109], [138].
Effect of Temperature. Most brewer's yeasts have a maximum growth temperature around 39 40 C [126]. The maximum
growth temperature reported for any species of yeast was 49 C for Kluyveromyces marxianus [149]. Mesophilic strains of
Saccharomyces have optimum cell yields and growth rates between 28 and 35 C. The optimum and maximum temperatures
for growth of thermophilic yeast are ca. 40 and 50 C, respectively; these strains have a high maintenance requirement and
more complicated nutritional requirements [109].
In batch processing, the optimum temperature for the complete utilization of glucose and the highest final ethanol
concentration is generally slightly below the optimum growth temperature. This is attributed to enhanced ethanol inhibition at
higher temperature [109], [150]. At higher temperature, the ethanol production rate is higher than its transport rate through
the cell membrane. The difference in these rates results in an increase of ethanol concentration in the cells, a subsequent
inhibition of some enzymes, and cell death. For continuous processes, the maximum process temperature (35 40 C) in the
absence of ethanol must be reduced by ca. 1 C for each percentage increase in ethanol concentration [109].
Some yeasts have an optimum fermentation temperature of ca. 40 42 C. They produce up to 12 % of ethanol with yields
>90 % of theoretical [126], [151], [152]. Because sugar fermentation is exothermic (586 J of heat produced per gram of
glucose consumed) [127], using yeasts that ferment at higher temperature substantially reduces cooling costs of fermentors.
5.1.4. Direct Fermentation

When polysaccharides (cellulose and starch) are used for ethanol production, they first must be hydrolyzed by means of
different physicochemical or enzymatic methods and then converted to ethanol. Combining this two-step process into a
single operation would be an advantage. This can be achieved by simultaneous saccharification and fermentation (SSF)
processes using cocultures of microbes that are able to hydrolyze polysaccharides and convert sugars to ethanol, or by
direct fermentation of polysaccharides to ethanol with monocultures.
In these cases the rate of enzymatic hydrolysis is increased by constant removal of the produced sugars through their
conversion to the product. This is particularly important in the case of cellulose hydrolysis where glucose and cellobiose
severely inhibit enzymes participating in the hydrolysis.
The coupled enzymatic hydrolysis of cellulose and simultaneous fermentation of the resulting sugars to ethanol by yeast
have been investigated [153-157]. Table 10 shows simultaneous hydrolysis of ball-milled cellulosic substrates by cellulases
of Trichoderma reesei QM 9414 and conversion of sugars to ethanol by Candida brassicae. Use of a thermotolerant yeast
and cellulolytic enzymes in a combined saccharification fermentation process is an advantage because the optimum
temperature for the hydrolysis of cellulose is 45 50 C, and cooling problems can be simplified during large-scale
fermentation [158].
Table 10. Simultaneous saccharification and fermentation (SSF) of various substrates with and without milling
pretreatment (Trichoderma reesei QM 9414 cellulase with Candida brassicae) [157]
Feedstock Conversion to ethanol
Unpretreated Pretreated
24 h 48 h 24 h 48 h
page 18 of 57
Eucalyptus grandis sawmill waste 0 0 33.44 38.64
Acacia mearnsii sawmill waste 0 0 36.73 45.71
Southern pine sawmill waste 0 0 38.01 42.59
Digester rejects 41.76 54.55 40.31 72.86
Primary clarifier sludge 51.22 66.93 57.49 84.24
Newsprint 34.61 45.40 54.25 60.34
Cardboard 35.12 42.96 53.45 77.93
Air classified sawmill waste 49.58 53.00 59.05 75.13
Cotton linters 10.41 8.74 18.37 16.59
Rice straw 81.49 110.06 57.66 85.47
Rice hulls 45.99 50.19 65.57 96.05
Corn stillage 14.37 16.69 45.57 65.92
A simultaneous saccharification fermentation process was also applied in the production of ethanol from starchy materials
(cassava and corn). Enzymes from Aspergillus niger, A. awamori, and Rhizopus sp. were used for hydrolysis of starch with
simultaneous yeast fermentation [159-161]. Ethanol yields in these processes ranged from 82 to 99 % of theoretical. In
certain cases ethanol concentration reached a level of 20 vol % in five days [159].
The yeast Schwanniomyces is able to hydrolyze starch directly and partially ferment it to ethanol [162]. Amylolytic systems of
S. castellii and S. alluvis have been reported [163-165]. Economic use of these and other amylolytic species is probably not
feasible because of their limited ethanol tolerance [162], [166].
5.2. Production by Bacteria

Among bacteria the gram-negative Zymomonas mobilis occupies an outstanding position as an excellent producer of ethanol
from glucose. This mesophilic organism was isolated at the beginning of the 20th century from palm wine and from the
Mexican beverage pulque [167], [168]. The pioneering work of ROGERS et al. showed that this bacterium has a number of
favorable characteristics for efficient production of industrial ethanol [169]. A major drawback of Z. mobilis, however, is its
incapability to ferment pentoses such as xylose and arabinose [167]. The lack of microorganisms that can ferment pure
ethanol from pentoses has given rise to a number of efforts to construct appropriate recombinant bacteria. By introduction of
the genes for ethanol production from Z. mobilis, the native abilities of Escherichia coli for pentose fermentation were
combined with an efficient ethanol-forming activity [170]. Alternatively, pentose-fermenting strains of Z. mobilis have been
developed by genetic engineering [171-175]. A number of facultative and obligatory anaerobes are capabable of fermenting
pentoses, but in addition to ethanol and carbon dioxide, they also synthesize large amounts of other fermentation products
(e.g., acetic acid, formic acid, lactic acid, acetone, and hydrogen) [176-181].
5.2.1. Ethanol Tolerance

The growth of most bacteria is inhibited by ethanol concentrations of 10 20 g/L. However, both Z. mobilis and yeast can
tolerate high ethanol concentrations. In fact, alcohol concentrations of 120 g/L have been attained by fermentations with Z.
mobilis [169]. High ethanol concentrations generally destroy the structure and function of the cell membrane [182]. The
cytoplasmic membrane of Z. mobilis contains various hopanoids 1 6 these membrane-stabilizing, pentacyclic triterpenoids
are present at concentrations of 30 mg/g cell dry weight, i.e,. they make up 40 50 % of the organism's total lipid content
[183-186]. Hopanoids play an important role in the ethanol tolerance of Z. mobilis by reinforcing the cytoplasmic membrane
against the fluidizing effects of high concentrations of ethanol [184], [187]. Hopanoids are synthesized in bacterial cells under
both aerobic and anaerobic conditions, whereas the biosynthesis of sterols in yeast requires oxygen. In yeast sterols are
essential membrane-stabilizing triterpenoids.
1
Diploptene
page 19 of 57
Diplopterol
3
Tetrahydroxybacteriohopane
4
Bacteriohopanetetrol ether
5
O- -D-glucosaminylbacteriohopanetetrol
6
O- -D-glucosaminyl-23-oxobacteriohopanetriol
5.2.2. Fermentation of Glucose by Z. mobilis

Zymomonas mobilis uses the 2-keto-3-deoxy-6-phosphogluconate or Entner Doudoroff pathway to metabolize sugar, in
which pyruvate is formed as an intermediate [167]. The formation of acetaldehyde by decarboxylation of pyruvate and its
subsequent reduction to ethanol are identical to the reactions in yeast: 2 mol of ethanol and 2 mol of carbon dioxide are
produced per mole of glucose. This homo-ethanol pathway involves two enzymes: pyruvate decarboxylase and alcohol
dehydrogenase (see Figs. 10 and 11). However, the energy (ATP) yield of this bacterium, based on the amount of glucose
metabolized, is only half of that obtained with yeast. Therefore, Z. mobilis can synthesize only half as much new cell mass
per mole of glucose degraded as yeast does, but this results in a higher ethanol yield (Table 11).
Table 11. Fermentation properties of the bacterium Zymomonas mobilis and of the yeast Saccharomyces
carlsbergensis
Property Zymomonas Saccharomyces

mobilis carlsbergensis
Doubling time, h 2.51 5.64

Ethanol production
rate qP/X,* g g1 h1 5.44 0.82
Cell yield YX/S,* g/g 0.028 0.043
Product yield YP/S,* g/g 0.465 ** 0.460 **
* Explanation of symbols:
page 20 of 57
** Max. theoretical product yield = 0.511 g/g.
Zymomonas mobilis, unlike yeast, does not require oxygen for growth, and this greatly simplifies fermentative ethanol
production. In contrast to a number of strictly anaerobic bacteria, Z. mobilis is relatively insensitive to oxygen. Indeed, it
possesses an electron-transport system similar to the respiratory chain of aerobic organisms [188-190] and can transfer
hydrogen, formed in the conversion of glucose to pyruvate, to molecular oxygen. These reducing equivalents are then not
available for reduction of acetaldehyde to ethanol; as a consequence, acetaldehyde accumulates and inhibits the growth of
Z. mobilis [188], [191].
A major consideration in achieving economic ethanol production is the cost of the sugar used as a raw material. For this
reason the fermentation of starch and waste starch has been investigated by two approaches: first, two-step processes,
involving separate saccharification and fermentation stages were developed [192], [193]. For the fermentative production of
ethanol from an industrial hydrolyzed waste starch fraction (B-starch) Z. mobilis was used in two 50 m3 continuous stirred-
tank reactors with a residence time of 15 h, and production of 10 000 L/d of ethanol (96 vol %) was achieved [194]. If
nonsterile B-starch hydrolysate was used, the process became unstable due to contamination by lactic acid bacteria. Lactic
acid bacteria with an acidic pH optimum of growth were able to use the pentoses in the hydrolysed waste substrate, in
contrast to Z. mobilis. It is known that lactic acid bacteria have a profoundly negative influence on the growth of Z. mobilis,
although the inhibition mechanisms are unresolved [195]. To overcome this problem, fluidized-bed reactors with immobilized
Z. mobilis cells in macroporous glass beads were used [193], [196]. The high concentration of immobilized Z. mobilis cells
permitted unsterile fermentation of hydrolysed B-starch to ethanol at short residence times, so that contaminants were
washed out. A cascade of two 55 L fluidized-bed reactors was operated stably at a mean residence time of 4.25 h and 99 %
conversion of the unsterile hydrolysed B-starch, resulting in an ethanol concentration of 50 g/L and an ethanol productivity of
13 g L1 h1 [193].
A second approach is simultaneous starch saccharification and ethanol fermentation where Z. mobilis cells and
amyloglucosidase are simultaneously present in the fermentor. With sago starch as substrate in a 500 L batch fermentor, the
final concentration of ethanol obtained by this technique was 82 g L1 with an ethanol yield of 0.457 g/g [197]. Whereas Z.
mobilis generally produces high yields of ethanol from glucose and fructose, the conversion efficiency from sucrose-based
substrates is reduced to 70 % due to the formation of byproducts such as levan and sorbitol [198]). A mutant of Z. mobilis
lacking the property of levan synthesis has been shown to produce 73.5 g L1 ethanol from 150 g L1 sucrose in fermentation
with immobilized cells [199]. The ethanol yield from sucrose by Z. mobilis could also be significantly enhanced (from 0.29 to
0.40 g/g) by addition of immobilized invertase to the fermentation broth [200]. Other co-immobilization systems of Z. mobilis
with other microbes or enzymes for fermentation of different substrates have been described (for reviews, see [175], [201]).
5.2.3. Fermentation of Other Sugars by Recombinant Z. mobilis Strains

Up to now no yeast or bacterial strains are available that can ferment pentose to ethanol as efficiently as these organisms
can ferment glucose or fructose. The price of the sugar substrate represents as much as 70 % of the total cost of ethanol
fermentation. Efficiency could, however, be improved considerably by using both the hexoses and the pentoses of the plant
biomass as substrates. A number of approaches have been made to genetically engineer Z. mobilis to broaden its range of
utilizable substrates. Genes encoding enzymes of several metabolic pathways have been introduced and successfully
expressed in Z. mobilis, and attempts have been made to construct new catabolic pathways (e.g. for L-arabinose, D-xylose)
[171-175] .
For the conversion of the pentose sugars commonly found in agricultural residues and other lignocellulosic biomass to
ethanol, the development of Z. mobilis strains with the capability of D-xylose and of D-arabinose fermentation was attempted.
By introduction of four genes encoding xylose catabolism (i.e. xylose isomerase, xylulokinase, transaldolase, transketolase)
into Z. mobilis, the organism was enabled to grow on xylose and ferment it to ethanol (Fig. 10) [202-204]. However, the
xylose-fermenting recombinant strains showed decreased growth and ethanol production rates and increased byproduct
formation [204], [205]. A 13C NMR flux analysis indicated that ribose 5-phosphate is synthesized via the pentose phosphate
pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the
level of heterologous xylulokinase [204]. As was shown by 31P NMR studies the levels of nucleoside triphosphates in cells
grown with xylose were lower than those in cells grown in glucose-containing media. This energy limitation probably
restricted the growth of the recombinant strain on xylose media [205].
Figure 10. Catabolism of D-xylose in recombinant strains of Zymomonas mobilis
When grown with glucose xylose mixtures, a recombinant, xylose-fermenting strain of Z. mobilis produced 62 g/L of ethanol
from 65 g/L of glucose plus 65 g/L of xylose with a yield of 0.46 g ethanol per gram of sugar [206]. With substrates derived
from lignocellulosic materials the ethanol-producing efficiency of Z. mobilis is substantially impeded by toxic substances
present in the woody biomass after chemical acid hydrolysis, such as acetic acid and furfural (a pentose degradation
product) [207], [208]. Nevertheless, it was shown that in a synthetic medium mimicing a dilute-acid hydrolyzate of yellow
popular, a recombinant xylose-utilizing Z. mobilis strain produced 0.48 0.50 g of ethanol per gram of sugar, equivalent to a
yield of 94 96 % of the theoretical maximum [209].
page 21 of 57
A second predominant pentose sugar found in agricultural residues (e.g., corn fiber) and other lignocellulosic biomass is l-
arabinose. Five genes from E. coli, encoding l-arabinose isomerase, l-ribulokinase, l-ribulose-5-phosphate-4-epimerase,
transaldolase, and transketolase, were introduced into Z. mobilis [210]. The engineered strain grew on and produced ethanol
from L-arabinose as sole C source at 98 % of the maximum theoretical ethanol yield, but arabinose was not completely
utilized [210]. A third abundant sugar in plant hemicelluloses, the hexose d-mannose, is not used as a growth substrate by Z.
mobilis, but is taken up via the glucose uptake system (glucose facilitator protein) of the organism [211]. It was shown that d-
mannose was phosphorylated by a side activity of the resident fructokinase to mannose 6-phosphate. By introduction of the
E. coli gene encoding phosphomannose isomerase, Z. mobilis strains were enabled to utilize mannose as the sole carbon
source [211].
5.2.4. Ethanol Production by Recombinant Enterobacteria

A variety of bacteria are able to metabolize and ferment both hexose and pentose sugars, but all produce a mixture of
fermentation products. Metabolic studies have shown that these bacteria use both the pentose phosphate pathway and the
Embden Meyerhof pathway to metabolize hexoses and pentoses to a common intermediate, pyruvic acid, which is
subsequently converted to a variety of fermentation products (Fig. 11). Members of the enteric group of bacteria, such as E.
coli, Klebsiella, and Erwinia, perform a mixed-acid fermentation which yields a nearly equal mixture of ethanol, acetic acid,
and formate (or carbon dioxide and hydrogen) to provide redox balance. INGRAM and collaborators focused on the genetic
engineering of enteric bacteria using genes encoding Z. mobilis pyruvate decarboxylase (PDC) and alcohol
dehydrogenase B (ADH B) [170], [212], [213]. The adhB and pdc genes were assembled to form an artificial operon (PET
operon) and were expressed to high levels as plasmid-encoded genes or as chromosomally integrated genes in E. coli [214],
[215]. These high levels of recombinant enzymes coupled with the low Km of PDC for pyruvate resulted in a redirection of the
carbon flux to ethanol and carbon dioxide, thus preventing the formation of lactic and acetic acids [216], [217].
Figure 11. Microbial pathways of sugar fermentations
A) Pyruvate decarboxylase (EC 4.1.1.1; yeast and Z. mobilis); B) Pyruvate ferredoxin oxidoreductase (EC 1.2.7.1;
Clostridia); C) Pyruvate formate lyase (EC 2.3.1.54; Enterobacteria)
FD = ferredoxin; P = phosphate.
Analogous plasmid constructs have been used to engineer Klebsiella oxytoca [218], [219] and Erwinia chrysanthemi [220]. In
addition to a wide substrate range of monomeric sugars K. oxytoca has the native ability to transport and metabolize
cellobiose and cellotriose, the soluble intermediates of the enzymatic hydrolysis of cellulose [221], [222]. During cellulose
bioconversion in simultaneous saccharification and fermentation processes, the use of K. oxytoca eliminated the need to add
supplemental cellobiase [223], [224]. Additional studies have added the PET operon to strains of E. chrysanthemi that have
the native ability to secrete endoglucanase and possess PTS systems for cellobiose and cellotriose utilization [220].
5.2.5. Comparison of Recombinant Bacterial Strains

Bacterial ethanol production from pentose sugars can be approached by either exploiting the native characteristics of Z.
mobilis for efficient ethanol fermentation, or the ability of E. coli to utilize a broad range of substrates. This can be brought
about by genetic engineering of Z. mobilis for pentose utilization and of E. coli for ethanol production (Table 12).
Table 12. Xylose fermentation properties of of recombinant strains of Z. mobilis and E. coli
Property Z. mobilis E. coli ATCC11303

ZM4 (pZB5) [205] (pLO1297) [215]
60 g/L xylose 80 g/L xylose
Ethanol production
rate qP/X,* g g1 h1 1.40 1.00
Cell yield YX/S,* g/g 0.010 0.050
Product yield YP/S,* g/g 0.410 ** 0.470 **
* Explanation of symbols:
** Max. theoretical product yield = 0.511 g/g.
5.3. Fermentation Modes

The two basic modes for production of ethanol by fermentation are the batch and continuous processes. A semicontinuous
mode, which is a combination of the two, is also used.
5.3.1. Batch Processes
page 22 of 57
Most fermentation ethanol is produced by batch processes [111], [225]. A batchwise process for the fermentation of corn is
shown in Figure 23. Typical kinetics in a batch process are shown in Figure 12.
Figure 12. Ethanol production by Kluyveromyces marxianus 10606 [226]
a) Sugar; b) Ethanol; c) Biomass
Conversion of sugars in a simple batch system is ca. 75 95 % of theoretical, with a final ethanol concentration of 10
16 vol % [225], [227]. The productivity of simple, conventional batch processes is usually 1.8 2.5 g of ethanol per liter of
fermentor volume per hour [111].
To increase productivity, a batch process with recycling of the yeast was developed; this is known as the Melle Boinot
process. Recycling of yeast cells creates a high biomass concentration at the beginning of the process, which reduces the
time for the conversion of substrate to ethanol (Table 13) [228]. This rapid fermentation also increases productivity.
Increasing the initial concentration of yeast cells decreases yield. With a yeast cell concentration of 21 g/L, no growth was
noticed; the existing cells used nutrients for both ethanol production and their own maintenance [228].
Table 13. Effect of initial cell concentration on time of fermentation during batch culture [228]
Initial cell concentration, g/L Time to develop 12 vol % ethanol, h
21.4 7
23.6 6
27.2 5
26.13 * 4.5
* With yeast vitamins.
Under rapid fermentation conditions, a high ethanol concentration is attained quickly and the death rate of yeast cells is quite
high. The death rate can be decreased by fermenting at lower temperature and by maintaining the dissolved oxygen
concentration in the medium at 10 20 % of oxygen saturation [145], [229]. The tolerance of some yeasts to ethanol is also
increased in the presence of yeast vitamins [228], [230].
Although classic batchwise processes for ethanol production are attractive because of their simplicity, they have many
disadvantages such as low productivity, difficulty in automating, long and frequent downtime, and significant labor cost.
5.3.2. Continuous Processes

Without Cell Recycle. Generally, continuous processes eliminate most of the disadvantages that are inherent in the batch
processes. They may be carried out for a long time period without shutdown, thus eliminating downtime between batches,
increasing overall productivity. Because of increased productivity, smaller volume reactors are required than in batch
production. Continuous processes can be fully automated and operated under conditions that give a uniform product.
Continuous stirred tank reactors are used for these processes (Fig. 13).
Figure 13. Continuous stirred tank reactor [111]
Simple continuous processes for ethanol production are characterized by continuous addition of feed (including oxygen) to a
reactor in which a desirable steady state has been established. Beer is also removed continuously from the reactor. The beer
contains ethanol, biomass, and unconsumed nutrients. The productivity of such a process (up to 6 g L1 h1 with highly
productive strains) is about three times higher than that of a batchwise process. Therefore, the reactor volume need be only
one-third that used to produce the same amount of ethanol in a batchwise manner. In this process, both specific ethanol
productivity and total productivity of the reactor are limited by ethanol inhibition. In addition, total productivity is also limited by
low biomass concentration (10 12 g/L) [111]. A sugar concentration of ca. 10 % in the feed gives the highest reactor
productivity (Fig. 14). Economic analysis showed a 53 % reduction in operating costs and 50 % in capital costs for this
process compared to classic batch processes [231].
page 23 of 57
Figure 14. Effect of glucose concentration on continuous fermentation [231]
a) Specific productivity; b) Fermentor productivity; c) Cell mass
Sometimes two or more continuous stirred tank reactors can be joined in series to increase productivity. With two reactors,
fresh medium is pumped to the first reactor; residence time is adjusted so that conversion of sugar is incomplete. This results
in a lower ethanol concentration in the first reactor compared to the final concentration and, therefore, less ethanol inhibition.
The outflow from the first reactor is fed to the second, where fermentation is completed. The productivity of the second
reactor is lower than that of the first, but the overall productivity is increased compared to the system with one reactor [136],
[232].
With Cell Recycle. Continuous systems with cell recycle were developed in order to increase productivity over systems that
did not recycle biomass. The biomass from the stream leaving the reactor is separated by centrifugation and returned to the
reactor. Yeast concentration as high as 83 g/L can be maintained during ethanol production [229]. Such high concentrations
permit rapid and complete fermentation of concentrated sugar solutions. Fermentor ethanol productivities of 30 51 g L
1 h1 have been achieved, which represents a more than tenfold increase over continuous fermentation without cell recycle
[228], [229], [233], [234].
Both investment and operating costs of these processes are higher because of the required separation of cell mass from
beer by centrifugation. Simpler and less expensive systems for cell separation are needed. Because of the low settling rate of
yeast (<1 cm/h), shallow-depth sedimentation of biomass with the use of plate or tube settlers has been proposed [235]. The
tube settler was found to be slower than the plate settler in reaching maximum effluent clarity. The plates and tubes are
inclined to allow self-cleaning by the yeast sliding down the collection surfaces. The sedimentation rate of the yeast cells
increases when evolution of CO2 is repressed. This repression can be obtained by heat treatment for 20 min at 50 C, which
does not seriously impair cell viability. These settlers help reduce bacterial contamination because of their lower settling
velocity.
Other separators, such as the whirlpool separator in which yeast cells are deposited in a central cone when the overflow is
pumped tangentially into a vertical cylindrical vessel, have also been developed [236]. Another method involves a partial
recycle fermentor that has a separate settling zone, which is not disturbed by agitation. This allows partial separation of cells
[237].
5.3.3. Other Processes

Flocculating Cells (Internal Recycle). High-ethanol-producing and flocculating yeasts are of particular commercial interest.
These cells can readily be concentrated and separated without the use of mechanical devices (e.g., centrifuges and
conventional settlers). This simplifies the process and makes it more economic [234], [238-240]. A highly flocculating yeast,
Saccharomyces diastaticus, was investigated for continous ethanol production from various substrates [234]. The process
involved one fermentor with internal cell settling and no cell recycle or cell concentration by centrifuges or other such
mechanical devices. A high specific ethanol productivity rate of up to 50 g L1 h1, with an inflowing sucrose concentration of
112 g/L (using fodder beet juice), was obtained at a dilution rate of 1.033 h1.
A semicontinuous (fill-and-draw) process was also developed using the flocculating yeast, S. diastaticus [241].
The yeast is allowed to grow to a high biomass concentration in an agitated bioreactor; this assures good mixing and mass
transfer during fermentation. The agitation is stopped after almost all the sugar has been consumed, and the yeast is allowed
to settle rapidly ( 1 min) in the bioreactor. The clear supernatant, which is the alcohol-containing liquid broth, is decanted and
sent to distillation. Next, fresh medium is added to the bioreactor, which starts a new fermentation batch. These cycles can
be repeated (ten times or more) without loss in productivity and cell viability. High ethanol productivity is achieved with a very
short fermentation time (Fig. 15).
Tower Reactor. The tower reactor is convenient for internal settling of flocculating yeast cells [242]. It consists of a vertical
cylindrical tower with a conical bottom (Fig. 16). Above the cylindrical section is a large, baffled settling zone (d), which has
the form of a yeast separator. The separator can have various forms and provides a space free from rising gas, which
enables yeast cells to settle and be returned to the main part of the reactor. An overall aspect ratio of 10 : 1, with an aspect of
6 : 1 on the cylindrical section, is used. The reactor does not have a mechanical agitator. High cell concentrations in excess
of 100 g/L are achieved without any auxiliary mechanical separator. Compared to simple batch processes, productivities are
32 80 times higher in the tower reactor [111], [239], [240]. Hydraulic retention time is less than 0.4 h, and conversion
efficiency is up to 95 % of theoretical [240].
Figure 15. Semicontinuous fermentation of fodder beet juice with Saccharomyces diastaticus [241]
a) Yeast ; b) Ethanol ; c) Sugar
page 24 of 57
Figure 16. Tower fermentor (APV Co.) [111]
a) Flocculating yeast plug; b) Baffles; c) Attemperator jackets; d) Yeast settling zone; e) Clarifying tube
Flocculation and sedimentation characteristics remain unchanged over several months of continuous operation. Selective
yeast retention is maintained even after moderate contamination, and any infecting organisms can be quickly washed out
without any subsequent effect on reactor performance. Cell viability of 90 95 % is attainable at biomass concentrations in
excess of 100 g/L and remains at this level when the substrate is limited. The viability drops to 70 80 % when the ethanol
concentration is allowed to exceed 75 85 g/L [240].
A major disadvantage of this type of reactor is the long time required (ca. two weeks) for the initial start-up.
Membrane Bioreactor. Buildup of a high yeast concentration can be achieved in a simple continuous dialysis fermentor. The
reactor has fermentation and nutrient zones to which fresh medium is fed and from which beer is removed. These two zones
are separated by a membrane, which prevents biomass escaping from the fermentation zone and allows transfer of nutrients.
At the same time ethanol passes through the membrane to the nutrient zone where it is recovered in an overflow [111], [243].
The rate of ethanol production in this bioreactor is limited by the rate of nutrient diffusion across the membrane. This problem
can be overcome by using a pressure dialysis reactor (Fig. 17) in which a pressure differential is applied across the
membrane (c) to force a flow of medium through the fermentation zone [111].
Figure 17. Pressure dialysis fermentor [111]
a) Low-pressure product recovery zone; b) High-pressure fermentation zone; c) Permeable dialysis membrane; d) High-
pressure feed pump; e) Pressure-regulating valve
Membrane fouling by proteins, which results from cell lysis and can affect flow, is a problem with this type of bioreactor. The
problem can be alleviated by replacing the fixed membrane with a rotating microporous membrane cylinder (rotofermentor)
enclosed in a stationary fermentor vessel. The rotofermentor allows continuous removal of metabolic products (including
ethanol) in the beer by filtration through the rotating membrane under a pressure of 115 170 kPa (Fig. 18). The cells are
retained in the annular part of the reactor outside the membrane [244]. Large molecules that tend to accumulate on the
membrane surface are sent back into the annular zone by the centrifugal force generated by rapid rotation of the membrane.
This cleans the membrane and allows diffusion through it. In the rotofermentor, the ethanol production rate reaches
26.8 g L1 h1 at concentrations of 24.8 and 50.4 g/L of yeast cells and ethanol, respectively [245]. The reactor has several
serious mechanical and operating disadvantages related to the complexity of the system and durability of certain parts of the
reactor (membrane and seals).
Figure 18. Schematic diagram of the rotofermentor assembly [244]
a) Rotor; b) Filtrate chamber; c) Fermentor; d) Rotating microporous membrane; e) Concentrated cell growth; f) Motor drive;
g) Recycle pump; h) Gas liquid separator
Membrane bioreactors in which the fermentation vessel is coupled in a semi-closed loop configuration to a membrane
filtration unit are available [246], [247]. The fermentation broth is pumped through the filtration unit. Inhibitory products
permeate through the membrane, while retained yeast cells are recycled to the fermentor. High cell concentrations (up to
100 g/L) and ethanol production rates (up to 100 g L1 h1) are achieved [246].
Vacuum Bioreactor. Because high concentrations of ethanol are toxic to yeast, a vacuum process for continuous removal of
ethanol from the beer has been developed [233], [248].
The bioreactor operates at a total pressure of 6.7 kPa. At that pressure, water and ethanol boil at a temperature that is
compatible with the yeast. The liquid level in the bioreactor remains constant during operation of the bioreactor. An ethanol
production rate of 82 g L1 h1 is achieved with a yeast density of 120 g/L. In the reactor, the ethanol concentration is kept at
35 g/L, while the ethanol concentration in the vapor product is > 200 g/L. Oxygen is sparged instead of air in order to satisfy
page 25 of 57
vacuum operation requirements [249]. Oxygen sparging can be omitted if the medium is supplemented with sterol and fatty
acids [248].
The compressors used in the process must be large and must operate at low pressure. Capital cost of the compressors is
quite high. Energy requirements exceed those of the normal ethanol fermentation process [228]. The probability of
contamination is increased in this process.
A modification of the process exists (Fig. 19) in which the reactor operates at atmospheric pressure and CO2 is released
without compression from the reactor [249]. Beer is circulated continuously through a flash vessel (d) where ethanol water
solution is stripped under vacuum.
Figure 19. Continuous flash fermentation [111]
a) Atmospheric pressure fermentor; b) Flow-regulating valve; c) Vacuum compressor; d) Flash vessel; e) High-compression
pump
Bioreactor with Solvent Extraction. The concentration of ethanol in beer can be reduced during fermentation by solvent
extraction. This reduces inhibition caused by the product, increases productivity of the reactor, and allows a higher
concentration of sugar to be fermented in a shorter period.
In some cases, after separation of the cells, the clear beer is treated with an immiscible solvent that removes most of the
ethanol. The resulting beer containing only small amounts of ethanol is returned to the reactor, and the saturated solution
undergoes ethanol separation [250].
In another system, both extractant and nutrients can be continuously fed to a reactor that contains immobilized cells. The
ethanol produced is extracted, which reduces its concentration in the aqueous phase, and is later recovered from the
extractant [251]. By using the technique of ethanol extraction from beer with a mixture of n-dodecanol (60 %) and n-
tetradecanol (40 %), the ethanol production is five times greater than in the same system without extraction. A solution of
407 g/L of glucose can be totally fermented by a yeast that cannot normally transform more than 200 g/L glucose [251].
The extractant used in this technology must have the following properties [252]:
1. it must be nontoxic to yeast;

2. it should be selective for ethanol in comparison with water and secondary fermentation products;
3. it must have a high distribution coefficient for ethanol; and
4. it should not form emulsions with fermentation broth.
Further development of this extractive technology is necessary to avoid inhibitory effects of ethanol and attain high rates of
ethanol production. If a good extractant is found, the cost of ethanol separation by means of a membrane technique will be
considerably decreased compared to the classic distillation of ethanol from beer.
Immobilized Cells. A high yeast concentration (5.41010 cells per milliliter) [253] in the fermentor can be obtained by various
cell immobilization techniques, e.g., by entrapment in a gel matrix, covalent binding to surfaces of various support materials,
or adsorption on a support. These systems do not require agitation. The immobilized cells are retained in the reactor;
therefore, cell separation devices and recycle are not needed. High dilution rates without cell washout can be achieved.
Immobilized cells can be used in fixed- and fluidized-bed reactors. In these cases, the substrate solution flows continuously
through the reactor, and the immobilized cells convert available sugar to ethanol. Calcium alginate [9005-35-0] can be used
to entrap yeast cells. In a system with calcium alginate beads, a maximum ethanol productivity of 53.8 g/L was achieved at a
dilution rate of 4.6 h1 and an initial glucose concentration of 127 g/L [254].
Carrageenan [9000-07-1] can also be used to immobilize yeast cells, giving ethanol production rates of 43 g L1 h1 at a
dilution rate of 1.0 h1 and sugar utilization of 86 % from the feed containing 100 g/L glucose [255].
Table 14 shows the ethanol production rates of Saccharomyces cerevisiae and Kluyveromyces fragilis cells immobilized by
entrapment, covalent binding, and adsorption.
Table 14. Ethanol productivity from immobilized systems (Saccharomyces cerevisiae and Kluyveromyces fragilis)
System Feed Feed sugar Feed Dilution Maximum Reference

sugar concentration, sugar rate, h1 ethanol
g/L used, productivity,
% g L1 h1
S. cerevisiae glucose 100 86 1.0 43 [255]

carrageenan
S. cerevisiae glucose 127 63 4.6 53.8 [254]
calcium alginate
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S. cerevisiae molasses 175 83 0.3 21.3 [256]
calcium alginate
S. cerevisiae molasses 197 74 0.35 25 [257]
carrier A.
S. cerevisiae glucose 150 99 43 [258]
glass beads
coated with
gelatin and
glutaraldehyde
S. cerevisiae glucose 120 94 53.1 [260]
anionic
exchange resin
XE-352
K. fragilis lactose 109 99 50.6 [259]
ceramic Intalox
saddles coated
with gelatin and
glutaraldehyde
(two fixed-bed
reactors)
A comparison between batch and continuous (free and immobilized) processes for ethanol production is shown in Table 15.
The ethanol production rate in an immobilized cell system had a lower optimum temperature (30 C) than a system with free
cells (37 C) [254]. The downshift of the temperature optimum was possible because of the effect of ethanol inhibition (which
increases with increased temperature) within the gel matrix.
Table 15. Ethanol productivity in free and immobilized systems (with Saccharomyces cerevisiae) [261]
Process Substrate Volumetric ethanol

production rate,
g L1h1
Batch molasses 2.0 *

Continuous (free cell) molasses 3.35
Continuous (immobilized)
molasses 28.6
* Excluding downtime of fermentor.
A diffusion limitation is imposed by the gel matrix, which produces a substrate and product gradient within the bead.
Therefore, the immobilized system should be operated at lower temperature than the free cell system to minimize inhibition
and maximize ethanol production.
The inhibition caused by high substrate concentration is excluded if the rate of sugar conversion to ethanol is higher or equal
to the rate of sugar diffusion into the matrix.
A difference between the optimum pH values for the production of ethanol by free and immobilized cells was also found.
Systems with free Saccharomyces cerevisiae cells have an optimum pH of 4.5, but the immobilized system showed a broad
optimum spectrum with a maximum activity between pH 3.0 and 7.5. The reason for this difference is a pH gradient within the
beads [254], [262].
5.4. Raw Materials and Processes

Raw materials for the production of ethanol by fermentation can be classified as:
1. readily fermentable carbohydrates that can be used directly, and

2. starch and other organic materials that must be converted to a fermentable form prior to fermentation.
The raw materials come from three major sources:
1. agricultural crops,
2. forest products, and
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3. industrial and agricultural byproducts and residues.
Depending on need, end use, and availability, the choice of raw material varies for different regions, countries, and
industries.
5.4.1. Readily Fermentable Carbohydrates

Various sugar crops, such as sugarcane, sugar and fodder beet, fruit crops, and crops based on crassulacean acid
metabolism (CAM), are in this category.
Sugarcane. Sucrose [57-50-1] ( -D-glucopyranosyl- -D-fructofuranoside) is the sugar obtained from cane or beet (
Sugar). Sugarcane is a tropical crop whose successful cultivation is limited to an area spanning 37 N to 31 S.
Although sugarcane is grown mainly for production of table sugar and molasses, it is also an excellent raw material for the
production of ethanol. The fermentable carbohydrates from sugarcane can be used either as the cane juice directly or as
blackstrap molasses (a sugar byproduct). A material balance shows that 160 kg of fermentable solids can be obtained from
1 t of cane [263].
The cane juice is prepared by crushing raw cane and extracting the sugar with water, followed by clarification using milk of
lime and H2SO4 to precipitate the inorganic materials [264]. The resulting extract is a green, sticky fluid, slightly more viscous
than water, with an average sucrose content of 12 13 % [265].
Blackstrap molasses is the residue remaining after sucrose has been crystallized from cane juice. Molasses is a heavy
viscous material, which contains sucrose, fructose, and glucose (invert sugar) at a total concentration of ca. 50 60 %
(wt/vol) [266]. In contrast to cane juice, molasses is stable on storage and is usually diluted to the desired concentration just
prior to fermentation.
A typical process for the production of ethanol from sugarcane is depicted in Figure 20. Production data are listed in
Table 16. Ethanol production reaches a maximum after 14 20 h and then decreases until ca. 95 % of the available sugar is
consumed. The process is usually batchwise, but some semicontinuous [232] and continuous operations [242], [267-269] are
also used.
Table 16. Yields in production of ethanol from sugarcane [267]
Alcohol, indirectly Alcohol, directly from

from molasses sugarcane juice
Sugarcane yield in 1.5 2-year cycle 63 63

(south-central region), t/ha *
Average sucrose yield (13.2 wt %), t/ha 8.32 8.32
Crystal sugar production, t/ha 7.0
Final molasses or cane juice production, 2.21 66.2
t/ha
Fermentable sugar, molasses, or juice, t/ha 1.32 8.73
Alcohol yield at 100 % global efficiency, 675 4460
kg/ha
Alcohol yield at reasonable 85 % global 11.5 75
efficiency, L per ton of cane
or in L/ha 730 4800
* Hectare (ha) = 104 m2.
Figure 20. Typical process for the production of ethanol from sugarcane [267]
In the batch process, several fermentors are usually operating at staggered intervals to provide a continuous feed to the
distillation columns. Overall productivity is ca. 18 25 kg of ethanol per cubic meter of fermentor volume per hour [225]. The
Melle Boinot process is used in most Brazilian distilleries (see Section Batch Processes).
Ethanol has been produced in a continuous process (using continuously stirred tank reactors) from molasses by Danish
Distilleries [268]. The process is shown in Figure 21, and performance data are given in Table 17.
Table 17. Performance data for the Danish Distilleries process * [268]
page 28 of 57
Fermentor 1 (f1) ** Fermentor 2 ( f2) **
Amount of yeast and dry

matter per liter, g 10 10
pH 4.7 4.8
Alcohol, vol % 6.1 8.4
Residual sugar, % 1.0 0.1
Temperature, C 35 35
* Residence time in each fermentor: 10.5 h; influx: 600 kg of molasses diluted in 22103 L/h.
** See Figure 21.
Figure 21. Continuous production of ethanol by Danish Distilleries [268]
a) Storage tank; b) Intermediate container; c) Metering pump; d) Regeneration section; e) Plate heat exchanger;
f) Fermentor; g) Yeast separator
According to this process, the molasses is stored in two or three 1500-m3 tanks from which it is pumped to intermediate
containers. The material is adjusted for pH and nutrients (nitrogen and phosphorus), sterilized at 100 C by using plate heat
exchangers, and then introduced to three fermentors with a total volume of 170 m3. The fermented wort is centrifuged after
fermentation, and the live yeast returned to the first fermentor. At the start, sufficient yeast propagation must be
accomplished by aeration (0.02 0.03 L of air per liter of liquid per minute). The yield is ca. 28.29 L of alcohol per 100 kg of
molasses, or a maximum of ca. 65 L of alcohol per 100 kg of fermentable sugar.
A continuous process for production of beer from sugar by use of tower fermentors is shown in Figure 22 [270], [271].
Figure 22. Commercial ethanol tower fermentation system (APV Company) [242]
a) Buffer storage tank; b) Divert line; c) Pasteurizer; d) Flow controller; e) Vertical cylindrical tower; f) Chiller; g) Yeast green
beer buffer storage; h) Centrifuge; i) Separator
The key to the process is a vertical cylindrical tower fermentor with a conical bottom. A baffled yeast settling zone constitutes
the upper part of the fermentor. The fermentor uses a flocculent yeast, which is pumped into the base of the tower. As the
reaction proceeds, the beer rises, and the flocculating yeast settles and is retained in the reactor. High cell densities of 50
60 g/L are achieved without the use of mechanical cell concentration or separation devices. Short residence time (<4 h) with
a sugar concentration of up to 12 % (wt/vol) sucrose, 90 % sugar utilization, and 90 % conversion to ethanol, produce up to
5 % ethanol in the final broth. The overall productivity of this system can be up to 80 times higher than that of the simple
batch system.
Sugar Beet. Like sugarcane, sugar beet produces carbohydrates that consist primarily of sucrose ( Sugar). Sugar beet is
a more versatile crop than sugarcane. It can tolerate a wide range of soil and climatic conditions, and is grown throughout
nearly half of the United States, Europe, Africa, Australia, and New Zealand.
In addition to sucrose, sugar beet contains sufficient nitrogen and other organic and microorganic nutrients [272] so that little,
if any, fortification is required prior to fermentation. Another benefit is the high yield of coproducts such as beet tops and
extracted pulp. The pulp has a high feed value, and the tops may be returned to the soil for erosion control and nutrient
replacement. The yield of fodder beets is high (ca. 50 150 t/ha); their composition is described in [273].
A new fodder beet crop, produced in New Zealand through a genetic cross between sugar beets and marigolds, gives
greater yields of fermentable carbohydrates per hectare than does sugar beet [274]. In addition, the sugar from fodder beet is
reported to be more resistant to degradation over long storage.
Processes for the production of alcohol from sugar and fodder beets are basically the same as from sugarcane.
Fruit Crops. Many crops (grapes, plums, peaches, apricots, pineapples, etc.) contain variable proportions of sugars (sucrose
plus fructose, usually 6 12 %). The fruit sugars can be readily fermented to alcohol, and this is done on a large scale for
production of alcoholic beverages. The alcohol content of the product, which basically is the liquid after fermentation,
separation of yeast, further treatment, and aging, depends on use and fermentation conditions. Table wines have <14 %
alcohol, whereas wines with >14 % alcohol fall in the category of desert wines and aperitifs ( ). Higher concentrations of
alcohol are achieved by means of distillation to produce strong alcoholic beverages (e.g., brandy, whiskey, gin, vodka) (
).
Alcohol for industrial or fuel use is seldom produced from fruit and vegetable crops. However, some fruit from tropical and
page 29 of 57
semiarid climates, such as dates [275], mohwa flowers [276], and rain tree fruit [277], have been investigated for fuel alcohol
production.
Crops Based on Crassulacean Acid Metabolism. Interest in using the agriculturally semi- or nonproductive regions of the
world to grow alcohol-producing crops has increased [278]. These regions could be used to grow plants that utilize
crassulacean acid metabolism (CAM) because their photosynthetic metabolism is extremely efficient with respect to irrigation
requirements. These plants exhibit above-average productivity (expressed as a function of biomass production per unit of
existing biomass) compared to other agricultural crops.
The CAM plants that are high in fermentable carbohydrates include various cacti (e.g., Opuntia sp.) and other plants such as
Euphorbia lathyrus and Agave sp. Few data are available on potential ethanol production from these crops and its economic
feasibility; however, an estimated 50 t/ha of these crops could be produced annually in subagricultural areas [279].
5.4.2. Starch
A variety of starch materials, such as grains, cassava, sweet potatoes, sweet sorghum, and Jerusalem artichoke, can be
used for fermentation to ethanol ( Starch). Selection depends on various factors, the major ones being climate and
availability for large-scale production. Corn, wheat, potatoes, and Jerusalem artichokes are the most common raw materials
in Europe and North America, whereas rice, cassava, sweet potato, and sweet sorghum are important in tropical countries.
Corn. Corn is the preferred raw material for conversion to alcohol in the United States and parts of Europe. It is available in
large quantity, and its price (especially for low-grade or distressed corn) is thus acceptable for conversion to ethanol.
Conversion to ethanol is efficient, and byproducts, such as corncobs, stalks, and leaves, are valuable as animal feed, energy
source, or fertilizer. About 66 % of corn production is used for food and feed, and ca. 5 % is used to make alcohol (
Cereals Maize (Corn)).
A number of batch and continuous processes have been developed for production of ethanol from corn. A conventional
fermentation plant producing 76103 m3 of anhydrous ethanol per year from 816.5103 kg of corn per day is shown in Figure
23 .
Figure 23. Flow diagram for a conventional fermentation plant producing anhydrous ethanol from corn [280]
In this process, corn is ground and cooked to dissolve and gelatinize the starch. The enzymes -amylase and glucoamylase
are then added to hydrolyze the starch to fermentable monosaccharides. After yeast fermentation for ca. 48 h at 32 C, about
90 % of the starch is converted to ethanol. The fermentation broth is fed to the beer still where alcohol (ca. 50 vol %) is
distilled. Subsequent distillation produces 95 % alcohol, which can be further concentrated by azeotropic distillation using
benzene. After centrifugation, the stillage is concentrated to ca. 50 % solids in a multiple-effect evaporator, further
concentrated in a fluidized-bed, transport-type dryer to ca. 10 % moisture, and then used as such for animal feed. This feed
contains all the protein originally present in the grain, plus the additional protein from the yeast, resulting in a product
containing 28 36 wt % protein.
In addition to alcohol and cattle feed, the original 816.5103 kg of corn yields 175103 kg of CO2 and 95 kg of byproduct
aldehydes, ketones, and fusel oils.
Alltech developed a method for an integrated grain-processing fermentation route [281]. The grain pretreatment step prior
to fermentation is shown in Figure 24.
Figure 24. Alltech process for continuous whole mash cooking [281]
a) Grain hopper; b) Screen; c) Magnets; d) Continuous weigher; e) Hammer mill; f) Slurry vessel with agitator, temperature
50 70 C; g) Rupture disk; h) Expansion vessel; i) Positive displacement pump; j) Continuous cooker tube, residence time
5 min, temperature variable up to 150 C; k) Pressure valve; l) Flash vacuum cooler to 66 76 C; m) Condenser; n) Open
impeller pump; o) Converter, residence time 20 min, agitator 1 rpm; p) Wort cooler
Two enzymes, alcoholase I (from Bacillus subtilis) and alcoholase II (from Aspergillus niger and Rhizopus niveus) are used to
hydrolyze the starch to fermentable sugars. Continuous whole mash cooking is applied. The ground starch is first mixed with
water and alcoholase I at 60 C, and then cooked at 93 165 C in a batch or continuous cooker. The cooked mash is then
cooled to ca. 66 76 C, and a second portion of alcoholase I is added; 20 min is allowed for conversion. After this first
hydrolysis step, the temperature is adjusted to 32 C and the mash, supplemented with alcoholase II, is fermented with yeast.
Cassava. Cassava, also known as manioc, mandioc, aipum, yuca, cassada, and tapioca, is second in importance only to the
sweet potato as a root crop throughout the tropics and in parts of South America where the plant originated. It was taken to
West Africa by the Portuguese around 1914, where it now seems to have replaced yams and cocoyams because it adapts
page 30 of 57
easily and requires less labor than other crops. Cassava is one of the highest yielding plants of the vegetable kingdom (10
30 t/ha ); it requires little cultivation and the tubers can be left in the ground until required without serious deterioration.
Cassava (genus Manihot) is in the family Euphorbiaceae, which belongs to the subdivision Angiospermae, class
Dicotyledoneae, order Geraniales. This large, widely spread family comprises 283 genera including 7300 species, with an
almost worldwide distribution [282].
Manihot esculenta, M. utilissima, and M. dulcis are some economically important members of a genus which includes over
150 species that are distributed throughout tropical countries. The species include herbs, shrubs, and trees, many of them
producing latex and some yielding rubber. Brazil, Indonesia, and Zaire are the largest producers of cassava.
Roots are generally of interest for alcohol production. They contain 20 35 wt % starch and 1 2 wt % protein, although
strains with up to 38 % starch have been developed [283]. The advantages of cassava for fuel ethanol production (which can
amount to 7600 L/ha) have been assessed [267], [284], [285]. The process used to obtain ethanol from cassava is shown in
Figure 25.
Figure 25. Production of ethanol from cassava root [267]
Fresh roots are washed, peeled, and ground into a mash. Part of this mash is dried; it can be stored in this form up to a year
and is used for animal feed. For fermentation to ethanol, the starch is hydrolyzed with -amylase, which is added in two
steps. The first addition decreases the vicosity of the mash and facilitates cooking. In the second addition, the enzyme
completes liquefaction of the starch. After that glucoamylase is added, which converts the liquefied starch to glucose and
prepares the mash for fermentation. The fermentation process is the same as the one used for production of alcohol from
sugarcane.
Alcohol yield from cassava is 165 180 L/t, which, on a mass basis, is higher than that obtained from sugarcane [285].
However, because sugarcane production can be as high as 90 t/ha, the alcohol yield per unit area is greater from cane under
present cultivation conditions. Another advantage of cane is its dry fiber content, which equals the amount of total sugar
present. This amount of fiber (bagasse) is sufficient to maintain the energy requirements of the plant; this is not the case with
cassava, which only contains ca. 3.5 % fiber. Another disadvantage of cassava is that it does not contain readily fermentable
sugars and, therefore, requires considerable processing of the roots prior to fermentation.
Sweet Sorghum. Sweet sorghum (Sorghum sacchartum) contains both starch and sugar. Its yield of ethanol from
fermentable sugars is ca. 3500 4000 L/ha; an additional 1600 1900 L/ha can be produced from stalk fibers. There are
more than 17 000 varieties of sorghum, and the yield is anticipated to increase by 30 % with some new hybrids [286]. The
plant is adaptable to most of the world's agricultural regions; it is resistant to drought, and its nutrients are efficiently utilized
by animals.
The fermentable sugars and starches are treated conventionally for ethanol production. The free sugars are fermented
directly, whereas the starches are hydrolyzed by use of amylases, as is the case with cassava.
Potato. The potato is a common starch crop worldwide ( Starch). The potato originated in South America (Chile and
Peru) and came to Europe through Spain at the end of the 16th century. It is now grown in almost all climates and almost all
types of soil, including dry and sandy soil [287].
Starch is the main carbohydrate component of potato (ca. 68 80 %). Depending on cultivation and variety of potato, starch
content can vary between 12 and 21 % in raw potatoes. Only small quantities of soluble sugars are present (0.07 1.5 %
sucrose, glucose, and fructose), as well as some rubber and dextrins (0.2 1.6 %) and pentosans (0.75 1.00 %).
The production of ethanol is based on fermentation of the available starch. A process developed by Danish Distilleries is
shown in Figure 26 [268]. The process is semicontinuous and is applicable to both potatoes and grain.
Figure 26. Danish Distilleries semicontinuous production of alcohol from potatoes or grain [268]
a) Preheater; b) Pulper; c) Enzyme treatment vessel; d) Flash cooler; e) Boiler tube; f) Holding tank; g) Condenser; h)
Liquefaction vessel
Potatoes are mashed and then treated with amylases to hydrolyze the starch. The treatment section involves rapid steam
treatment at 150 C for ca. 3 min. The mash is cooled to 70 C for liquefaction with commercial amylase preparations of
bacterial origin; it is then cooled further to 30 C and used for alcohol fermentation in the customary manner.
Jerusalem Artichoke. The Jerusalem artichoke (Helianthus tuberosus) is a member of the Compositae family and is closely
related to the sunflower (Helianthus annus), earning it the nickname wild sunflower. About 102 different names are
synonymous with the name H. tuberosus.
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The plant is native in North America. It was originally grown by the Cree and Huron Indians who called it askipaw and
skibwan, respectively. The plant was introduced to Europe at the beginning of the 16th century where it rapidly spread
through the Mediterranean countries. The addition of Jerusalem to the name is most likely the result of an English version
of Girasole, the Italian name for this plant [288].
The plant grows 1.5 2.5 m tall for wild strains and up to 3.7 m under cultivation. Top growth accounts for 40 56 % of the
total plant biomass. The tubers are of greatest interest as a raw material for fermentation to ethanol.
The main soluble carbohydrate in the Jerusalem artichoke is inulin, which is composed of a homologous series of
polyfructofuranose units. These units consist of linear chains of D-fructose molecules joined by -2,1-linkages. The chains
are terminated by a D-glucose molecule linked to fructose by an -1,2-bond as in sucrose [289].
A process to produce 360103 kg/a of ethanol from Jerusalem artichoke tubers is presented in Figure 27 [290].
Figure 27. Production of ethanol from Jerusalem artichoke tubers [290]
In this process, the juice is expressed from the tubers and extracted with water to obtain a carbohydrate concentration of
about 20 %. The carbohydrates (predominantly inulin) are then hydrolyzed enzymatically by activating the endogenous
inulinases at ca. 50 60 C; acid hydrolysis (pH ca. 1) of inulin is also effective. The resulting fermentable sugars are then
converted to ethanol by a conventional route.
Novel routes for conversion of the juice to ethanol have also been explored; the flocculating yeast Saccharomyces
diastaticus has been used in semicontinous and continuous modes [234].
5.4.3. Lignocellulosic Materials

Lignocellulose is the largest terrestrial source of biomass that is renewably produced through photosynthesis ( Biomass
Chemicals; Cellulose Cellulose; Lignin). The solar energy reaching the earth surface is 3.671021 kJ/a [291]. Gobal
photosynthesis (with an efficiency of 0.07 %) could convert 2.571018 kJ of that energy to cellulose-containing biomass. This
would result in a net production of 1.81011 t/a of biodegradable material, 40 % of which is cellulose [292]. Estimates are that
1 1.251011 t/a of terrestrial dry mass is produced together with 0.44 0.551011 t/a in the oceans [293]. Present removal
of this potential energy source is ca. 0.5 % of the total growing stock on a global basis [294].
The fermentation potential of lignocellulose is based mainly on the cellulose content of the biomass. Chemically, cellulose is
similar to starch. It is a polymer of glucose in which the glucose units are linked by -1,4-glucosidic bonds, whereas the
bonds in starch are predominantly -1,4-linkages. The degree of polymerization (DP) varies for different sources of cellulose;
for example, newsprint cellulose has a DP of 1000, whereas cotton has a DP of ca. 10 000 [295].
The cellulose molecule is more resistant to hydrolysis compared to starch. This resistance is due not only to the primary
structure based on glucosidic bonds, but also, to a great extent, to the secondary and tertiary configuration of the cellulose
chain, as well as its close association with other protective polymeric structures such as lignin, starch, pectin, hemicellulose,
proteins, and mineral elements.
The lignin molecule seems to be primarily responsible for difficulties in hydrolyzing the lignocellulosic material, because it
forms a protective sheath around the cellulose microfibrils. Lignin is a macromolecule of phenolic character and can be
viewed as a dehydration product of three monomeric alcohols: trans-4-coumaryl alcohol, trans-coniferyl alcohol, and trans-
sinapyl alcohol. The relative amount of each varies with the source [296].
When cotton cellulose is treated with dilute acid, partial hydrolysis occurs rapidly, and ca. 15 % of the cellulose chain is
degraded to glucose. The remaining 85 % is more resistant to hydrolysis, possibly because this portion of the cellulose exists
in a highly crystalline order [295].
In order to use lignocellulosic materials for fermentation to ethanol, they must be pretreated and then hydrolyzed to
fermentable sugars. Pretreatment may be physical or chemical, e.g., milling, steam explosion, or use of solvents and various
swelling agents.
In steam explosion green wood chips are heated to ca. 180 200 C for 5 30 min in a continuous operation (Stake
process), or to a temperature of 245 C for 0.5 2 min (Iotech process) [297]. The acids formed from hemicellulose under
these high-temperature and high-pressure conditions start to autohydrolyze the cellulose and intact lignin. Lignin is
sufficiently softened at the end of the steaming period, so that when the vessel is suddenly depressurized to atmospheric
pressure, an explosion occurs within the woody cells. This partially disrupts the close association of cellulose with lignin and
consequently increases the surface area available for further hydrolysis. The effect of steam pretreatment on the enzymatic
hydrolysis of various cellulose-containing materials is shown in Table 18.
The pretreated lignocellulosic material is then subjected to further hydrolysis, which can be acidic or enzymatic (
Cellulose Cellulose; Enzymes Cellulases). A comparison of enzymatic and acid hydrolysis for cellulose
degradation is given in Table 19. Cellulose and its degradation products are the only materials considered for fermentative
purposes.
page 32 of 57
Table 18. Effect of steam pretreatment on the enzymatic hydrolysis ** of cellulosic substrates [298]
Substrate Pretreat- Total reducing

ment sugars, mg/mL
4h 24 h
Hardwoods
Poplar none 1.4 2.4
steam 15.3 25.8
Aspen none 1.8 3.0
steam 12.8 24.8
Agriculture residues
Corn stover none 4.9 7.8
steam 15.7 22.5
Sugarcane bagasse none 1.7 2.5
steam 9.5 16.1
Urban waste none 10.5 18.0
steam 6.2 10.8
Softwoods
Eastern spruce none 2.0 3.8
steam 3.5 6.4
Douglas fir none 1.6 3.2
steam 2.8 4.3
* Trichoderma reesei cellulase (QM9414), 19 IU (International Units) per gram of substrate; 5 % substrate slurries, pH
4.8, 50 C; steamed substrates washed prior to enzymatic hydrolysis.
Table 19. Comparison between enzymatic and acid hydrolysis of cellulosic materials [227]
Acid Enzyme
Nonspecific catalyst; therefore, will Specific macromolecular catalyst; therefore, extensive

delignify material as well as hydrolyze physical and chemical pretreatment of material
cellulose necessary to make cellulose available for degradation
Decomposes hemicellulose to inhibitory Produces clear sugar syrup ready for subsequent
compounds (i.e., furfural) anaerobic fermentation
Harsh reaction conditions necessary; Run under mild conditions (50 C, atmospheric pressure,
therefore, increased cost of heat- and pH 4.8)
corrosion-resistant equipment
High chemical cost requires catalyst Cost of producing cellulases is the most expensive
recovery and reuse process step; therefore, recycle is necessary
High rate of hydrolysis Lower rate of hydrolysis
Low overall yield of glucose because of High glucose yield depending on system and
degradation pretreatment
An example of a semicontinuous process for ethanol production from wood is shown schematically in Figure 28; this process
uses dilute sulfuric acid for cellulose prehydrolysis.
Figure 28. Ethanol production from wood [299]
Optimum conditions for this process are: acid concentration in total water, 0.53 %; maximum temperature of percolation,
page 33 of 57
196 C; rate of temperature rise, 4 C/min; percolation time, 145 190 min; ratio of total water to oven-dried wood, 10;
percolation rate, 8.69 14.44 L min1 m3
a) Digester; b) Flash evaporator; c) Furfural tower; d) Neutralization vessel; e) Clarifier; f) Fermentor; g) Yeast separator; h)
Alcohol stripper; i) Extraction tower; j) Rectifying tower; k) Evaporator; l) Vapor compressor
The hydrolysate percolates through a bed of wood chips. Optimum conditions for the process are described in [299]. After
acid digestion (a), the effluent passes through a flash evaporator (b), which separates the vapors containing furfural and
methanol from the underflow condensate containing the sugar solution. The acid hydrolysate solution is further neutralized
with a lime slurry, and the precipitated calcium sulfate is separated in a clarifier (d) as a 50 % solids sludge. The neutralized
liquor is blended with recovered yeast (Saccharomyces cerevisiae) from previous fermention and is fermented to ethanol (e),
which is further concentrated to 95 % by distillation (i).
The bottom material from the alcohol stripping column (g), which contains pentose sugars, is further concentrated in multiple-
effect evaporators (j) to a 65 % solution, that can be used as animal feed or for chemical conversion to furfural.
Figure 29 shows a strong acid hydrolysis process.
Figure 29. Ethanol production from wood by use of strong acid hydrolysis [300]
a) Feed hopper; b) Feeder; c) Digester; d) Neutralization vessel; e) Multiple-effect evaporators; f) Dryer; g) Electrodialysis
membrane; h) Filter; i) Fermentor; j) Carbon dioxide scrubber; k) Seed fermentor; l) Centrifuge; m) Yeast wash vessel;
n) Surge vessel; o) Beer still; p) Alcohol column
The air-dry wood is first pretreated with dilute sulfuric acid (c). Complete hydrolysis is accomplished in a subsequent strong
acid cycle in which cellulose is hydrolyzed at room temperature with 70 80 % H2SO4. The glucose, retained by the dialysis
membrane (g), is neutralized, deionized, and then sent to fermentation (i). The sulfuric acid permeate from the dialysis unit is
evaporated and reconcentrated for recycle. Lignin is separated from the concentrated acid by filtration (h) and washing.
To illustrate enzymatic hydrolysis of cellulose for alcohol production, a process used by the Natick Development Center is
shown in Figure 30. A large part of this process involves the preparation of the cellulase enzyme. Newspaper is the cellulose-
containing substrate.
Figure 30. Enzymatic hydrolysis of newsprint by Natick Development Center (NDC) [298]
A) Pilot plant process for cellulase production:
a) Production vessel (vertical filters); b) Inoculum vessel; c) Filter; d) Harvest storage; e) Ultrafilter; f) Concentrate
storageB) Pilot-plant process for newspaper hydrolysis: a) Ball mill; b) Solids metering; c) Solids transfer; d) Bioreactor;
e) Enzyme storage; f) Metering pump; g) Harvest pump; h) Crude filter; i) Polish filter; j) Evaporator
5.4.4. Waste Materials and Residues

Various types of agricultural, industrial, or municipal refuse and waste can be used as substrates for ethanol fermentation.
The fermentation is based on available sugar, starch, or cellulose in the waste material. The major advantage of this route
lies in coupling waste treatment with the production of a higher value product. Both environmental pollution abatement and
process economics are thus improved.
Cornstalks. Cornstalks are available in large quantities as a byproduct of corn agriculture. This material is predominantly
composed of lignocellulose. A two-stage process using dilute acid treatment followed by concentrated acid impregnation of
the lignocellulosic material is shown in Figure 31.
Figure 31. Production of ethanol from cornstalks [301]
A) Acid hydrolysis: a) Prehydrolysis tank, 4.4 % H2SO4; b) Filter; c) Rotary dryer; d) Impregnator; e) Hydrolysis tank, 8.0 %
H2SO4; f) Filter; g) Electrodialysis unit
B) Fermentation: A) Acid hydrolysis process; h) Fixed film of Fusarium oxysporum; i) Centrifuge; j) Fixed film of
Saccharomyces cerevisiae; k) Distillation column
In this process, ground corn stover (841 nm, 20 mesh) is treated with 4.4 % H2SO4 at 100 C for 50 min (a). The mixture is
then filtered (b) and the xylose-rich liquid is processed by electrodialysis (g) for acid recovery. The solids are dried further (c)
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and impregnated with 85 % H2SO4 (d), followed by dilution with water to give a H2SO4 concentration of 8 % (e). Subsequent
hydrolysis is carried out at 110 C for ca. 10 min, and acid is again recovered by electrodialysis. The combined yield of xylose
is 94 %, and the yield of glucose is 89 %. Glucose is converted to ethanol by Saccharomyces cerevisiae (j) and xylose by
Fusarium oxysporum (h), both in immobilized cell reactors. The overall annual capacity of the plant is 17103 m3.
Domestic Refuse. Domestic refuse contains a complex variety of materials that come mainly from cellulosic-type residues. A
large quantity of this material, ca. 1.3 2.2 kg per person, is generated daily. A process for the production of 36.5 t/d of
ethanol from domestic refuse is illustrated in Figure 32 [302].
Figure 32. Flow diagram for continuous production of ethanol from refuse with 60 % content of cellulose [302]
* Biochemical oxygen demand.
The refuse is separated into dense and light fractions by the use of a flotation separator or a special pulper. The pulped
fraction, which contains cellulose, is first subjected to removal of fines and plastics, and then introduced into a reactor where
it is hydrolyzed with 0.4 % H2SO4 for ca. 1.2 min at 230 C. This process is followed by flash cooling, neutralization with
CaCO3, and filtration. Fermentation of the sugar solution takes ca. 20 h at 40 C and yields ca. 1.7 % aqueous ethanol
solution, which is further concentrated by distillation to ca. 95 % ethanol.
Waste Liquor from the Pulp and Paper Industry. Two chemical pulping methods are predominant in the pulp and paper
industry: the sulfate (Kraft) and the sulfite processes ( Paper and Pulp). The basis of the these operations is treatment of
the lignocellulosic material (wood, straw, etc.) with highly concentrated acid or base, which should dissolve the lignin portion
of the wood and leave cellulose fibers that are processed into the final paper product. Depending on conditions (temperature,
pressure, concentration of chemicals, chemical to wood ratio, and time of digestion), more or less delignification and
breakdown of the original cellulose occur. As a result, a product pulp is produced as well as a waste chemical liquor, which
basically consists of spent cooking chemicals. The more drastic the delignification conditions (low yield process), the better is
the quality of the paper obtained. The high-yield process refers to milder delignification and a pulp that still contains a
considerable amount of lignin. Low-yield processes are characterized by waste liquors with a high concentration of chemicals
and a higher organic content.
The organic content of these liquors is primarily sulfonated lignin (e.g., 43 % of organic dry substance in a spent spruce
sulfite liquor). However, cellulose and hemicellulose are also partially hydrolyzed during digestion so that waste liquors
contain a certain proportion of free sugars (hexoses and pentoses in ca. 2 4 % concentration and ca. 14 % total solids)
[303].
Tremendous quantities of waste liquor are generated in a pulp and paper mill; they amount to ca. 9180 L per ton of pulp
produced [304]. Consequently, a chemical pulping process with a medium capacity of 500 t/d of pulp produces 4.59106 L/d
of waste liquors. Release of this liquor into natural waters is prohibited because both organic and toxic pollution result.
The sulfate (Kraft) process is designed so that the majority of the waste liquor can be recycled and its organic value
converted to energy by combustion in a specially designed steam boiler (recovery furnace).
Previously, the majority of pulping mills worldwide were sulfite mills. Because of economic and environmental problems,
sulfite pulping is gradually being phased out and the process converted to sulfate pulping or modified in other ways.
Recovery of chemicals in the sulfite process is not as feasible as in the Kraft process, so large quantities of waste sulfite
liquors (WSL) are discharged to the environment.
Because WSL contain fermentable sugars, these liquors have been used efficiently as fermentation substrates for alcohol
production. The process is relatively old (1908 in Sweden) but is still in operation in some mills (e.g., the Ontario Paper
Company, Canada). A typical process for fermentation of WSL is shown schematically in Figure 33.
Figure 33. Production of ethanol from waste sulfite liquors (WSL) [305] *
a) Digester; b) Blowpits; c) Storage; d) Stripper; e) Screen; f) Storage; g) Flash cooler; h) Barometric condenser; i) Ejectors;
j) Fermentor; k) Yeast separator; l) Storage; m) Preheaters; n) Beer still; o) Rectifying column; p) Oil washer; q) Fusel oil;
r) Purifying column; s) Vaporizer; t) Condenser; u) Alcohol; v) Heads
* Reprinted with permission of American Institute of Chemical Engineers.
The waste liquor is first stripped of SO2 with a conventional steam stripper. This is necessary because SO2 would inhibit
subsequent fermentation. The liquor is adjusted to give a ca. 10 12 % concentration of sugars, the pH is adjusted to 4.5,
and nitrogen and phosphorus nutrient sources are added (e.g., urea and phosphate). The fermentation is conventionally
carried out with yeast (Saccharomyces cerevisiae) at 30 C for ca. 20 h. The yeast is usually concentrated and recycled, and
the broth containing ethanol is sent to the distillation section.
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Cheese Whey. Whey is a byproduct of cheese production ( Cheese, Processed Cheese, and Whey Introduction,
Processed Cheese, and Whey). An estimated 74106 t of whey is produced annually worldwide. This amount of whey
contains ca. 0.7106 t of milk protein and 3.2 t of lactose [306].
Whey with its protein, carbohydrate, and vitamin content is a valuable, nutritious material; its composition is described in
[307]. Whey is used in various forms as a component of either animal or human food. However, the utilization and recycling
of whey nutrients depend on many factors, one of which is the size of the cheese factory. The smaller the factory, the less
recycling of whey is practiced.
The alcohol produced from whey is derived mainly from the fermentation of available lactose. However, only a few
microorganisms can convert lactose to ethanol, and conventional yeast (Saccharomyces cerevisiae) is not among them. The
most efficient lactose-utilizing organism is reported to be S. fragilis [308].
A process using Kluyveromyces fragilis yeast was developed in Denmark (Fig. 34) [309-311]. The whey is first concentrated
by reverse osmosis and ultrafiltration, and then introduced into fermentation vessels. The yield based on lactose is about
80 % of theoretical. About 42 L of whey, containing 4.4 % lactose, is required to produce 1 L of absolute ethanol.
Figure 34. Continuous production of ethanol from whey [311]
a) Acid; b) Storage tank; c) Heat exchanger; d) Control; e) Antifoam; f) Chemicals; g) Fermentor; h) Substrate; i) Propagation
plant; j) Storage; k) Separator; l) Buffer tank; m) Distillation; n) Alcohol storage
A better substrate for industrial fermentation of whey is enzymatically hydrolyzed lactose. -Galactosidase-treated whey
yields a mixture of monosaccharides, glucose, and galactose, which can be efficiently fermented by high-alcohol-producing
yeasts [308], [312].
[Top of Page]
6. Recovery and Purification

Otto Goebel
Over the past 20 years a series of distillation systems have been developed for the efficient recovery of ethanol from
synthetic and fermentation feedstocks. These units produce high-grade industrial alcohol, anhydrous alcohol, alcoholic
spirits, and ethanol for motor fuels. Ethanol quality and recovery have been improved; energy consumption has decreased.
Distillation. Synthetic ethanol is purified in a simple three-column distillation unit (see Sections Production Process and
Production Process). Recovery is 98 %, and the high-grade product contains less than 20 mg/kg of total impurities and has a
permanganate time of over 60 min.
The following are key features of the efficient recovery of high-grade ethanol from fermentation feedstocks:
1. Extractive distillation results in a higher degree of purity than is possible in conventional purification columns; both
investment and operating costs are reduced.
2. Pressure-cascading installations and heat pumps permit substantial heat recovery and recycling, thus minimizing heat
loss and steam consumption. Virtually all (95 99 %) the ethanol in the crude feed is recovered as high-grade
product.
3. Advanced control systems ensure stable operating conditions. Product quality can be maintained with a total impurity
content of less than 50 mg/kg and a permanganate time of over 45 min.
4. Energy requirements are minimized. The flash heat recovered from the grain-cooking system is used to heat the
ethanol distillation unit, thus reducing the energy consumption for ethanol production by ca. 10 %. Use of a vapor
recompression technique can reduce the energy required for the evaporation of stillage to as little as one-tenth of that
required in a triple- or quadruple-effect evaporator.
Dehydration. To produce anhydrous ethanol, the water ethanol azeotrope obtained from distillation of the crude synthetic
or fermentation feedstock must be dehydrated. For economic reasons, large distilleries rely mostly on azeotropic distillation
for ethanol dehydration. Benzene has been used as an azeotropic dehydrating (entraining) agent in many plants, but some
concern exists about its carcinogenicity and toxicity ( Benzene Toxicology and Occupational Health). However, proper
design and control minimize benzene loss and exposure of operating personnel to this substance. Cyclohexane and ethylene
glycol are used in some distilleries; they are also effective dehydrating agents.
Some smaller ethanol plants use molecular sieve adsorption techniques to dry the ethanol azeotrope. Pervaporation through
semipermeable membranes or use of a solid dehydrating agent may reduce energy and equipment costs.
Concentration Units. Several systems are used to define ethanol concentration in addition to those based on conventional
units (e.g., weight percent, volume percent).
Proof. In Canada, Great Britain, and the United States, the ethanol concentration of beverage spirits is expressed in terms of
proof. United States law defines proof as follows: Proof spirits shall be held to be that alcoholic liquor which contains one-
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half of its volume of alcohol of a specific gravity of 0.7939 at 15.5 C. More simply, the figure for proof is twice the
percentage of ethanol content by volume. For example, 100 proof means 50 vol % alcohol.
In Great Britain as well as Canada, proof spirit is such as at 10 C weighs exactly twelve-thirteenths of the weight of an
equal bulk of distilled water. A proof of 87.7 therefore signifies an ethanol concentration of 50 vol %. British proof can be
converted into U.S. proof by multiplying by 1.11.
Degree Gay-Lussac. The degree Gay Lussac (GL) is measured with a hydrometer, which reads the volume percentage of
ethanol in a mixture of ethanol and water at 15 C.
Grades of Ethanol. Several grades of ethanol are available.
Industrial ethanol (96.5 vol %) is used for industrial and technical purposes as a solvent and a fuel, and is also converted into
many other products. Industrial ethanol is usually denatured with 0.5 1 wt % of crude pyridine and is sometimes colored
with 5104 wt % methyl violet for easy recognition.
Denatured spirit (88 vol %) is a term used in some countries to describe industrial 88 vol % ethanol, which has been
denatured and colored, and is generally used for heating and lighting.
Fine alcohol (96.0 96.5 vol %) is a purer type of ethanol used mainly for pharmaceutical and cosmetic preparations and for
human consumption.
Absolute or anhydrous ethanol (99.7 99.8 vol %) is the term given to very pure, extremely dry ethanol, which is used in the
food and pharmaceutical industries and also in the manufacture of aerosols. Anhydrous denatured ethanol mixed with 70
80 vol % of gasoline is used as a fuel for internal combustion engines. The specifications of anhydrous ethanol obtained by
azeotropic distillation are given in Table 20.
Table 20. Specifications for anhydrous ethanol (99.6 vol %)
Parameter Specification Typical value Test method
Purity, vol % 99.8 min. >99.9 ETM 240.03 *

0.7911 max. 0.790 ASTM D 268
Acidity, wt % acetic acid 0.002 max. 0.001 ASTM D 1613
Color, Pt Co 10 max. 5 ASTM D 1209
Nonvolatile matter, mg/100 mL 2.0 max. 1.0 ASTM D 1353
Distillation temperature, C
initial 77.5 min. 78.2
dry point 79.0 max. 78.4

Permanganate time, min 25 min. 45 ETM 110.77 *
Residual odor nil nil ASTM D 1296
Appearance clear and free of clear and free of ETM 80.65 *
suspended matter suspended matter
* ETM = Ethanol testing method (CORA Engineering).
Motor fuel ethanol refers to fermentation ethanol that is used as an anhydrous or hydrous fuel or as a blending agent to
improve the octane number of gasoline ( Automotive Fuels).
6.1. Distillation
Ethanol is recovered as an azeotrope from ethanol water mixtures by means of distillation [313], [314]. The boiling point
diagram for this system is shown in Figure 35; the pure water azeotrope at the azeotropic point (a) contains 95.57 wt %
(97.3 vol %) ethanol and has a boiling point of 78.15 C.
Figure 35. Boiling point diagram of ethanol water mixtures
a) Azeotropic point
To obtain anhydrous ethanol, the pure ethanol water azeotrope must be dehydrated. This is generally accomplished by
azeotropic distillation with an entraining agent, usually benzene. The water is thus removed in the form of an overhead
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ternary benzene ethanol water azeotrope.
6.1.1. Distillation of Azeotropic Ethanol

The crude ethanol synthesized by the direct or indirect hydration of ethylene contains ca. 50 vol % ethanol, whereas the
crude product obtained after fermentation contains no more than 10 vol % ethanol. Consequently, the distillation of synthetic
ethanol requires less energy and is less expensive than that of fermentation ethanol.
The distillation of synthetic azeotropic ethanol obtained by the direct and indirect hydration of ethylene is described in
Sections Production Process and Production Process, respectively. The principles on which it is based are the same as
those used in the distillation of fermentation ethanol described in this section.
The low-energy distillation of ethanol from a fermentation feedstock is shown schematically in Figure 36 [314-316].
Fermented mash containing ca. 10 wt % ethanol and ca. 10 wt % total solids is preheated to near saturation temperature,
degassed to remove CO2, and fed to the stripping column. The overhead product leaving the stripping column contains 75
85 vol % ethanol. The bottom liquid (stillage) contains less than 0.02 wt % ethanol and is sent either for disposal or for
animal feed production. A vapor recompression system may be used to heat the reboiler of the stripping column for steam
economy.
Figure 36. Distillation of 95 vol % ethanol from a fermentation feedstock
a) Degasser; b) Stripping column; c) Extractive distillation column; d) Rectifying column; e) Fusel oil washer; f) Concentrating
column; g) Charcoal filtration; h) Storage tank
The overhead distillate from the stripping column is mixed with recycled ethanol from the concentrating column and fed into
the extractive distillation column, which operates at a pressure of 0.6 0.7 MPa. This column removes essentially all
fermentation byproducts, mainly aldehydes, ethers, methanol, and higher alcohols, from the ethanol. The aldehydes, ethers,
and methanol are more volatile than ethanol and leave the top of the column. However, the higher alcohol byproducts, known
collectively as fusel oil, are normally less volatile than ethanol. The average composition of fusel oil from fermentation ethanol
derived from different raw materials is shown in Table 21 [317]. The technique used to extract fusel oil from ethanol exploits
the fact that higher alcohols are more volatile than ethanol in solutions containing a high concentration of water. They can,
therefore, also be steam distilled and removed in the overhead steam to leave a virtually pure ethanol water mixture.
Table 21. Average composition of fusel oils (in wt %)
Fusel oil 1-Propanol 1-Butanol (n- 2-Methyl-1- 2-Methyl-1- 3-Methyl-1-

[71-23-8] butyl alcohol) propanol butanol (active butanol (isoamyl
[71-36-8] (isobutyl amyl alcohol) [ alcohol) [
alcohol) [ 137-32-6] 123-51-3]
78-83-1]
Molasses 13.2 0.2 0.7 15.8 28.4 37.4

Wheat 9.1 0.2 0.7 19.0 20.0 51.2
cereals
Potatoes 14.0 0.5 15.5 15.0 55.0
Sulfite 7.0 22.0 13.0 55.0
waste
liquor
Fruit 8.0 2.0 19.0 14.0 57.0
Dilute ethanol is then sent from the bottom of the stripping column to the rectifying column where it is brought up to strength.
The rectifying column is heated by overhead vapors from the extractive distillation column and the concentrating column. The
ethanol (95 vol %) is withdrawn as a side stream from one of the upper trays; it is then filtered through charcoal and stored.
The water that collects at the bottom of the rectifying column contains traces of ethanol and is recycled to the extractive
distillation column. Fusel oil is withdrawn as a side stream from one of the lower trays of the rectifying column and fed to the
fusel oil washer.
In the concentrating column, overhead vapor from the extractive distillation column, which contains aldehyde, ether, and
alcohol impurities, is separated into low-boiling and high-boiling fractions. Ethanol is also recovered and recycled to the
extractive distillation column. A small stream is taken from the overhead condensate (low-boiling fraction), which contains
acetaldehyde and a small amount of ethanol. It may be sold as a byproduct or burned as a fuel. A side stream containing a
high concentration of fusel oil is sent through a cooler to the fusel oil washer. In the washer, ethanol is extracted from the
fusel oil with water, and the washings are recycled to the concentrating column. High-boiling fusel oil is also run off from the
bottom of the concentrating column. The combined fusel oil (high-boiling) fractions may be sold as a byproduct.
6.1.2. Dehydration by Azeotropic Distillation

Azeotropic distillation systems are designed for the production of pure, anhydrous ethanol (99.98 vol %), which contains
<200 mg/kg of water and <20 mg/kg of total impurities (for other specifications, see Table 20) [314-316]. A flow scheme for
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the two-column azeotropic distillation of anhydrous ethanol is shown in Figure 37. The dehydrating column and the
hydrocarbon stripping column operate at atmospheric pressure. Therefore, they may be heated with low-pressure steam, hot
condensate, or hot waste streams from other parts of the ethanol processing plant, thus minimizing steam consumption.
Figure 37. Production of anhydrous ethanol by azeotropic distillation
a) Dehydrating column; b) Decanter; c) Condenser; d) Cooler; e) Hydrocarbon stripping column; f) Entrainer tank
Water is removed from the ethanol water azeotrope in the form of a ternary azeotrope which is produced by adding an
entraining agent such as benzene, heptane (C6 C8 cut), or cyclohexane. The 95 vol % ethanol feed enters the dehydrating
column near the midpoint. The anhydrous ethanol product collects at the bottom of the tower and is sent through a cooler
prior to storage. The ternary azeotrope leaves the column as an overhead product which is condensed and then separated
into an organic and an aqueous phase in the decanter. The upper organic layer containing the entrainer is returned to the top
of the dehydrating column. The lower aqueous layer is pumped to the hydrocarbon stripper, where the hydrocarbon
entrainer, the ethanol, and some water vapor are recovered overhead and sent to the condenser decanter system. Water
from the stripper is pumped off as waste; if it contains a substantial amount of ethanol, it may be recycled to the ethanol
distillation unit.
The overall efficiency and reliability of the anhydrous ethanol system are the result of the following special features:
1. The use of a common condenser and decanter for the two columns reduces capital costs.
2. The use of very efficient column trays which can operate at a low throughput.
3. The BOD of the wastewater is low.
4. Consumption of the entraining agent is low.
5. Steam consumption is low: 1 1.5 kg is required per liter of anhydrous ethanol depending on the quality of the end
product.
In some plants that produce motor fuel ethanol, gasoline is substituted for benzene as the entrainer. Since gasoline is
required in the end product, the hydrocarbon stripping step is eliminated. However, gasoline dehydrating agents give
products of varying composition because the gasoline contains a number of different components.
6.1.3. Motor Fuel Ethanol

Two types of ethanol for motor fuel are produced industrially [314-316], namely, anhydrous and hydrous motor fuel ethanol (
Automotive Fuels).
Anhydrous Motor Fuel Ethanol. The distillation and dehydration of motor fuel ethanol from a fermentation feedstock are
shown schematically in Figure 38.
Figure 38. Production of anhydrous motor fuel ethanol
a) Degasser; b) Stripping rectifying column; c) Fusel oil washer; d) Filter; e) Dehydrating column; f) Decanter; g) Entrainer
tank; h) Entrainer stripping column; i) Cooler; j) Condenser
Stripping Rectifying. Ethanol is distilled in a single stripping rectifying column where the fermented mash is separated into
an overhead, ca. 95-vol % ethanol fraction and a bottom liquid fraction (stillage) containing <0.02 wt % ethanol. The mash is
preheated at its saturation temperature and degassed to remove residual CO2 before it enters the stripping rectifying
column. The column operates at a pressure of ca. 0.3 MPa and is heated with steam by a forced circulation reboiler. This is
the only part of the system that uses steam. The stillage leaving the bottom of the column is cooled to its boiling point at
atmospheric pressure by heat exchange, the extracted heat being used to preheat the mash feed. The temperature of the
resulting stillage is such that it can be subjected to vaporrecompression evaporation without preheating or flashing in an
evaporator.
The pressurized overhead vapors from the stripping rectifying column are used to preheat the mash feed and to heat the
reboilers of the dehydration column and of the entraining stripping column. The condensed vapors are returned to the top of
the stripping rectifying column. A small overhead stream is drawn off to remove volatile acetaldehyde from the system; it
contains <1 % of the product ethanol and is either added to the purified anhydrous ethanol product or burned as a fuel in the
plant boiler.
A side stream containing fusel oil is also taken from the rectifying section of the column and sent to a fusel oil washer. The
aqueous washings are returned to the stripping section of the column; the decanted, washed oil is combined with the ethanol
product stream and fed to the dehydration column. Fusel oil has a higher energy value than ethanol and acts as an agent for
blending ethanol with gasoline.
Dehydration. After leaving the stripper rectifier, the 95 vol % ethanol and fusel oil are filtered and fed to a dehydrating
column, which operates at atmospheric pressure. Here, water is removed from the feed by use of benzene, heptane (C6 C9
cut), cyclohexane, or some other entraining agent. The bottom stream from the dehydrating column consists of anhydrous,
99.5 vol % ethanol and is cooled prior to storage. The overhead fraction from the dehydrating column is a ternary azeotrope,
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which is combined with overhead vapors from the entrainer stripping column. The combined vapors are condensed and
cooled, forming two phases that are separated in a decanter. The upper entrainer layer from the decanter is pumped to the
top of the dehydrating tower, while the lower aqueous layer is fed to the entrainer stripping column for recovery of the
entrainer and ethanol.
Steam consumption in this system is 1.8 2.5 kg per liter of ethanol produced, depending on the alcohol concentration of the
mash.
Hydrous Motor Fuel Ethanol. A low-energy system has been developed for distillation of the 85 95 vol % motor fuel
ethanol used in engines that require neat alcohol and not gasoline. This distillation process is used mainly in Brazil; a flow
scheme is shown in Figure 39. For maximum steam economy, the fermentation feed is fed into two stripper rectifiers, the
first of which operates at a pressure of ca. 0.4 MPa, the second at atmospheric pressure. Steam is used only in the high-
pressure column, which takes 55 60 % of the fermentation feed. The steam consumption of this system is 1.2 1.5 kg per
liter of 85 95 vol % motor fuel ethanol. The overhead and fusel oil byproducts are removed and processed in a fashion
similar to that described for production of anhydrous motor fuel ethanol.
Figure 39. Distillation of hydrous motor fuel ethanol
a) High-pressure stripping rectifying column; b) Reboiler; c) Fusel oil washer; d) Stripping rectifying column (operating at
atmospheric pressure)
6.1.4. Reduction of Energy Costs

Distillation of fermentation ethanol requires large amounts of energy. Energy costs have been cut by reducing steam
consumption and by using vapor recompression systems.
Earlier columns for the distillation of ethanol from corn, wheat, or molasses were operated at atmospheric pressure. The
development of new multiple-stage, high-pressure systems has reduced steam consumption by 40 % compared to previous
systems. The new commercial installations are based on a pressure-cascading technique and consume 3.0 4.2 kg of
steam for every liter of 96 vol % ethanol produced. Earlier distillation systems required 6 kg of steam per liter of ethanol.
A modern motor fuel ethanol plant has a total energy consumption of 1.1 1.4 MJ per liter of ethanol. Steam consumption
figures for the systems described in Sections Dehydration by Azeotropic Distillation and Motor Fuel Ethanol are summarized
in Table 22 [318-320].
Table 22. Steam consumption and ethanol concentration in the low-energy distillation of ethanol
Parameter Distillation product
Ethanol water Anhydrous Anhydrous motor Hydrous motor

azeotrope ethanol fuel ethanol fuel ethanol
Maximum ethanol 10 96 10 10
concentration in the feed,
vol %
Ethanol concentration of
the product,
vol % 96 100 99.5 95
proof (U.S.) 192 200 199 190

Steam consumption per 4.1 1.4 2.2 1.2
liter of ethanol, kg
Vapor Recompression Systems. Energy costs can be reduced by up to 80 % in low-pressure distillation columns by using a
vapor recompression system [321].
In this system, compressed overhead vapor is used as the heat source for the reboiler, instead of expensive steam. The
temperature of the column overhead vapor is increased by compression.
Vapor recompression can be applied to distillation columns operating at or below atmospheric pressure. It is used mainly in
the distillation of motor fuel ethanol. Investment costs for ethanol distillation units equipped with a vapor recompression
system are almost 50 % higher than those for conventional distillation.
6.2. Nondistillative Methods

The energy required to remove water from ethanol can be reduced significantly by using methods that do not rely on
distillation.
6.2.1. Solvent Extraction
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Ethanol dissolves in some liquids that are virtually immiscible with water. These solubility differences can be exploited to
recover ethanol from an aqueous solution by means of solvent extraction [322].
In the United States, the Energol Corporation employs liquid liquid extraction with a proprietary solvent to separate ethanol
from water. The solvent is then removed by distillation. This method does not require energy-intensive azeotropic distillation
and thus has a low energy consumption. The energy budget for the entire plant is 3500 3700 kJ per kilogram of ethanol
produced. In 1987, Energol's technique was used in four 40 000 to 45 000-L/d plants which came into operation in the mid
1980s.
The University of Pennsylvania and General Electric have developed a process that uses dibutyl phthalate [84-74-2] as a
water-immiscible solvent for purifying ethanol. This solvent has a much higher boiling point than ethanol, and ethanol can
therefore be separated in a single distillation step; solvent losses are low.
6.2.2. Carbon Dioxide Extraction

Another type of solvent extraction makes use of a critical fluid, i.e., a gas compressed to the point at which the distinction
between gas and liquid disappears.
Ethanol is selectively extracted from grain mash with carbon dioxide close to its critical point of 7.3 MPa and 31 C [321]. The
ethanol-rich stream is flashed at ca. 4.8 MPa to remove the carbon dioxide, leaving ethanol separated in the liquid phase.
This route requires a third to a half of the energy needed for conventional ethanol distillation, but the capital cost is 20 %
higher. A further advantage of this method is that the carbon dioxide used as solvent is obtained as a low-cost byproduct of
fermentation. This is important because some solvent inevitably escapes during ethanol recovery and has to be replaced.
6.2.3. Adsorptive Dehydration

Molecular Sieves. Ethanol azeotropes are dehydrated industrially by adsorption with molecular sieves whose pores are
permeable to water but not to ethanol [323-325]. The molecular sieve may be a synthetic or naturally occurring zeolite (e.g.,
clinoptilolite) or a proprietary resin. The 95 vol % ethanol is dehydrated in molecular sieve columns; 75 % of the adsorbed
material is water and 25 % ethanol. When the column is saturated, the stream is directed to a fresh column and the saturated
column is regenerated. The regeneration stream containing 25 vol % ethanol is fed back to the ethanol distillation system.
Solid Agents. Ethanol can also be dehydrated by adsorption with solid agents [326]. Less energy is required to vaporize
water from cellulose or corn-starch than from calcium hydroxide, because of their low heats of adsorption. Therefore, ground
cornmeal is used as a dehydrating agent for removing water from an 85 % ethanol feed stream. The cornmeal adsorbent can
be recycled ca. 20 times before being used as animal feed.
6.2.4. Membrane Technology

Pervaporation. A new method of ethanol purification based on pervaporation has been developed [327], [328] and is shown
schematically in Figure 40. The pervaporator consists of a number of semipermeable membrane modules made of poly(vinyl
alcohol) resins.
Figure 40. Production of anhydrous ethanol by pervaporation
a) Pump; b) Heater; c) Pervaporator; d) Condenser; e) Vacuum pump
The 94 vol % ethanol feed is preheated to 60 C and pumped to the semipermeable membrane modules of the pervaporator.
Water permeates the membrane down its concentration gradient; a phase change occurs from the liquid phase at the
membrane inlet to the vapor phase in the permeate. Water is thus separated without azeotrope formation. The driving force
for permeate flow is provided by a vacuum of less than 1 kPa at the permeate condenser inlet. The total energy consumption
is the sum of the evaporation and the condensation enthalpies.
The condensed permeate contains a small amount of ethanol and can be recycled to a rectifying or distillation tower for
recovery of ethanol. Pervaporation of 1096 L of a 91 wt % (94 vol %) ethanol feed yields 1000 L of anhydrous ethanol
(99.85 wt %, 99.9 vol %) and 107.5 L of permeate byproduct containing 23 wt % ethanol. The production of 1000 L of
anhydrous ethanol requires 135 kg of steam (200 kPa), 10 m3 of cooling water (20 C), and 15 kW h of electricity.
Reverse Osmosis. Purification of ethanol by reverse osmosis employs membranes that are relatively impermeable to
ethanol but permeable to water [329]. A pressure of 4 7 MPa is usually applied to remove the water by forcing it across the
membrane. The ethanol retention of new noncellulosic membranes is much higher than that of the cellulose acetate
membranes used earlier (80 % compared to 50 %). Reverse osmosis may prove useful for savings in energy costs by
concentrating ethanol to about 10 % prior to distillation.
6.2.5. Yarn-Filled Column

Textile yarns, such as rayon, retard the movement of water vapor but allow ethanol vapor to pass. This phenomenon has
been exploited to develop a technique for separating ethanol water mixtures [330].
Water containing 12 wt % ethanol is vaporized by injecting it into a stream of air; the ethanol water air mixture is then sent
through a yarn-filled column equipped with heating elements. A zone of water builds up at the beginning of the column; air
and ethanol pass through and are cooled to condense the ethanol. Before the water zone moves too far into the column, the
page 41 of 57
direction of flow is reversed and the water is flushed out with air. The cycle takes about 25 s; flow occurs for 10 s in the
forward direction and 15 s in the reverse direction.
A continuous version of the process is being developed in which the yarn is made into a continuous belt by tying the ends
together. The belt moves slowly through the column, in the opposite direction to the flow of the ethanol water air vapor.
Ethanol is then produced continuously at one end of the column. The yarn leaves the other end of the column and passes
through a heating zone where water is driven off.
6.3. Storage and Transportation

Ethanol is shipped in railroad tank cars, 200-L tank trucks, 20-L drums, and smaller glass or metal containers. In the case of
special quality requirements, the drums may be lined with phenolic resin.
The hazard classifications of ethanol are as follows:
GGVE/GGVS 3/3 b
IMDG Code 3.2, 3.3, UN No. 1170
United States CFR 49 : 172.101, flammable liquid
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7. Comparison of Process Economics for Synthetic and Fermentation Ethanol

Otto Goebel
The figures and calculations in this chapter are based on data taken from [331-333].
7.1. Summary of Cost Analysis for Synthetic and Fermentation Ethanol

The cost of producing ethanol by ethylene hydration (synthetic ethanol) or fermentation (fermentation ethanol) depends
primarily on the price of the raw material used. Fermentation ethanol became economically competitive with synthetic ethanol
when crude-oil prices rose in the late 1970s and early 1980s, because ethylene is derived from refinery products. Production
costs for fermentation ethanol may be further reduced by introduction of new processes and improved technologies that use
raw materials such as wood, waste, and other low-cost feedstocks.
The feedstock price for synthetic ethanol production is high, but the capital cost requirement is low. Even if fermentation
ethanol is produced from an inexpensive feedstock, the operating and capital costs are higher than those for synthetic
ethanol.
The following analysis shows that the production costs for synthetic ethanol based on 1986 ethylene prices of $ 350/t are in
the same range as those for fermentation ethanol produced from molasses costing $ 45/t.
Production figures are given in liters and metric tons. To convert liters to gallons, divide by 3.785; 1 t = 1260 L.
7.2. Production Costs of Synthetic Ethanol

The synthesis of ethanol from ethylene is divided into three processing steps: hydration, purification, and distillation (for
details, see Chaps. Synthesis and Recovery and Purification). Approximately 87 major pieces of processing equipment are
required.
Investment Costs. To calculate the production costs of synthetic ethanol, the number of installed pieces of major equipment
and their costs were estimated in U.S. dollars based on 1986 cost levels. Figure 41 shows the estimated total investment
costs for hypothetical plants and offsites (auxiliary equipment, primarily storage tanks) with capacities of 25 000, 50 000,
100 000, and 150 000 t/a. The breakdown of the investment for the main processing plant the total inside-battery-limit
investment is as follows:
1. Equipment and machinery, including piping, insulation, painting, and electrical installations, 50 %
2. Construction and erection, 25 %
3. Civil engineering work, 10 %
4. Engineering, costs for equipment purchase and start-up, 15 %
License fees are considered separately.
Figure 41. Investment costs for a plant producing synthetic ethanol
a) Hydration; b) Purification; c) Distillation
Production Costs. The annual costs (in 106 dollars) of synthetic ethanol produced in a 50 000-t/a plant from ethylene
costing $ 350/t are as follows (with the assumption that 0.625 t of ethanol can be obtained from 1 t of ethylene):
Raw materials 11.26 (59 %)

Utilities 4.18 (22 %)
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Labor 0.24 (1 %)
Depreciation, interest, maintenance 3.59 (18 %)
Total production costs 19.27 (100 %)
Payback Time. The payback time (in years) for a 50 000-t/a plant can be calculated by using the following figures (given in
106 $/a, with the assumption that ethanol sells at $ 450/t and that ethylene costs $ 350/t):
Annual turnover 22.50

Total production costs 19.27
Difference 3.23
Depreciation 1.65
Cash flow 4.88
For a plant with double this capacity (100 000 t/a), the payback time is reduced from 3.4 to 2.5 years. Figure 42 shows the
payback times for synthetic ethanol production in a 50 000-t/a plant as a function of the selling price for ethanol at various
ethylene costs. The 1986 prices for naphtha and gasoline are indicated for comparison.
Figure 42. Price of anhydrous ethanol as a function of the payback time in a synthetic alcohol plant
Cost of ethylene (per ton): a) $ 300; b) $ 350; c) $ 400; d) $ 450Production capacity: 50 000 t/a (19 300 L/d)
7.3. Production Costs of Fermentation Ethanol

The processing scheme of ethanol fermentation from regenerable resources is divided into five major steps (for details, see
Chaps. Fermentation and Recovery and Purification):
1. Handling of raw materials

2. Hydrolysis pretreatment
3. Fermentation
4. Distillation and dehydration
5. Processing of stillage and production of optional byproducts
Approximately 140 major pieces of process equipment are required for a molasses plant and almost 200 for a wood
saccharification plant.
Investment Costs. Figure 43 shows the estimated investment costs for a fermentation-based ethanol plant with a capacity of
52 000 t/a (200 000 L/d); costs are given for each processing step and for the entire processing plant and offsites. The total
investment costs are indicated for plants using sucrose, starch, or cellulose-containing raw materials such as molasses,
cassava/manioc, or wood.
Figure 43. Investment costs for a plant producing fermentation ethanol from molasses, cassava, or wood *
a) Handling of raw materials; b) Hydrolysis pretreatment; c) Fermentation; d) Distillation and dehydration; e) Stillage
processing
* Production capacity: 52 000 t/a (200 000 L/d).
The breakdown of the inside-battery-limit investment is similar to that for synthetic ethanol plants.
Production Costs for Three Different Raw Materials. The annual production costs of fermentation ethanol produced in a
52 000-t/a (200 000-L/d) plant from three basic raw materials (sugarcane molasses, cassava, and eucalyptus wood) are
listed in Table 23.
Table 23. Production costs of fermentation ethanol obtained from sugarcane molasses, cassava, and eucalyptus
page 43 of 57
wood
Cost, 106 $/a
Molasses Cassava Eucalyptus

wood
Raw materials 9.27 14.77 7.19

Utilities 3.77 7.66 12.32
Labor 0.76 1.10 1.18
Depreciation,
interest, maintenance 7.06 7.75 11.58
Total production costs 20.86 31.28 32.27
Both wheat and corn (maize) are used by large ethanol-producing companies in the corn-belt region of the United States.
The economics of ethanol production from these raw materials are similar to those of molasses-based ethanol production,
but are strongly influenced by byproduct utilization and market values. The wet-milling process facilitates the production of
high-quality ethanol, and retains the corn oil and corn protein in the food chain. In this process, the corn kernel is separated
into starch, corn oil, gluten, and fiber, which are converted into a variety of other products. Utility costs are reduced by the
use of coal-based energy. In some plants, the enzymatic hydrolysis is designed so as to permit production to be switched
seasonally from ethanol to high-fructose corn syrup.
Payback Time. The payback time (in years) can be calculated by using the following figures (given in 106 $/a, with the
assumption that ethanol sells at $ 450/t and that molasses cost $ 45/t):
Annual turnover 24.90

Total production costs 20.86
Difference 4.04
Depreciation 3.18
Cash flow 7.22
If the selling price of ethanol is higher, e.g., $ 550/t, the payback time will be reduced to 2.8 years.
Figure 44 shows the payback times for fermentation ethanol production as a function of the selling price for ethanol.
Sugarcane molasses, cassava, and eucalyptus wood have been considered as raw materials, their costs being $ 45, $ 30,
and $ 15 per ton, respectively. In the early 1970s, synthetic ethanol was less expensive than fermentation ethanol because
crude-oil prices, and thus ethylene prices, were low. Increases in the price of oil and, therefore, ethylene were accompanied
by drops in the price of corn and sugarcane molasses; this meant that fermentation ethanol was less expensive to produce
than synthetic ethanol. Fermentation ethanol can be expected to maintain a slight price advantage over synthetic ethanol as
long as the raw material prices remain low. If ethanol is blended with gasoline for use as a motor fuel, i.e., gasohol (
Automotive Fuels), a payback time of 3 4 years is acceptable. The minimum selling prices for ethanol which would
economically justify its production from molasses, cassava, and eucalyptus wood would therefore be $ 500, $ 675, and $ 775
per ton, respectively (see Fig. 44). In 1986, Western European prices were $ 300 400 per ton for raw naphtha and $ 500
600 per ton for gasoline from the refinery (tax not included).
Figure 44. Price of anhydrous ethanol as a function of the payback time in a plant * producing fermentation alcohol
Raw material and price (per ton): a) Sugarcane molasses, $ 45; b) Cassava, $ 30; c) Eucalyptus wood, $ 15
* Production capacity: 52 000 t/a (200 000 L/d).
Stillage Processing Options. If stillage is used directly as a fertilizer without further processing (as in developing countries),
the economic feasibility of fermentation ethanol production is increased by about 15 %.
Stillage can also be processed further in four main ways:
page 44 of 57
1. Multiple-effect evaporation, drying of the concentrated stillage, and production of distillers' dry grain (a byproduct,
which is used as an animal feed)
2. Mechanical vapor recompression, syrup evaporation, drying of the concentrated stillage, and production of distillers'
dry grain
3. Evaporation and syrup combustion
4. Anaerobic digestion with generation of biogas
Analysis of the economic merits and drawbacks of these four schemes with regard to the production of fermentation ethanol
reveals that system (2) is the most advantageous; systems (1), (3), and (4) are less favorable, but equal. The disadvantage
of schemes (1) and (2) is the unstable selling price of distillers' dry grain. Scheme (3) is expensive because an off-gas
purification system is required. The advantage of scheme (4) is the production of energy in the form of biogas.
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8. Analysis
The classic physical methods for the determination of ethanol concentration in a sample are based on measurements of
relative density (with a calibrated pycnometer, hydrometer, or density meter) [334]; boiling-point depression of alcohol
water mixtures (using an ebulliometer) [335]; and refractive index [334], [336]. In addition, gas chromatography is quick and
reliable. Various internal standards, such as acetone, ethyl acetate, n-propanol, 2-propanol, and ethylene glycol monoethyl
ether, have been used [334], [337], [338].
Chemical methods include oxidation to acetic acid by dichromate in the presence of sulfuric acid and titration of unreacted
dichromate with ferrous ammonium sulfate (sodium diphenylamine 4-sulfonate or 1,10-o-phenanthroline are added as
indicators):
The pH of the reaction is critical because ethanol could be oxidized either to acetaldehyde or to a mixture of acetaldehyde
and acetic acid [335], [339], [340]. If the ethanol water mixture is denatured, other compounds can interfere.
The chemical purity of ethanol is often determined using acetylation or phthalation reactions in which ethanol reacts with a
defined amount of either acetic or phthalic anhydride in pyridine solution. The decrease in acidity of the anhydride solution
(after hydrolysis and comparison with a standard solution) corresponds to the reaction of the hydroxyl groups. Any reactive
hydroxyl group can take part in these reactions, which are not specific for ethanol. Other functional groups do not interfere
[341].
Trace amounts of ethanol can be determined by colorimetric methods using compounds such as 8-hydroxyquinoline or
vanadic acid, which form colored complexes with alcohols [342].
In addition to physical and chemical methods, a biochemical method is used to determine ethanol concentration [343]. This
method is based on the oxidation of ethanol to acetaldehyde by nicotinamide adenine dinucleotide (NAD+ ). The enzyme
alcohol dehydrogenase catalyzes the reaction:
To bring the reaction to completion, semicarbazide, (aminooxy)acetic acid, or the enzyme aldehyde dehydrogenase is added
to remove acetaldehyde from the reaction mixture. The reaction is followed spectrophotometrically at a wavelength of 340 nm
where the absorbance is proportional to the increase in NADH concentration [335]. Despite the fact that some other alcohols
can also react, the method is good and can be used to determine very small ethanol concentrations.
Some enzymatic methods use enzymatic probes with immobilized enzymes [344]. Electrode probes with whole microbial
cells have also been developed for alcohol determination [344].
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9. Uses
Ethanol is an organic chemical with many applications, e.g.,
1. alcoholic beverage ( Beer; ; ),

2. solvent,
3. raw material in chemical synthesis, and
4. fuel ( Automotive Fuels).
In most countries, ethanol produced by fermentation has been used for beverages and specialty chemicals, and ethanol
produced by chemical synthesis has been used for industrial purposes. Certain countries, such as Brazil and India, use
fermentation ethanol for industrial purposes.
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For many years, alcoholic beverages have been taxed worldwide. However, the price of taxed ethanol was too high for its
use as an industrial raw material. To enable industry to obtain alcohol at a lower price, the Tax-Free Industrial and Denatured
Alcohol Act of 1906 was passed in the United States. Similar laws have been enacted in other countries.
The U.S. government implemented financial, administrative, and chemical controls to prevent the utilization of tax-free
ethanol in beverages. These regulations established the following four distinct categories of industrial ethanol:
1. Completely denatured alcohol

2. Proprietary solvents and special industrial solvents
3. Specially denatured alcohol
4. Pure (absolute) ethanol
Pure ethanol and slightly denatured ethanol are under strict control, whereas highly denatured ethanol has the fewest
financial and administrative controls. A wide range of chemicals can be used for ethanol denaturation [345].
Solvent. Ethanol is the most important solvent after water. Its major commercial applications are in the manufacture of
toiletries and cosmetics, detergents and disinfectants, pharmaceuticals, surface coatings, and in food and drug processing.
Both synthetic and fermentation ethanol can be used for these purposes; however, fermentation ethanol is preferred
(particularly in Europe) for applications involving human consumption (or body use) such as cosmetics, toiletries, and
pharmaceuticals.
The amount of denatured ethanol used as a solvent is increasing. For example, in the United States 197 000 and 340 000 t
of ethanol were used in 1960 and 1979, respectively. In Japan, between 1974 and 1978 the amount of ethanol consumed as
a solvent increased by 28 % [346], [347]
Raw Material. Ethanol is also used to produce various chemicals (see Fig. 2):
1. Acetaldehyde ( Acetaldehyde Production)

2. Butadiene ( Butadiene Production)
3. Diethyl ether ( Ethers, Aliphatic Diethyl Ether )
4. Ethyl acetate ( Acetic Acid Ethyl Acetate)
5. Ethylamines ( Amines, Aliphatic Production from Alcohols )
6. Ethylene ( Ethylene Other Processes and Feedstocks)
7. Glycol ethers and other products formed by reaction with ethylene oxide or epoxides ( Epoxides Reactions with
Compounds Containing Ionizable Hydrogen; Ethylene Oxide Chemical Properties; Propylene Oxide)
8. Vinegar ( )
Table 24 shows the most important products derived from ethanol in the United States. The amount of specially denatured
ethanol used as a raw material in the manufacture of chemicals was 37.6 % of the total amount of denatured ethanol used in
the country during the specified period of time. In the United States, consumption of ethanol as a raw material reached a
peak in 1960 (627 000 t) and then decreased substantially. The amount of ethanol used to manufacture chemicals in 1979
was only one-third of the amount used in 1960. The main reasons for this decline are the use of ethylene glycol rather than
ethanol in antifreeze, replacement of ethanol by ethylene and ethane in the production of acetaldehyde, and production of
ethylhexanol and butyraldehyde from other raw materials. Certain trends indicate that further decline in the use of ethanol to
manufacture chemicals may be reversed [346].
In some countries, such as Brazil, the production of a variety of chemical products is based on ethanol. Among these
products are those, such as acetaldehyde, that are made from petrochemicals in countries richer in crude oil. In the United
States and elsewhere, ethylene is used to produce synthetic ethanol, whereas in Brazil, fermentation ethanol is used for
ethylene production.
The example of Brazil is stressed because it is the world's largest producer of ethanol from sugar. Table 25 shows the
amount of sugar used for ethanol production in the period from 1981 1982 to 1985 1986 in Brazil and in all other
countries, except the United States in which production of ethanol is based on corn. In 1985 1986 Brazil's production
represented 95 % of world production. Brazil almost doubled its ethanol production in the tabulated five year period [348].
Most of the ethanol is used as a fuel; only a small fraction is employed as a chemical feedstock for the production of
acetaldehyde, acetic acid, butanol, octanol, chlorinated ethylenes, glycols, polyethylene, styrene, vinyl acetate, and other
chemicals [346], [349].
In 1977, 158106 L of ethanol was used as a raw material for chemical manufacturing in the EEC. A much larger amount was
used as a solvent. In these countries, ca. 55 % of the ethanol was derived from fermentation [346].
More than 50 % of the total ethanol produced in India in 1978 was used to produce chemicals. The total production of
acetaldehyde, acetic acid, acetic anhydride, and DDT (dichlorodiphenyltrichloroethane) was based on ethanol, as was a
significant fraction of the production of organic acetates, acetone, butanol, and a certain amount of polyethylene and poly
(vinyl chloride) and styrene [346].
Table 24. Ethanol used as a raw material in chemical manufacturing in the United States (from July 1, 1978 to
June 30, 1979) [347]
Product Ethanol
consumption, L
page 46 of 57
Vinegar 73 853 912

Acetic acid 1 570 148
Ethyl acetate 33 190 215
Ethyl chloride NA *.
Other ethyl esters 84 679 089
Sodium ethoxide 4 180 679
Ethylenamines for rubber processing 35 208 990
Dyes and intermediates 1 357 936
Acetaldehyde NA *.
Ether, diethyl 327 524
Ether, glycol, and others
(excluding diethyl ether) 11 419 306
Xanthanes 752 425
Drugs and medicinal chemicals 2 518 511
Organosilicon products 126 683
Other chemicals 9 733 531
Synthetic resins 12 609 456
Total 271 528 405
* Not available.
Table 25. Sugar consumed to produce ethanol [348]
Crop years Sugar consumption, 106 t
Brazil Other Worldwide

countries *
1981 1982 4.8 0 ** 4.8

1982 1983 4.9 0 ** 4.9
1983 1984 5.9 0.1 6.0
1984 1985 9.1 0.2 9.3
1985 1986 8.5 0.5 9.0
* Does not include the United States.

** Negligible amount.
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10. Economic Aspects

Otto Goebel
The data given in this chapter are taken from [350-352].
10.1. Worldwide Production of Synthetic and Fermentation Ethanol

The capacities and annual production figures for already installed plants producing synthetic and fermentation ethanol in the
developed and developing countries are listed in Table 26. Only industrial installations have been taken into consideration;
small-scale production units with capacities of less than 5000 L/d (e.g., small units on farms and in local communities) have
been ignored.
Table 26. Ethanol plant capacities and annual production
Synthesis Fermentation
Installed Number Production, Installed Number Production,

plant of plants 106 L/a plant of plants 106 L/a
capacity, capacity,
106 L/d 106 L/d
page 47 of 57
Developed
(industrialized)
countries
Australia, New 0.270 9 40
Zealand
Eastern Europe 1.100 28 165
Western Europe 2.283 8 610 4.327 123 974
Israel, Japan, 0.231 4 69 0.918 31 138

South Africa
North America 3.262 10 880 9.569 74 2153
Soviet Union 0.200 2 55 0.850 75 128
Total 5.976 24 1614 17.034 340 3598
Developing
countries
Africa 1.035 35 155
Asia 0.230 4 62 4.608 163 691
Asia (centrally
planned
economies) 2.240 605
Central America 1.931 67 290
South America 7.704 116 1156
Oceania 0.036 2
Total 2.470 4 667 15.314 383 2297

Total worldwide 8.446 28 2282 32.348 723 5895
The total worldwide production capacity for synthetic ethanol is 8.446106 L/d and annual production is 2282106 L. Most of
the 28 plants producing synthetic ethanol are located in industrialized countries; the developing countries produce mainly
fermentation ethanol. In Italy, the Federal Republic of Germany, the United Kingdom, and Canada, ca. 50 % of the ethanol is
produced by ethylene hydration; the corresponding figure for the United States is ca. 35 %. The developed and developing
countries produce approximately equal amounts of fermentation ethanol in a total of ca. 720 plants.
The total worldwide production capacity for fermentation ethanol far exceeds that for synthetic ethanol and amounts to more
than 32106 L/d; the average production is, however, less than 20106 L/d. The reason is that fermentation plants in most of
the developing countries are only supplied with raw materials derived from sugarcane for part of the year.
Fermentation ethanol producers in the industrialized nations import some of their raw materials in the form of molasses from
the sugar-producing industries of developing countries.
10.2. Major Producers of Fermentation Ethanol from Regenerable Resources

More than 60 % of the total world production capacity for fermentation ethanol is shared by Brazil, the United States, and
India. These countries all have favorable climates, good soil conditions, and enough land to produce the necessary raw
materials.
Figure 45 shows the development of production capacities for fermentation ethanol in these countries and worldwide from
1970 to 1986. Since the first large increase in price of crude oil in the early 1970s, worldwide capacities of fermentation
ethanol plants have more than doubled. The rate of capacity increase is, however, slowing down. Capacities of fermentation
ethanol plants can be expected to decrease because of reduced investments and closure of obsolete plants. Drastic
increases in the price of oil may, however, change this situation.
Figure 45. Development of production capacities for fermentation ethanol
Brazil. Brazil's 65 industrial fermentation ethanol plants have a total capacity of over 6.7106 L/d but produce only 3.410
page 48 of 57
6 L/d. Raw materials for the production of fermentation ethanol are molasses and sugarcane juice; cassava and roots
(manioc) have also been tested. Ethanol, therefore, originates from sugar factories and distilleries. The sugar industry has
traditionally played a major role in the economic and social development of Brazil.
In 1975, the following two factors encouraged Brazil to set up a national program, called Proalcool, for the production of
fermentation alcohol:
1. the fluctuations and sudden decline in international sugar prices, and

2. the increase in price of crude oil.
The establishment of new agroindustrial complexes requiring large investments and close coordination between agricultural
and industrial planning is perhaps the major challenge facing the Proalcool program. The Brazilian automobile industry has
already spent $ (30 40)106 in development costs for the alcohol engine.
India. The production of ethanol from agricultural raw materials (primarily molasses) in India has a long tradition. The present
green revolution is in favor of establishing more sugarcane plantations.
Until recently, India was the only country to use ethanol as a raw material for producing chemicals customarily derived from
ethylene, but Brazil is now also adopting this approach.
The total capacity of India's approximately 80 industrial ethanol plants is over 3106 L/d. Unfortunately, most of these plants
operate at less than full capacity and for only five to six months of the year, so that actual production is ca. 1.2106 L/d.
United States. Because of its vast cornfields, the United States uses corn as the primary raw material for production of
fermentation ethanol. Some companies use corn to produce both ethanol and high-fructose corn syrup. Most fermentation
ethanol producers are located in the corn-belt states of Iowa, Illinois, and Indiana.
Sugarcane molasses is used as a raw material for ethanol production primarily in the Southern States and the coastal areas.
A few paper-manufacturing companies also use sulfite waste liquor as a raw material.
Tax reductions offered by the U.S. government as an incentive for gasohol utilization have led to the creation of more than 70
fermentation ethanol projects with an estimated total capacity of 13106 L/d. The number of government-supported projects
has, however, been reduced to about 45 because of budget cuts.
The total production capacity for fermentation ethanol in the United States is 9.5106 L/d, and annual production is about
2109 L.
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11. Toxicology
Dieter Mayer
The acute oral toxicity of ethanol in rats (LD50) is 11.5 [353] 13.7 g/kg [354]. In mice [355], guinea pigs [353], rabbits [356]
and dogs[355] the LD50 values are 9.5, 9.6, 9.9, and 6 g/kg, respectively.
Ethanol can cause mild to severe irritation in the rabbit eye depending on the concentration and amount used [357], but it is
not appreciably irritating to the intact skin [357], [358].
Inhalation of ethanol causes irritation of the mucous membranes, excitation, ataxia, drowsiness, narcosis, and death due to
respiratory failure. Lethal concentrations are in the range of 20 000 40 000 ppm in the mouse, guinea pig, and rat after
inhalation for several hours. Concentrations of less than 6000 ppm can be tolerated without symptoms of intoxication [359],
[360].
Repeated oral administration (12 weeks) of 10 g kg1d1 to juvenile rats resulted in low weight gains and fatty liver
degeneration [361]. Triglyceride, cholesterol, and phospholipid metabolism were disturbed in monkeys fed a diet in which
40 % of the total calories were replaced by ethanol. Fatty degeneration occurred in the liver and the myocardium [362].
Ethanol increased embryolethality in pregnant rats and retarded fetal development when administered at levels of 5 mL/kg
[363], [364]. Major malformations were not induced in the fetus.
Ethanol doses of 1 1.5 g/kg cause dominant lethal mutations in male mice [365], but not in female mice [366]. In the Ames
test, ethanol does not exhibit mutagenic activity [367]. An increase of chromatid breakage was observed in human fibroblast
cultures, but this is considered to be a cytotoxic effect rather than evidence of mutagenicity [368].
Ethanol is oxidized to acetaldehyde by means of the following enzyme systems [369]:
1. Alcohol dehydrogenase [370]

2. Catalase acting as a peroxidase and coupled to a system supplying oxygenated water [371]
3. A microsomal ethanol oxidizing system requiring NADPH as a coenzyme [372].
In the liver 80 % of the absorbed ethanol is metabolized first to acetaldehyde [371] and then to acetic acid. Acetic acid is
page 49 of 57
finally degraded to CO2 and water. The oxidation to acetaldehyde seems to be much slower than that of acetaldehyde to
acetic acid [373].
General symptoms produced by intake of ethanol in humans are listed in Table 27 [359]; however, considerable variations
are observed between individual subjects.
Table 27. Symptoms produced after ethanol intake in humans
Symptom Blood ethanol

concentration, %
Beginning of uncertainty 0.06 0.08

Slow comprehension 0.10
Stupor 0.11 0.15
Drunkenness 0.16
Severe intoxication 0.2 0.4
Death 0.4 0.5
In humans suffering from alcoholism, fatty liver degeneration is thought to be due to increased fatty acid synthesis from
acetate, increased transportation of lipids from peripheral fat depots to the liver, and decreased oxidation of fatty acids [374].
Fatty infiltration of the myocardium and chronic leptomeningitis are also reported in chronic alcoholism and are responsible
for the well defined symptomatology of this condition [375].
The ACGIH and OSHA exposure limits for ethanol are both 1000 ppm; its MAK value is also 1000 ppm (1900 mg/m3).
[Top of Page]
References
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2. W. Gorianoff, A. Butleroff, Justus Liebigs Ann. Chem. 180 (1876) 245.
3. C. R. Nelson, M. L. Courter, Chem. Eng. Prog. 50 (1954) 526. Links
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5. D. S. Tsiklis, A. I. Kulikova, Zh. Fiz. Khim. 35 (1961) 954; Chem. Abstr. 58 (1963) 6660g.
6. Z. Novosad, Chem. Prum. 5 (1955) no. 30, 72.
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Figure 1. Industrial sources of ethanol

At 100 C 799.05 kJ/kg

Table 1. Freezing points of ethanol water mixtures

Ethanol, fp, C Ethanol, fp, C

Table 2. Flash points of ethanol water mixtures

Ethanol, Flash Ethanol, Flash

Ethanol in liquid, Ethanol in vapor, bp, C

Table 4. Volume contraction for 1 mol of ethanol water mixture at 15C

1.00000 0.79354 0.0000

* Mole fraction of ethanol.

Figure 2. Chemicals produced from ethanol

4.1. Direct Catalytic Hydration of Ethylene

The mechanism of ethylene hydration is assumed to consist of four steps [11].

1. Formation of a -complex by the addition of a proton to the ethylene molecule:

2. Conversion of the -complex to a carbocation:

3. Addition of water to the carbocation:

4. Removal of a proton from the protonated ethanol:

4.1.3. Production Process

Figure 3. Synthesis of ethanol by direct hydration

Hydrolysis involves three steps:

4.2.2. Production Process

Figure 4. Synthesis of ethanol by the sulfuric acid method

4.3. Other Methods

Table 5. Homologation of methanol to ethanol a, b

Catalyst Additive Ethanol selectivity, % Reference

Co(OAc)3 tetrahydrofuran 72 [56]

4.3.2. Carbonylation of Methanol and Methyl Acetate

The net reaction is:

4.3.3. Conversion of Synthesis Gas

4.3.3.1. Synthesis by Heterogeneous Catalysis

4.3.3.2. Synthesis by Homogeneous Catalysis

5.1. Production by Yeast

Table 6. Ability of Saccharomyces and Kluyveromyces species to ferment sugars [109]

Carbon Type of Sugar Basic unit Yeast e

of basic subunit S. S. uvarum K.

Ion Growth Cellular concentration, CSTR inlet concentration for a cell

B+ 0.0004 0.001 0.5103 0.0004 0.001

a CSTR = continuous stirred tank reactor.

5.1.2. Fermentation Pathways

In addition, biomass and energy are also generated.

Figure 6. Embden Meyerhof Parnas scheme of glycolysis [113]

Glycolysis: glucose + 2 P 2 lactate + 2 ATP (classical mammalian muscle or brain)

glycerol + Pi (third form, no net ATP)

Table 8. Reactions and thermodynamics of glycolysis and alcoholic fermentation [113]

Reaction Equation a Name of enzyme Characteristic G0 b,

1 Glucose + ATP glucose 6-P + ADP hexokinase 14.32

cosubstrate: Mg2+ glucokinase

cosubstrate: Mg2+ nonspecific

cosubstrate: Mg2+ or Mn2+

Sums: (glucose)n + H2O 2 lactate + 2 H+ + (glucose)n1 219.40

(glucose)n + 3 Pi + 3 ADP 2 lactate + 3 ATP + (glucose)n1 glycolysis

Product yield YP/S,* g/g 0.465 0.460

Product yield YP/S,* g/g 0.410 0.470