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J. of Supercritical Fluids 46 (2008) 299–321
Review
From plant materials to ethanol by means of
supercritical fluid technology
Christian Schacht, Carsten Zetzl, Gerd Brunner

Institute for Thermal and Separation Processes, Hamburg University of Technology,
Eissendorfer Strasse 38, D-21073 Hamburg, Germany
Received 14 September 2007; received in revised form 9 January 2008; accepted 10 January 2008
Abstract
Plant and waste material from agriculture or food industry represents on of the the worlds largest resources of ligno-cellulose and therefore
fermentable sugars. Conversion of these sugars to ethanol is one way to take optimized profit of the solar energy incorporated in the plant growth.
For the target product of ethanol of >99.8 wt.%, there are several plant material sources available. The carbohydrate compounds of these
materials can be pretreated and partly hydrolyzed by nearcritical water. CO
2
dissolved in water may be used as catalyst. Hydrolysis is favorably
accomplished by enzymatic catalysis. The product streams from the hydrolytic treatment are fermented. The resulting diluted ethanol solution is
processed by multistage counter-current supercritical carbon dioxide extraction to ethanol of 99.8 wt.% concentration. Non-fermentable residues
may be subjected to a second hydrolysis or transferred to a biogas production. Solid residues of the biogas reactor, in particular lignin containing
fractions, can be oxidized with near and supercritical water to mainly gas and a smaller fraction of mainly short chain fatty acids, which can be
reintroduced to the biogas reactor.
© 2008 Elsevier B.V. All rights reserved.
Keywords: Bio-ethanol; Renewable resources; Ligno-cellulose; Hydrolysis; Supercritical; Hot water
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
2. The starting material (ligno-cellulosic material) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
3. The goal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
4. State of the art: conventional processes of the 1st and 2nd generation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
4.1. Pre-treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
4.2. Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
4.2.1. Kinetic modeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
4.2.2. Enzymatic saccharification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
4.3. Fermentation of C5 and C6 sugars to ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
4.4. Separation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
5. Supercritical/near critical fluid technology contributions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
5.1. Pre-treatment with CO
2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
5.2. Hydrolysis of starch and ligno-cellulose compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
5.2.1. Type of reactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5.2.2. Influence of solids concentration in the feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5.2.3. Influence of linear velocity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5.2.4. Influence of pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5.2.5. Inhibiting degradation products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5.2.6. Carbonic acid addition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305

Corresponding author. Tel.: +49 40428783040; fax: +49 40428784072.
E-mail address: brunner@tu-harburg.de (G. Brunner).
0896-8446/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.supflu.2008.01.018
300 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
5.3. Contributions of TUHH laboratory. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5.3.1. Hydrolysis of starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
5.3.2. Liquefaction kinetics and conversion of cellulose and starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
5.3.3. Ligno-cellulose and lignin conversion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
6. Application: hydrolysis of rice bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
6.1. Influence of hot water treatment on pH-value . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
6.2. Formation of sugar monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
6.3. Concentration of oligomers after treatment with hot pressurized water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
6.4. Monomers and oligomers: kinetic modelling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
6.5. Formation of degradation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
6.6. Enzymatic hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
7. Fermentation to ethanol and separation with CO
2
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
7.1. Estimation of the necessary number of theoretical equilibrium plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
7.2. Experimental reliability tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
7.2.1. Counter current SFE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
7.2.2. Hydrodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
7.2.3. Mixer settler SFE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
7.3. Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
1. Introduction
The ongoing worldwide discussion about fuels from renew-
able resources has several products in mind, which shall
contribute to the replacement of fossil fuels. One of them is
bio-ethanol, i.e. ethanol from plant material with a concentra-
tion of >99.8 wt.%. Ethanol is only one product option of many
which can be produced by different pathways.
Biomass can be converted for fuel purposes to heat by
combustion, to a product mix consisting of char, oil, and gas
by pyrolysis, to oil by chemical processes (reduction), to gas
(synthesis gas) by gasification, to methane by anaerobic diges-
tion, and to ethanol by disintegration and fermentation [1].
For motor fuels, gasification to synthesis gas with subsequent
Fischer–Tropsch reaction to liquid products, and fermentation
of sugars to bio-ethanol seem to be the most favored production
lines. This contribution will be restricted to bio-ethanol and to
aspects of high-pressure technology only.
The efficiency of plants to convert radiation energy from the
sun into useful biomass is not overwhelming. C4-plants (named
from the photosynthetic process, like wheat, rice, sugar beet,
potato) have an efficiency between 1.6%and 2.9%(5.7%in the-
ory) and C3-plants (corn, sugar cane) have an efficiency between
0.5%and0.9%(3.2%intheory) inpercentage of the total amount
of incident radiation [1]. But the available amount of biomass is
enormous. The harvestable dry matter for sugar cane is between
35 t ha
−1
year
−1
and 90 t ha
−1
year
−1
, for sugar beet between
8 and 18, and for temperate grass between 7 t ha
−1
year
−1
and
25 t ha
−1
year
−1
, [1].
The major disadvantage for the time being is the higher
production cost for bio-ethanol as compared to gasoline. Bio-
ethanol production costs are (to be looked at with care, as all
general cost data): 0.78 D /L (24 D /GJ) from sugar, 0.72 D /L
(22 D /GJ) from starch, 0.31 D /L (10 D /GJ) from sugar cane
(Brazil), and from ligno-cellulose 0.98 D /L (30 D /GJ). For com-
parison: Bio-diesel 0.69 D /L (19 D /GJ) (from rape seed), biogas
0.74 D /L (21 D /GJ), BtL-fuel 1.03 D /L (30 D /GJ) [2]. Fossil
fuels have a heating value of about 42.7 GJ/t, the lower heating
value of Ethanol at ambient temperature is about 26.7 GJ/t.
2. The starting material (ligno-cellulosic material)
Ligno-cellulosic material, or plant biomass, is the world-
wide greatest resource of sugars and thus, for the production
of ethanol. The ligno-cellulosic material contains mainly cellu-
lose (35–50%), hemi-cellulose (20–35%), and lignin (10–25%)
[3]. The composition varies in dependence on the type of sub-
strate and the processing. In addition, the material may contain
starch (which usually is used separately), ash (mineral matter),
proteins (e.g. gluten), oils, and other minor compounds.
Physico-chemical behavior of ligno-cellulose shall not be
mixed up with starch. Starch is an alpha-linked polysaccha-
ride. It is composed of two components with different molecular
weights: 20–30% of linear amylose and 70–80% of branched
amylopectin.
The structure of cellulose and ligno-cellulose as well as its
biological degradations can be reviewed at [4–9].
In plants, ligno-cellulose is forming a complex crystalline
structure that is resistant to enzymatic attack and insoluble in
water. It consists of cellulose, hemicellulose and lignin. The
molecules are held together by covalent bonding, various inter-
molecular bridges, and van-der-Waals forces.
Cellulose is a linear polymer consisting of ␤-1,4-glycosidic
bound glucose molecules. Each glucose unit is turned around by
180 degrees relatively to the neighboring molecules. Therefore,
the repeating units are cellobiose, the double sugar of glucose.
The chain length of cellulose ranges from about 100 to 14000
units [5].
Hemi-cellulose consists of pentoses (xylose, arabinose),
hexoses (mannose, glucose, galactose) and sugar acids, with dif-
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 301
fering concentrations depending on the type of substrate. Mostly
1,4-bound xylose units form a basic structure with different side
chains. These hetero-xylanes are bound together and onto the
lignin molecules by ferula acid bonding. They form a network,
in which the cellulose micro-fibrils are inserted [8].
Lignin is a complex, aromatic polymer compound consist-
ing of p-hydroxyphenylpropanoid units, which are connected by
C–CandC–O–Cbridges. Ligninmostlyis integratedintothe cell
walls. It stabilizes them and makes them resistant against water.
Structure and composition of the lignin molecules vary with the
substrate. Lignin encloses the micro-fibrils and strengthens the
fiber [10].
3. The goal
In order to convert ligno-cellulosic material into ethanol, a
hydrolysis of the polymers to the mono-sugars must be carried
out first. These mono-sugars can then be converted to ethanol in
a fermentation process by means of micro-organisms or yeast.
An efficient process should also make use of the pentose-sugars
being bound within the hemicellulose, (xylose and arabinose)
since the yield of ethanol may be doubled by their conversion.
The ethanol is then separated fromthe fermentation broth, where
a maximumconcentration of about 10%ethanol can be achieved
[11]. Higher concentrations of ethanol may inhibit the fermen-
tation. Ethanol for use as motor fuel must then be concentrated
to >99.8%, beyond the azeotrope (95.57 wt.% at 1 bar, 78.2

C).
The process, nevertheless, comprises some steps, which have
to be improved for the efficient use of ligno-cellulosic material,
e.g. pre-treatment, hydrolysis, fermentation, and the concen-
tration step to absolute alcohol. This will be discussed in the
following. More questions are still open for the use of lignin,
which mostly is used for energy supply, for the waste water, and
for the solid residues.
4. State of the art: conventional processes of the 1st and
2nd generation
The conversion of biomass to ethanol generally includes three
steps: The hydrolysis of the cellulose and hemi-cellulose into
monomer sugars, the fermentation of the sugars to ethanol as
well as the purification of ethanol. Although different ways of
hydrolysis have been studied, enzymatic treatment provides the
greatest potential to lead the technology towards a successful
competition with conventional fuel technologies [12–14].
Unlike 2nd generation processes, 1st generation processes
basically use starch, contained in grains or seeds (e.g. corn,
wheat, rye, etc.) as a starting material for ethanol production.
2nd generation processes try to make use of the whole plant,
e.g. corn stover, rye stover, bagasse or grass. Processes, which
directly use sugar solutions, e.g. from sugar cane or sugar beet,
start with their process line after the saccharification. 1st gen-
eration processes can be subdivided into dry milling and wet
milling processes [15].
Wet-milling plants, which comprise about 25% of the capac-
ity in the USA, produce ethanol together with a variety of
co-products. In contrast, dry grind plants are designed for the
production of ethanol and produce animal feed as co-product.
Capital cost are lower than for wet milling plants.
Ligno-cellulosic biomass can be pre-treated and enzymati-
cally hydrolyzed to yield a mixture of sugars including glucose,
galactose, arabinose, and xylose [16].
So far, no commercial plant is operating on the basis of cellu-
losic material. There is a demonstration plant, operated by Iogen
Corporate, Ottawa, Canada. The process is basically the same
as for starch-based plants with appropriate steps for the pre-
treatment of the biomass. Technological innovations claimed by
Iogen include [17]: Pre-treatment by a modified steamexplosion
process, new, highly potent and efficient cellulase enzyme sys-
tems, separate hydrolysis and fermentation using a multistage
hydrolysis process, advanced micro-organisms and fermenta-
tion systems that convert both C6 and C5 sugars into ethanol,
energy efficient heat integration, water recycling and co-product
production.
4.1. Pre-treatment
An effective pre-treatment includes the decrease of the cel-
lulose crystallinity by the removal of hemi-cellulose and lignin
as well as the increase of the cellulose porosity [18,19]. Fur-
thermore, the formation of decomposition products, that inhibit
further process steps, have to be avoided, size reduction of the
feed stocks has to be obviated and energy demands as well as
costs have to be minimized.
Duringthe last 25years plentyof pre-treatment-methods have
been developed, which can be reviewed at other places [12–14].
Whereas dilute acid pre-treatments are already commercially
available, treatment of biomass with liquid hot water (LHW)
is still being worked at on laboratory scale [11,20]. However,
due to its simplicity, low generation of inhibiting by-products
and projected yields of up to 98% LHW pre-treatment exhibits
a great potential, and Hamelinck [11] points out the need to
perform further research on this pre-treatment method.
Lignin hinders water-molecules to enter the cellulosic micro-
fibrils andthus inhibits the actionof enzymes. Inaddition, crystal
structure of cellulose reduces the surface available for contact
with the enzymes [13].
Pre-treatment of ligno-cellulosic biomass should conse-
quently eliminate steric hindrance and enlarge porosity of the
substrate. Hereby, reaction of mono-sugars to degradation prod-
ucts must be avoided since it reduces yields and produces
inhibiting compounds.
Dilute acid pre-treatment was a long-time preferred pro-
cedure and has been reviewed repeatedly [12,18,21–23]. The
substrate is contacted with dilute sulphuric acid (0.5–1.5 wt.%),
or other acids like nitric acid or hypo-chloric acid, at tempera-
tures of 160–220

C and reaction times ranging from seconds to
several minutes [11,24]. Hemi-cellulose can be fully dissolved
(xylose yields of 75–90% are reported) and the downstream
enzymatic hydrolysis of cellulose reaches values up to 90%[13].
Disadvantageous are the corrosive medium, the neutralization to
adjust the pH for the following process steps, (e.g. with calcium
hydroxide, which produces gypsum [25]), and the formation of
inhibiting degradation products [13].
302 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Steam pre-treatment was first used in the Masonite-process
for fibreboard [26]. The advantage that no toxic or inhibiting
substances are added, is obvious. Biomass is contacted with sat-
urated steam at elevated pressures (7–50 bar) and temperatures
(160–260

C) for several minutes and then suddenly depres-
surized. Thermal, chemical, and mechanical effects change the
structure:
1. Hydrolysis reactions produce small amounts of acids (i.e.
acetic acid), which themselves act then catalytically. The low
pH of water at the elevated temperatures [27] also enhances
hydrolysis.
2. The sudden evaporation of water after pressure reduction
only loosens the structure of the substrate [28].
Enhanced enzymatic conversion of cellulose (up to 90%) is
attributed to the removal of hemi-cellulose [13]. The method
is particularly suitable for leaf-wood. Disadvantages are the
limited decomposition of the ligno-cellulosic structure, the for-
mation of xylose degradation products and the lower yield in
pentose monomers of about 65% [29].
4.2. Hydrolysis
Hydrolysis of ligno-cellulosic material can be carried out
as acid catalyzed or as enzymatically catalyzed process. Acid
catalysis encounters the disadvantages of adding a substance,
which is corrosive and must be recovered, enzymatic catalysis
leads to long residence times and to additional biomass, which
has to be disposed of. The relatively small pore size within the
lignocellulose (about 51
˚
A, [30]) is the reason for the long reac-
tion time during enzyme catalysis [28], while in acid catalysis
the active species, protons of 4
˚
A diameter, can easily dif-
fuse through the substrate. Pre-treatment of the ligno-cellulosic
material, as already discussed, leads to much shorter reaction
times for enzymatic decomposition. Reaction conditions must
be chosen in a well defined interval, since hemi-cellulose decom-
poses rapidly into mono-sugars, while cellulose needs a much
harsher treatment for substantial yield of mono-sugars [12,13].
It has been shown experimentally that the mono-sugars of hemi-
cellulose will subsequently convert to degradation products at
elevated process temperature resp. residence time in the appa-
ratus.
The molecular mechanism of acid catalyzed hydrolysis
includes three steps [31]. First, a conjugated acid is formed by
the addition of a proton onto the oxygen atom connecting both
sugar-units. Then, the C–O-linkage between the glycosidic oxy-
gen atom is split and a cyclic carbonium ion in shape of a chair
is formed. This step is rate determining [32]. By a fast addition
of water, this ion is transferred into glucose in the last step of
the reaction mechanism.
The reaction rate is very high at first and after a certain
reaction time and degree of conversion, decreases. The initially
high reaction rates are attributed to the amorphous and surface
parts of the ligno-cellulose, the lower rates are explained by the
crystalline structure of the remaining substrate.
4.2.1. Kinetic modeling
Kinetic modeling of the decomposition of ligno-cellulose
is complicated by the structure of the substrate, where lignin
inhibits the path of the protons. Accessibility of the glycosidic
linkages is changing with reaction place and reaction progress.
Several types of linkages (e.g. between xylose, the acetyl groups,
linkages in lignin) are stronger than the glycosidic linkages.
The number and variety of modeling approaches is enormous
and may vary according the specific compounds inside the ligno-
cellulose system. An overview can be found by Jacobsen and
Wyman [33].
Sasaki et al. [34] studied the kinetics of cellulose hydrolysis at
elevated temperatures (above 300

C) and applied the shrinking
core model for the numerical description.
A common model, initially designed for Douglas fir wood,
is based on two first order reactions [35].
Cellulose
k
1
−→glucose
k
2
−→degradation products.
This model could also be applied with success to the decom-
position of hemi-cellulose. It can be generalized to
Polymers
k
1
−→monomers
k
2
−→degradation products
wherein k
1
and k
2
are the reaction rate constants. Integration of
the differential equations leads to an equation for the amount of
monomers [36,37]
M = M
0
e
−k
2
t
+P
0
k
1
k
2
−k
1
(e
−k
1
t
−e
−k
2
t
) (1)
with M for the monomer concentration, and P for the polymer
concentration, and t as the reaction time. The index 0 indicates
the initial values.
The model may be broadened by assuming a reactive type of
polymers as a fraction α of the total amount of substrate [38],
and a non-reactive type, thus incorporating the decomposition
behavior of hemi-cellulose and cellulose.
M = M
0
e
−k
2
t
+αP
0
k
1
k
2
−k
1
(e
−k
1
t
−e
−k
2
t
). (2)
The combined influence of temperature and residence time
can be shown by introducing a severity coefficient, which makes
possible to consider both influences at the same time. This coef-
ficient R
0
was introduced by Overend [39]
R
0
= t exp

T −100
14.75

, (3)
with t as reaction time (min) and T as temperature (

C).
4.2.2. Enzymatic saccharification
Due to the disadvantages of acid catalyzed hydrolysis,
enzymatic hydrolysis is nowadays preferred. The reaction mech-
anism of the enzymatic hydrolysis is discussed in the literature
([5,10]), but not fully understood, yet. To enable the reac-
tion, the enzyme (cellulase) must be coupled to the substrate,
which requires the substrate to exhibit a certain size and type
of the three-dimensional structure. Different types of cellulases
are known. Exogluconases act at the end of the chains, while
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 303
endogluconases split the chains. Both actions lead to water
soluble oligomers which are transformed by ␤-glucosidase or
cellobiase to glucose [40,41].
The costs for the enzymes are a substantial part of pro-
duction costs for bio-ethanol. For recycling the enzymes, the
enzymes must be separated from the product flow, for repeated
use, enzymes must remain in the reactor and the product flow
must be removed. A detailed discussion is beyond the scope of
this contribution.
4.3. Fermentation of C5 and C6 sugars to ethanol
Saccharomyces cerevisiae (bakers yeast) produces adenosin-
triphosphate (ATP) during anaerobic fermentation via glycoly-
sis. Pyruvate is formed and is transformed to ethanol. In total,
2 mol ethanol and 2 mol CO
2
are formed per mol of glucose. To
make use of most of the ligno-cellulosic material, pentose sug-
ars must also be transformed to ethanol. Genetically modified S.
cerevisiae is developed in order to transformglucose and xylose
at the same time [42–44]. So far, glucose is transferred at much
higher rates than xylose.
4.4. Separation
The common process uses for separation of the ethanol a first
distillation column, called beer column. Remaining solids and
the major part of water is separated in this column. Ethanol is
removed, together with CO
2
which was produced during the
fermentation, over top. CO
2
is washed out with water and this
stream is recycled to the bottom. The bottom flow contains
all the solids which are separated with a centrifuge. The liq-
uid flow in the centrifuge is concentrated with heat from the
distillation. Condensed water is returned into the process. The
product flow from this column contains about 37% ethanol.
Ethanol is enriched in another distillation column up to the
azeotropic concentration of 95%. To produce absolute alcohol,
various distillation methods have been developed and employed,
partially with compounds like benzene or tri-chlor-ethylene. A
more recent and more environmentally friendly process is the
removal of water by molecular sieve adsorption [25].
5. Supercritical/near critical fluid technology
contributions
Recently, research has been carried out to compare hot liquid
water (LHW) pre-treatment to the treatment of biomass with
steam. The concept is a treatment of ligno-cellulose by sub-
critical pressurized water, eventually assisted by CO
2
-enhanced
hydrolysis.
Utilization of a steam treatment could significantly lower the
water usage. However, due to the advantage of significantly
lower hydrolysate inhibition, the main attention is being paid
on the development of LHW processes, which promises also
a higher conversion. Higher xylan recovery suggesting lower
generation of degradation products could be recognized for the
LHW treatment [45,46].
Fig. 1. Treatment of ligno-cellulosic material with liquid hot water.
Generally, an aqueous suspension of ligno-cellulose
(10–20%) is treated at elevated pressures, to keep the water
in the liquid phase (Fig. 1). For an industrial application the
following scenario was developed for a LHW pre-treatment, in
which the products are already separated into different streams.
The biomass is contacted with water at temperatures from
170–230

C and at pressures of 25–100 bar (compare Fig. 1).
The effluent is separated in a cyclone into dissolved monomers,
oligomers and a first residual suspension. This suspension is
heated to 210–250

C and partially hydrolyzed. The effluent is
again separated by a cyclone into a liquid with dissolved mono-
and oligomers, nowmostly of cellulose. The fully liquid streams
from the cyclones are transferred to an enzymatic hydrolysis for
saccharification and further to ethanol fermentation. The suspen-
sion fromthe second cyclone is again heated to 300

Cor higher.
Some oxygen is added (e.g. by in-line high-pressure electrolysis)
and the effluent streams are processed further.
Generally no particle size reduction is required for this kind
of pre-treatment [13,19,47]. The pioneer work regarding pre-
treatments with hot liquid water has been performed by Bobleter
and co-workers, who found out that hemi-cellulose could be
completely separated from the ligno-cellulose and enzymatic
digestibility of cellulose can be significantly increased by treat-
ing the ligno-cellulosic material with hot compressed water in
flow through systems [48,49].
The contact time is one of the main variables determining
the efficiency of the pre-treatment in which the hemi-cellulose
is removed from the biomass and the structure of the cellu-
lose is changed enabling accelerated enzymatic hydrolysis. The
feasibility of breaking chemical bonds with water are facili-
tated by the fact that water develops acidic characteristics at
high temperatures. The ion product K
w
of water increases with
the temperature up to a maximum of 6.34 ×10
−12
at 250

C
resulting in a pH of 5.5 for water at a temperature of 220

C
[27].
By percolating biomass at temperatures of 200–230

C and
residence times between0 minand15 min, Antal andco-workers
were able to achieve a total decomposition of hemi-cellulose and
90% yield of pentose sugars [50,51].
In Table 1 an overviewshows work carried out with hot liquid
water for pre-treatment of biomass [21,29,45,46,49–59]. The
304 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Table 1
Bibliography on liquid hot water pretreatment
Author Year Parameters Flow (mL/min) Operation Substrate Yield (% of the theoretical maximum)
Temperature (

C) Res. time (min) Xylose Glucose
Liquef. Recovery
S.G. Allen 1996 220 2 – FT Bagasse >80
a
N/A N/A
S.G. Allen 2001 215 2 – B Corn Fiber 82
a
N/A N/A
H. Ando 2000 180 rising 60 10 FT Hardwood N/A N/A N/A
T.H. Kim 2006 190 30 5 FT Corn Stover 86
a
90
a
94
b
M. Laser 2002 220 2 – B Bagasse <83
a
N/A 96
b
W. Mok 1992 230 2 – FT Bagasse 99
a
99
d
N/A
N. Mosier 2005 190 15 – B Corn Stover N/A 82
b
90
b
M. Sasaki 2003 200 rising 10 FT Bagasse N/A N/A N/A
H.K. Srenath 1999 220 2 – FT Alfalfa 70
a
75
d
91
a
G.P. Walsum 2004 180 32 – B Corn Stover 64
a
N/A N/A
S.E. Jacobsen 2002 200 10 – B Bagasse 86
a
N/A N/A
C. Liu 2003 220 16 10 FT Corn Stover 97
a
99
d
N/A
C. Liu 2004 200 24 10 PFP Corn Stover 89
a
95
d
90
c
C. Liu 2005 200 24 10 FT Corn Stover 96
a
99
d
95
c
a
Recovery from fluid phase after pretreatment FT flowthrough.
b
After enzymatic hydrolysis; B: batch.
c
Referring to the cellulosis content in feed PFP: particle flowthrough.
d
Sum of xylanes in the fluid phase and in solid residue.
most important parameters influencing the quality of the pre-
treated product stream are temperature, residence time, and the
type of the reactor. Pressure is adjusted to be higher than the
vapor pressure of water, since its influence is small [60], unless
CO
2
is dissolved in water, the concentration of which depends
on pressure.
Generally, the amount of dissolved hemi-cellulose rises with
increasing temperature and time of the treatment. However, the
hydrolyzed hemi-cellulose monomers (or some liquefied cel-
lulose) can further react to hydroxy-methyl-furfural (HMF) or
other by-products, most of which are inhibitory for one of the
following process steps of the ethanol generation. Addition-
ally, any kind of by-product reduces the final ethanol yield
as it is produced by the degradation of the fermentable sug-
ars. Thus, an optimum set of time and temperature parameters
can be found for each substrate, maximizing the final ethanol
yield.
A systematic investigation of the decomposition of ligno-
cellulosic material according to reaction kinetics could not be
found in the literature, yet. It would greatly contribute to the
understanding of the design of reactors.
5.1. Pre-treatment with CO
2
Disadvantages emerging from the pretreatment including
mineral acids could be avoided by the substitution of the acidic
compounds with CO
2
. While keeping the advantage of an
acid catalyzed process, corrosiveness is therefore significantly
reduced. At the same time CO
2
is a green process component,
can be easily removed by depressurization and creates no waste
products. If pre-treatment of lignocellulose with CO
2
could be
applied at lower temperatures than a similar process without
CO
2
addition, xylose degradation could be avoided [61] and
yield enhanced. Pure supercritical CO
2
did not enhance yield
at conditions of 214–275 bar, 112–165

C and reaction times
of 10–60 min [19]. With water added, about 70% saturation,
a substantial increase of the yield in mono-sugars could be
achieved with leaf-wood: 85% of the theoretical value, as com-
pared to 16% for the untreated sample. The efficient application
of water and carbon dioxide at higher temperatures and pres-
sures seems to be very promising. Sun [12] even expects lower
cost.
Results in the literature differ on the effect of CO
2
. Van Wal-
sum [29,62] found for corn stover that CO
2
in the pre-treatment
process enhanced the xylan- and the furan-concentration, and
increased the pH-value. Therefore, it was suggested that CO
2
hinders the formation of organic acids. All experimental results
cited have been obtained in batch-reactors, which renders them
problematic for transfer to other types of reactors.
5.2. Hydrolysis of starch and ligno-cellulose compounds
The first question which arises is to which extent the compo-
nents of ligno-cellulosic material undergo hydrolysis and yield
fermentable sugars or other products. In chapter 5.3 the influence
of LHW-treatment on the hydrolyses of starch, cellulose, lignin
and a lignocellulosic material (rice bran) will be investigated,
respectively.
Studies on the hydrolysis of glucose have been reported in
a range of temperature from 150

C to 300

C [63–65] and a
detailed kinetic investigation of the hydrolytic reactions of glu-
cose near the critical point was published by Kabyemela [66].
Also studies on hydrolysis of cellulose were available, but to our
knowledge the hydrolysis of starch had not been investigated
until end of the 90s.
Sakaki et al. [67] described the saccharification of cellulose,
starting from temperatures at 180

C, increasing to values to
above 280

C.
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 305
5.2.1. Type of reactor
For pre-treatment with hot liquid water three types of reactors
can be used: Batch, semi-continuous, and continuous reactors. In
batch-reactors the solid substrate (ligno-cellulosic biomass) as
well as the reaction medium (water) is reacted together without
adding or removing products during reaction time. In semi-batch
reactors a fixed bed of biomass is contacted by a flowof the reac-
tion medium, which is constantly added and removed, and may
be additionally recycled. Data from semi-continuous reactors
may be transferred to industrial scale reactors.
Continuous processes can be operated in a co-current and
a counter-current mode. Ligno-cellulosic biomass is mostly
contacted co-currently. A slurry of biomass and water is con-
tinuously pumped first through a heat-exchanger and then kept
at constant reaction conditions in a flow-through reactor, ideally
at plug-flow conditions. Short residence times are easily real-
ized, but longer residence times can only be achieved with long
tubular reactors or a high recycle ratio. The problems for this
operation mode are connected with the continuous handling of
solid material, especially if higher pressures are involved. But
ground biomass (about 1 mm) can be pumped into the reactors
under any condition. High solids concentrations are also not easy
to achieve. Most experimental data are obtained therefore at low
concentrations and are not relevant for industrial application. A
solids concentration of at least 10 wt.% must be achieved for
practical and economic purposes.
5.2.2. Influence of solids concentration in the feed
Since most experiments have been carried out at low con-
centration, the concentration dependence of the ligno-cellulose
decomposition is not clear. Most results indicate that an
increased biomass concentration results in a lower pH-value.
Results on xylan yield are differing. In a batch reactor no influ-
ence on yield of xylans could be found by Jacobsen and Wyman
[56], while Laser et al. [46] found an increased concentra-
tion of xylanes (beside higher concentration of furfural, and
an increased ethanol-yield after fermentation). Allen et al. [45]
determined a decreased decomposition with increasing biomass
concentration. All investigations agree on the decrease of pH
with increasing concentration of ligno-cellulose, which may
be explained by the increasing amount of organic acids being
formed.
5.2.3. Influence of linear velocity
An increased flow rate in the reactor enhances degrada-
tion of the ligno-cellulosic substrate. This result was confirmed
by several groups [50,54,57] and may be considered as result
of the higher mass transfer rates at higher Reynolds-numbers.
Increasing (e.g.) the linear velocity from 2.8 to 10.7 cm/min,
the resulting xylans conversion increased from 60% to 82%
[58].
To avoid enhanced water consumption and decreased con-
centration of biomass, Liu and Wyman [59] used a fixed-bed
reactor with periodically changing fluid flow velocity. After a
period of zero flow rate, the fixed bed was contacted with a hot
water flow, which was shut down after the pre-designed reac-
tion time and followed by another batch-reaction phase. The
result is in context with a normal extraction curve from solid
material. For the reduction of the specific solvent consumption
and increase of product concentration, it is to be preferred to
use several fixed beds which are operated in a counter-current
mode.
5.2.4. Influence of pressure
The influence of the pressure on hydrolysis of biomass is basi-
cally limited to keeping the water liquid according to the process
temperature. Liu [60] could show that the pressure influence on
the monomer concentration after pre-treatment can be neglected.
5.2.5. Inhibiting degradation products
During the decomposition process of biomass, degradation
products are formed, which inhibit the normal action of the
micro-organisms. Such degradation products are acetic acid,
which stems from the decomposition of hemi-cellulose [68],
hydroxy-methyl-furfural (HMF) and furfural. For these sub-
stances a strong inhibiting influence on yeast could be shown.
Micro-organisms, active in ethanol fermentation from pentoses,
are more inhibited than those which ferment hexoses [69]. The
mechanism is explained by Palmquist [70,71]. Decomposition
of lignin produces phenolic compounds, which strongly inhibit
the action of yeast [69] and the enzymatic hydrolysis of ligno-
cellulose.
5.2.6. Carbonic acid addition
Dilute acid pre-treatment processes are applied on large scale
for biomass to ethanol processes [11]. Hemi-cellulose yields as
well as glucose yields after enzymatic treatment reach up to 90%
of the possible maximum (in [57]). The main disadvantages of
this process can be found in the application of the acid itself.
It results in a higher corrosion and the neutralization leads to
the formation of waste particles which have to be separated and
disposed. These drawbacks could be overcome by introducing
carbon dioxide to the HLWprocess instead of acid, because CO
2
can be neutralized by a pressure reduction. Van Walsum [62]
presented an increase of xylan hydrolysis by adding pressurized
CO
2
to the HLWtreatment. These results could not be replicated
with aspen wood being the feedstock in 2002 [72].
However, utilising corn stover an increase of xylose and furan
concentration was demonstrated by van Walsum and Shi [29].
Also, an increase of the final pH was noticed in the LHW
treatment to which CO
2
was introduced. These results are in
agreement with the measurements from 2002, implicating the
assumption that the presence of carbonic acid represses the
formation of side products.
5.3. Contributions of TUHH laboratory
In this part a continuous process for the treatment of biomass
has been established, exhibiting the advantage of a process
without interruptions and frequent start-ups. The experimental
results are presented in this paper. Furthermore the effect of
simultaneous CO
2
supply has been investigated regarding xylan
yields as well as cellulose digestibility.
306 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
5.3.1. Hydrolysis of starch
The hydrolysis of starch with water and carbon dioxide at
elevated pressure and temperature was carried out with the aim
of optimizing the reaction conditions to obtain a maximumyield
of sugars, such as maltose, glucose and fructose. The hydrolysis
of starch is an acid catalyzed hydrolytic decomposition where
a C–O–C linkage is cracked between two glycopyranose units
and a water molecule is inserted.
Experiments on starch were carried out in a tubular reactor of
4.02 m length an inner diameter of 6 mm and an outer diameter
of 10 mm. The used Inconel 600
®
tubes were made of a corro-
sion resistant and high temperature resistant nickel-based alloy
(2.4816). A detailed description of the experimental setup can
be found elsewhere [57].
Corn starch with concentrations varying from 0.2 up to
10 wt.% with residence times from 0.4 min to 20 min were
applied. Temperature was kept in the range from 170

C to
300

C and the pressure varied between 60 bar and 240 bar. Car-
bon dioxide influence is expressed in degree of saturation, which
is the ratio of the added carbon dioxide to the maximum soluble
quantity of carbon dioxide in water at given conditions. Water
was saturated with nitrogen to exclude the influence of oxygen.
Initial starch concentration was 0.2 wt.%, if not stated otherwise.
Fig. 2 shows the results of the hydrolysis of starch at 230

C and
240 bar.
The influence of the concentration of carbon dioxide on the
formation of glucose is shown in Fig. 3.
Glucose yield is significantly higher than without the use of
carbon dioxide. With log(R
0
) =4.5 the yield can be increased
from 5% to 60% by the addition of CO
2
. Increasing the initial
starch concentration up to 10 wt.% did not affect the glucose
yield significantly (Fig. 4). Therefore, the results obtained with
a low initial concentration, seem to be transferable to higher
initial starch concentrations.
5.3.2. Liquefaction kinetics and conversion of cellulose and
starch
The overall degree of conversion of the biopolymer as well as
the yield and selectivity of the main reaction products are deter-
mined experimentally in a flow through type reactor. Additional
means like the acidification by carbon dioxide [60] to lower
Fig. 2. Product yields for conversion of starch under influence of carbon dioxide
as a function of residence time at T=230

C.
Fig. 3. Glucose yields with different CO
2
concentrations as a function of resi-
dence time.
the pH and promote acid catalysed hydrolysis steps are being
investigated.
Micro-crystalline cellulose as purchased from Merck
(Avicel
®
) was used. Real biomass samples were obtained from
a mesophilic biogas reactor run at a hydraulic residence time of
20 days, the biomass being the non-biodegradable residues of
the process. The solid residues were ground in a rotary cutter to
a particle size of less than 250 ␮m prior to the experiments.
The experimental set-up is schematically illustrated in Fig. 5.
The main building blocks of the apparatus are the feed supply
vessel, the feed pump, the tubular reactor and the down-
stream processing units, which consist of a double-pipe heat
exchanger, an expansion valve and the effluent collection sys-
tem. The high-pressure reaction unit is designed as a tubular
reactor made of high temperature resistant steel (o.d. =6.35 mm,
i.d. =3.05 mm).
Cellulose degradation experiments using pure micro-
crystalline cellulose were conducted to gain information about
Fig. 4. Glucose yields at different initial starch concentrations.
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 307
Fig. 5. Flowscheme of experimental set-up for continuous hot pressurized water
hydrolysis.
the rate of liquefaction and the yield of main degradation prod-
ucts for this most abundant plant constituent.
In this context, the degree of liquefaction was calculated as
the ratio of dissolved organic carbon (DOC) in the effluent to the
total carbonof the influent suspension. The results reveal a strong
increase of the rate of liquefaction with increasing temperature
in the range of 250–310

C, leading to a complete conversion
to soluble products in less than half a minute at 310

C. A
further increase in temperature results in an even more rapid
degradation, yielding a complete conversion within seconds.
This behavior is illustrated in Fig. 6, which depicts the degree
of liquefaction on a carbon balance versus process severity at
constant pressure.
Fig. 6. Cellulose: degree of liquefaction f at different temperatures and residence
times at P=250 bar.
The calculated curves were derived assuming a first order
kinetic for the cellulose decomposition for each applied
temperature. For this approach, the degree of conversion can
be described by Eq. (4):
ln(1 −f) = −kτ, (4)
where k denotes the reaction rate constant of the biomass
hydrolysis.
A common approach to express the temperature dependence
of the reaction rate constant is the Arrhenius lawwith k
S,j,0
being
the pre-exponential factor and E
A,j
the activation energy of the
reaction.
k
j
(T) = k
j,0
exp


E
A,j
RT

. (5)
The reaction rate constant for the degradation of cellulose
and of corn starch (obtained in an analogous manner) are stated
in Fig. 7.
For cellulose conversion, one may obtain
k
C,0
= 7.7 ×10
13
s
−1
E
A
= 163.9 ×10
3
J/mol.
For corn starch conversion, one may obtain
k
S,0
= 5, 3 ×10
12
s
−1
E
A
= 147.9 ×10
3
J/mol.
Regarding the temperature dependence, the reaction rate con-
stant at 250

C and short residence times reveals a significant
deviation from the linear relationship derived from the other
data points. This behavior of decreased values might be con-
tributed to some limitation, which is due to the crystalline
structure.
It can be inferred fromFig. 6 that the simulation curve derived
by linear regression reflect the respective data points reasonably
well, thus justifying the application of a first order approach.
The influence of acidification by carbon dioxide on both the
hydrolysis kinetics and the product formation was investigated
Fig. 7. Reaction rate constant of starch and cellulose in function of temperature.
308 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Fig. 8. Liquefaction of cellulose at 240

C, 260

Cand 280

Cin pure and CO
2
-
saturated water.
and compared to the hydrolysis in pure water. For that reason,
the density and solubility of carbon dioxide under the conditions
applied were taken from literature data [8].
Fig. 8 shows the effect of carbon dioxide on the liquefac-
tion of cellulose exemplarily for 240

C, 260

C, and 280

C.
A marked rate enhancement of cellulose hydrolysis by carbon
dioxide addition could be determined at temperatures of 240

C
and 250

C (results not shown). The catalytic effect dimin-
ishes with increasing temperature, as can be inferred from the
respective degrees of liquefaction at 260

C and 280

C. There
is still a slightly increased rate of hydrolysis at 260

C, while
at 280

C no differences in hydrolysis kinetics could be deter-
mined.
The kinetic modeling of cellulose liquefaction in the system
water/CO
2
was done in accordance with the method applied for
pure water.
Thus, CO
2
was believed to promote the acid-catalyzed reac-
tion pathway of biopolymer hydrolysis by the formation and
dissociation of carbonic acid according to Eq. (6).
CO
2
+H
2
O ⇔ H
2
CO
3
⇔ H
+
+HCO
3

⇔ 2H
+
+CO
3
2−
.
(6)
Besides the kinetics of substrate hydrolysis, the investiga-
tion of product formation was of special interest. A detailed
knowledge of product formation is important to optimize reac-
tion conditions with respect to the selective production of desired
compounds, e.g. glucose or amino acids. Their product yields
are strongly affected by the choice of the reaction temperature as
well as by the residence time. For all temperatures a maximum
in yield is observable, which shifts to lower residence times as
the temperature increases.
The reaction rate constants of formation and decomposi-
tion can be determined by fitting Eq. (7) to the experimentally
determined yields. With c
i,0
denoting the initial substrate con-
centration, the concentration of glucose can be calculated as
Fig. 9. Glucose yields after subcritical water hydrolysis of cellulose at different
residence times and temperatures at P=25 MPa; effect of dissolved CO
2
.
follows (Fig. 9):
c
i
=
k
f
i
k
d
i
−k
f
i
c
i,0
[exp(−k
f
i
t) −exp(−k
d
i
t)]. (7)
The rate constants for the formation and decomposition of
glucose (both from starch and cellulose) were obtained in an
analogous manner and are also stated in Fig. 10.
The constants for glucose confirm that its formation from
starch passes on faster than from cellulose due to the above
mentioned reasons. Furthermore, glucose is obviously insta-
ble under the conditions applied and accordingly consumed
by consecutive reactions. Different secondary degradation and
isomerization products were detected in this work, including
pyruvaldehyde, levoglucosan and HMF (from glucose) as well
as several carboxylic acids (from amino acids). However, a
Fig. 10. Rate constants of formation and decomposition for glucose (fromstarch
and cellulose).
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 309
closer inspection of these degradation products goes beyond
the scope of this work. The same applies for the different
by-products (mainly maltose, and oligomers and oligopep-
tides) formed by parallel reactions of the substrate according
to Fig. 10.
The optimumglucose yields, however, are obtained with resi-
dence times in the range of a few seconds up to several minutes,
depending on the reaction temperature. The maximal glucose
yield is reached at increased temperatures in combination with
short residence times. This finding is in accordance with lit-
erature results on cellulose hydrolyses at temperatures up to
400

C. The reported glucose yields amounted to maximal 50%
at 400

Cand 25 MPa. This value was, however, stated for a very
short residence time of 0.00025 s [5].
Based on the results of Fig. 10, the kinetic parameters for the
formation and decomposition of glucose can be evaluated.
For glucose yield conversion from starch, one may obtain:
k
G,S
(formation) = 1.1 ×10
11
s
−1
k
G,S
(decomposition) = 1.8 ×10
5
s
−1
E
AG,S
(formation) = 134.4 ×10
3
J/mol
E
AG,S
(decomposition) = 75.5 ×10
3
J/mol.
For glucose conversion from cellulose, one may obtain:
k
G,C
(formation) = 1.7 × 10
13
s
−1
k
G,S
(decomposition) = 4.6 ×10
14
s
−1
E
AG,S
(formation) = 160.7 ×10
3
J/mol
E
AG,S
(decomposition) = 168.7 ×10
3
J/mol.
5.3.3. Ligno-cellulose and lignin conversion
Hydrothermal treatment of ligno-cellulose becomes partic-
ularly attractive, when implemented into a complete cycle of
all components, as might be achieved in future including,
micro-organisms and higher plant based life support systems
[73]. In such applications, biomass offers the potential of an
autonomous system allowing for almost complete recycling.
It enables us to harvest its products, followed by a sub- or
supercritical water treatment of the wastes generated and the
subsequent, renewed build-up of plant biomass, thus closing the
cycle.
Hydrothermal conversion of ligno-cellulosic biomass was
investigated on the compounds cellulose, lignin, the complex
waste specified by European Space Agency (ESA) (wheat straw
and indigestible residues from a methane reactor) and rice bran.
Lignin was chosen as a model compound, since as one of the
main components in plant biomass, it proved to be the most per-
sistent component. Beside the conversion of lignin by hydrolysis
in near-critical water, the oxidative treatment using hydrogen
peroxide was also investigated (Table 2).
Treatment of ligno-cellulose was carried out continuously
in a plug flow reactor at pressures from 15 MPa to 25 MPa
Table 2
Composition of model waste and respective suppliers
Component Portion
(wt.%)
Source Dry mass content
(wt.%)
Wheat straw 23.3 Local farmer 94.5
Cabbage 23.3 Market 9.7
Soya 23.3 Oil-mill 91.1
Algae 10 BlueBioTechGmbH 95.5
Faecal material 20 – 27.4
and at temperatures from 240

C to 500

C with residence
times ranging from a few seconds to 3 min. Pure lignin
and ligno-cellulosic biomass could be liquefied by hydrol-
ysis up to 70–80%. Effluents were subsequently treated by
biological degradation. Overall efficiency of DOC (Dissolved
Organic Carbon) removal increased to 90–95%. No toxic
effects on the micro-organisms were observed. The oxidation
of ligno-cellulose in near-critical water by hydrogen peroxide
converted all carbonaceous material to mainly gaseous prod-
ucts. Only about 10% of the initial carbon load remained
in the aqueous phase, with the main product being acetic
acid.
In contrast to the hydrolysis of pure cellulose, the hydrother-
mal degradation of the biomass residues from the methane
reactor yielded an incomplete liquefaction, which can probably
be attributed to the lignin present in the samples.
As a result, the model waste specified by ESA can be readily
converted by hydro-thermolysis without the addition of a cata-
lyst or an oxidant. Degrees of liquefaction up to 90–95% could
be obtained on a carbon basis. Carbon in form of gaseous com-
pounds had a minor contribution and amounted to less than three
percent. Regarding the nitrogen balance, even higher degrees
(Table 2) of liquefaction up to 100% could be achieved. Both
the carbon and the nitrogen balance could be closed satisfacto-
rily. Based on these findings, the conclusion is drawn that the
hydrothermal conversion of complex biomass is a suitable pro-
cess for the production of soluble hydrolysates, which can be
utilized by subsequent biological treatment.
In this context, the degree of liquefaction was calculated as
the ratio of dissolved carbon in the effluent to the total carbon
of the influent suspension. The results reveal a strong increase
of the rate of liquefaction with increasing temperature in the
range of 250–310

C, leadingtoa complete conversiontosoluble
products inless thanhalf a minute at 310

C. Afurther increase in
temperature results in an even more rapid degradation, yielding
a complete conversion within seconds (Fig. 11).
As in the case of cellulose, the addition of carbon dioxide
leads to distinctly increased rate of reaction. For the continu-
ous water oxidation of ligno-cellulosic biomass, the set-up was
partially modified. Due to the corrosive atmosphere of high-
temperature water in the presence of an oxidant, a new reaction
coil made of corrosion-resistant nickel alloy (Inconel
®
, Alloy
600, o.d. 6,35 mm, i.d. 2,13 mm) with an internal volume of
20 mL was installed downstream of the mixing point. The reac-
tor was placed in a 4 kW oven (Heraeus RO 7/75), which in
principle allows temperatures up to 1000

C. Under operating
conditions, reactor outlet temperatures up to 500

C could be
310 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Fig. 11. Experimental results on model waste specified by ESA, computation
of carbon balance, P=25 MPa, initial solid concentration: 1 wt.%.
accomplished. Hydrogen peroxide was used as an oxidant. It was
directly introduced into the feed vessel and delivered to the reac-
tor along with the biomass in order to facilitate the experimental
procedure.
Water-soluble alkali lignin was used in these studies, since the
continuous processing of insoluble organosolv lignin was ham-
pered by the formation of lignin coagulates in the feed vessel and
lignin deposition in the reactor. As for the Cellulose, hydrogen
peroxide was used as the oxidant. It was directly introduced to
the feed vessel and delivered to the reactor together with lignin.
Time in the reaction zone was between 5 to 17 s for all experi-
ments. The conversion progress and the deduced rate constant,
considering a 1st order gasification of alkali lignin on a carbon
basis is shown in Fig. 12.
It can be concluded that the stoichiometric and over-
stoichiometric oxidant supply leads to an almost complete
oxidation of lignin in less than 20 s. About 10% of the influent
Fig. 12. Conversion of alkali lignin to gaseous species on a carbon basis, influ-
ence of amount of oxidant and temperature, initial lignin concentration: 1 wt.%.
carbon remains in the liquid effluents as DOC at temperatures
up to 390

C.
Experiments on the hydrolysis of lignin were conducted
with organosolv lignin, which is essentially water insoluble at
ambient conditions. The results show that organosolv lignin
completely dissolves in near-critical water and undergoes sig-
nificant chemical modification.
The results discussed in literature and own experiments point
to the conclusion that a complete liquefaction of isolated lignin
by non-catalyzed hydrolysis in pure high-temperature water is
not feasible. At lower temperatures inthe well-subcritical region,
where the cleavage of the most widespread linkage, the ␤-O-4-
bond, by hydrolysis should yield a high degree of degradation,
the portion of insoluble residues is still in the range of 30–40%.
The formation of insoluble residues cannot be prevented even
under optimized conditions. Furthermore, the treatment in pure
water does not lead to the selective production of valuable com-
pounds but to a broad product spectrum. Treatment of lignin
in high temperature water led to the build-up of carbon-rich
residues, which eventually resulted in reactor clogging and pre-
vented the attempted continuous processing. Summing up these
aspects, the non-catalyzed hydrolysis of lignin in pure water
does not appear to be a promising approach.
This finding was derived from mass spectroscopy analyses
of soluble reaction products, which are illustrated exemplarily
by the total ion chromatogram in Fig. 13.
The chromatogram shows the existence of numerous degra-
dation products in considerable concentrations. Although some
major classes of degradation products could be identified, e.g.
phenols, phenol derivatives and other aromatic substances, a
complete identification and quantification of all reaction prod-
ucts is virtually impossible.
Comparing the results of ESA biomass with those from the
studies on lignin (and wheat straw), it can be seen that the
hydrolysis of the model waste yields much higher degrees of
liquefaction than in case of lignin and wheat straw, which can
be attributed to the much lower content of ligno-cellulose in
the model waste. Some ingredients, e.g. algae, do not have
any ligno-cellulosic structures at all. This repeatedly shows that
ligno-cellulose is the most persistent fraction of biomass and
points towards the potential of algae as a feedstock for bio-
ethanol production.
Fig. 13. Total ion chromatogram of products from Lignin degradation after
hydrolysis, T=417

C, P=25 MPa, t =29 s.
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 311
Table 3
Composition of model waste and respective suppliers
Fraction (mass%) Fraction (mass%)
Glucose
a
28.2 Sugar
a
39.9
Xylose
a
5.5 Lignin
a
13
Arabinose
a
4.7 Ash
b
18
Galactose
a
1.1 Protein
b
16
Mannose
a
0.3 Water
c
5.2
Ramnose
a
0.1 Sum 92.1
a
Analysis of the Bundesforschungsanstalt f¨ urForst-und Holzwirtschaft.
b
Analysis report Euryza GmbH (10.06.2004).
c
Inhouse analysis.
6. Application: hydrolysis of rice bran
Due to its low lignin content compared to other biological
waste products, rice bran, milled and defatted (27% cellulose,
37% hemi-cellulose, 5% lignin), is an interesting representative
of the group of ligno-cellulosic biomass [20]. Rice bran produc-
tion is in the range of 40 megatonnes/year. Defatted rice bran is
mainly used for cattle feed.
Rice bran was obtained from a rice mill (Euryza Ham-
burg GmbH). Non-polar substances like fat and vitamins where
extracted in a supercritical fluid extraction with CO
2
[74]. To
ensure delivery of a water rice bran suspension by a membrane
pump (LEWA EL1), the residue was ground to a particle size
smaller than 500 ␮m (Ika MF10 basic) and passed through a
180 ␮msieve. Finally, the composition was determined (Table 3)
and the rice bran was stored at −18

Cuntil usage. The mannose
and ramnose contents are neglected in the following.
Hot pressurizedwater treatment was investigatedfor rice bran
in order to evaluate the formation of fermentable mono-sugars or
sugar-oligomers which are easily transformed to mono-sugars
by enzymes.
6.1. Influence of hot water treatment on pH-value
During hydrolysis of hemi-cellulose and cellulose, as well as
lignin, organic acid may be formed. This influences the pH-value
of the treated product solution or suspension. In general, the pH-
value decreases with increasing temperature and residence time
(Fig. 14), in accordance with results published by van Walsum
[29], and can be explained by the progress of the hydrolysis
reactions.
The dissolved carbon dioxide reduces the pH-value due to its
dissociation and in this way catalyses the hydrolysis reaction.
In addition, it can be noticed that increasing residence times
lead to smaller deviations between the pHof the products treated
with pure water and the products treated with a mixture of water
and CO
2.
This indicates that the influence of CO
2
decreases with
increasing process severity.
6.2. Formation of sugar monomers
Sugar monomers, directly fermentable to ethanol seem to be
the main goal of hydrolysis. The higher the monomer concen-
trations after the pre-treatment, the more fermentable sugars are
already present in the suspension. Hereby, it should be taken
into account that in a real substrate part of the carbohydrates
may be already monomer sugars. For rice bran, 225 mg/L glu-
cose, 290 mg/L xylose and 135 mg/L arabinose are already in
the feed solution.
During the pre-treatment the dissolved oligomers will react
to monomeric sugars, which can further degrade depending
on temperature and residence time. Thus, the sugars can be
protected from decomposition by keeping them in oligomeric
form. It should therefore be the target to minimize the reaction
towards the monomeric sugars and maximize the dissolution of
oligomeric sugars. Taking a closer look at the sugar monomer
concentration will give an idea of the influence of the process
parameters on the degradation behavior.
If a fermentative saccharification is carried out after the pre-
treatment, oligomers, are a target product as they readily react
in the presence of enzymes. Even the changes in structure of the
substrate, enabling enzymes to access the polymer molecules,
are advantageous.
In the course of the treatment, the xylose/galactose as well
as the glucose monomer fractions decrease rapidly with increas-
ing pre-treatment time. Increasing temperature accelerates this
process. Obviously, these monomeric sugars undergo decompo-
sition reactions, consuming the present amount of monomeric
sugars. Aconcentration raise can not be seen, indicating that the
decomposition reaction is very fast compared to the reactions
towards the monomeric sugars.
In contrary, the arabinose monomer concentrations increase
as a function of the severity parameter R
0
. Arabinose is prefer-
ably found in the side chains of the hemi-cellulose [10].
Therefore, the accessibility of arabinose towards hydrolysation
reactions is increased and one would expect fast release and
degradation of arabinose in comparison with xylose/galactose.
And yet, a decrease of the maximal arabinose monomer concen-
tration at higher temperatures cannot be detected. This indicates
significantly slower (degradation) reactions. This assumption is
Fig. 14. Dependency of pH-value of hydrolysis product on temperature and
residence time (a) P=75 bar and (b) liquid solution saturated with CO
2
(P see
original table).
312 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
confirmed by Garrett and Dvorchik [75], who found that the acid
degradation of xylose proceeds 5.4 times faster than the degrada-
tion of arabinose. This observation adds significant importance
towards the recovery of arabinose during pre-treatment pro-
cesses and further utilization of arabinose after the pre-treatment
processes.
The effect of CO
2
is definitely smaller for arabinose than
for xylose and glucose. Arabinose is bound mainly in the side-
chains, from where it can be easily contacted and transformed
into the monomer. Xylose and glucose are mainly fixed in the
main chain of the polymer und are therefore more difficult to
access and react slower.
By plotting the monomer concentration (xylose) versus the
coefficient log(R
0
), the experimental data can be represented
by one linear line, which proves once more R
0
to be a useful
parameter.
6.3. Concentration of oligomers after treatment with hot
pressurized water
A substantial part of the polymers is not transformed to
monomers but to oligomers. According to literature, up to 70%
of dissolved sugars may be oligomers [51]. The quantity of
oligomers was determined by difference from the amount of
total sugars and monomer sugars, which could be determined
directly. Due to that procedure, the values are uncertain and
definitely somewhat too high.
From the data can be concluded that there is an optimum for
the thermal hydrolysis, which is in the range of log(R
0
) =4.0–4.5
(230

C, 2–5 min) for xylose and arabinose, and at 170–200

C,
1–2 min for the glucose (due to the original content of starch
in the rice bran). The influence of CO
2
is in the range of 10%,
which is non-significant for these type of experiments.
6.4. Monomers and oligomers: kinetic modelling
The reactions are modeled in adaptation of the proposition in
chapter 4.2.1 as a sequential reaction, assuming two reactions
of first order according Eqs. (1) and (2).
For the definition of the parameters k
1
and k
2
, a non-linear
regression has been executed. The sum of deviations of the
calculated concentration results from five experimental val-
ues for each temperature program has been minimized via the
Newton-Approach. The model accordingEq. (1) was insufficient
when focusing on Glucose conversions, therefore the extension
according Eq. (2) has been chosen.
As the substrate type was identical, the adapting factor a must
be kept constant. Due to this, the average value of calculated
results for the fast-reacting fraction has been defined and fixed.
Then, the regression has been repeated for obtaining the opti-
mized values k
1
and k
2
. Secondly, the error R
2
of the linear
regression for the logarithmic approach of the reaction constant
on the inverse temperature scale has been minimized.
The result of the modeling is presented in the following tables
and figures (Figs. 15–21).
The reactionrate of the degradationis about one order of mag-
nitude higher than the rate for the formation of monomers. From
Fig. 15. Xylose concentration during hydrolysis; influence of carbon dioxide
(a) P=75 bar without CO
2
and (b) P: saturation pressure (see original table).
that can be concluded that mono-sugars are rapidly reacting to
degradation products. In order to produce oligomers, which can
be treated by enzymes, the reaction rate constants should be kept
low. The lowest values are obtained at 200

C. The addition of
CO
2
significantly enhances the ratio of k
1
/k
2
. Secondly, Fig. 22
Fig. 16. Glucose concentration during hydrolysis; influence of carbon dioxide;
P=75 bar without CO
2
, P: saturation pressure (see original table).
Fig. 17. Arabinose concentrationduringhydrolysis; influence of carbondioxide;
P=75 bar without CO
2,
P: saturation pressure (see original table).
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 313
Fig. 18. Concentration of xylose in dependency of the coefficient R
0
.
shows that the addition of CO
2
increases k
2
only marginally,
while k
1
is enhanced.
The concentration dependence of the oligomers shows the
hydrolysis of hemi-cellulose to soluble oligomers and the
sequential reaction to xylose monomers. The first reaction step is
much faster than the formation of monomers. The reaction from
oligomers to monomers is the rate determining step. A reac-
tion temperature of 230

C seems to be the optimum. If CO
2
is
Fig. 19. Xylose monomers and oligomers: total concentration.
Fig. 20. Glucose: monomers and oligomers: total concentration .
Fig. 21. Arabinose: monomers and oligomers: total concentration .
added, the reaction rates seem to be slowed down, contradicting
the findings for the monomers. This can be explained by the con-
centration dependence of the reaction rate on CO
2
which makes
the model not applicable. Therefore, the rate constants for both
cases can not be compared.
In the following, the same modeling is carried out for glu-
cose. The fraction α for the fast reacting part was determined to
α=0.588. Results are shown in the diagrams of Figs. 24 and 25.
They are basically similar to that obtained for xylose.
The significant information which can be concluded fromthe
experiments mentioned above is the optimum temperature for
the controlled hemicellulose and cellulose conversion.
Hemicellulose converts faster to xylose oligomers than the
degradation of the oligomers to monomers. The degradation of
the monomers, however, is considerably faster than its produc-
tion. Consequently, the second oligomer hydrolysis reaction step
is defining the overall reaction rate. In order to optimize the yield
of the total process, it is considereduseful toavoidthis hydrolysis
towards the monomers. In consequence moderate temperatures
(below 260

C) shall be applied. The existing oligomers remain
in solution, and can be easily converted to monomeric sugars
Fig. 22. Reaction rates k1 for modeling the reaction of xylose oligomers to
xylose monomer and k2 for the degradation of xylose.
314 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Fig. 23. Reaction rate constants k1 and k2 for the reaction of hemicellulose to
xylose oligomers (k1) and the hydrolytic reaction to xylose (k2).
in a subsequent enzymatic hydrolysis step without additional
degradation losses.
On the other hand, the conversion of hemicellulose to xylose
monomer shall be enhanced, this will lead to an optimum reac-
tion temperature of about 230

C.
The addition of carbon dioxide for the treatment of hemicel-
lulose (Fig. 23) will lead to a decrease of both process reaction
velocities. This finding contradicts to the observation of Fig. 22
(xylose oligomer conversion). One maydeduce a direct influence
of the carbon dioxide saturation on the reaction rate. Conse-
quently, the reaction rate constants are not directly comparable
for the application with and without carbon dioxide saturation.
Analogous observations can be found for the hydrolysis of
cellulose. In the same way, a non-desired – slow-conversion of
oligomers to monomers and direct – fast-degradation to side
products shall be avoided by the application of moderate tem-
peratures.
6.5. Formation of degradation products
The hydrolysis of hemi-cellulose and cellulose to mono-
sugars may yield degradation products, which are at least
reducing the yield of fermentable sugars. HMF and furfural
could not be detected in the product flow (limit of detection:
40 mg/L).
Temperature has a dominant influence on the formation of
organic acids. The addition of CO
2
inhibits the formation of
organic acids, as has also been reported by Walsum [29].
For an ethanol process, the total concentration of sugars is of
major important, since an enzymatic saccharification will fol-
low the thermal hydrolysis. The total concentration of sugars
is shown in the following diagrams, including the role of CO
2
(Figs. 24 and 25).
The picture of degradation products changes if residence
time is increased substantially. Rice bran was treated in a batch
reactor from 20 min to 60 min. The degree of liquefaction is
increasing even at low temperatures (160–180

C), but in addi-
tion to the formation and degradation of mono-sugars, several
Fig. 24. Glucose: reaction rate constants for the reaction of oligomers to glucose
(k1) and the reaction of glucose to degradation products (k2).
degradation products in a relatively high concentration, includ-
ing HMF and furfural, were detected after the reaction as shown
in Figs. 26 and 27 [20].
The conclusion is straightforward: Hot pressurized water
hydrolysis for itself –evenwhencatalyzedwithdissolvedcarbon
dioxide – is not sufficient to produce mono-sugars for ethanol
Fig. 25. Glucose: reaction rate constants for the reaction of cellulose to glucose
oligomers (k1) and the reaction of glucose oligomers to glucose monomer (k2).
Fig. 26. Formation of acetic acid during treatment of rice bran with hot pressur-
ized water.
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 315
Fig. 27. Formation of formic acid during treatment of rice bran with hot pres-
surized water.
fermentation, since the formation of by-products reduces the
yield. On the other hand, for hemi-cellulose and in particular
for cellulose, no really satisfying biological methods are avail-
able which are really competitive to a hot water treatment. From
that follows, that only a combination of both methods will be
successful. In concurrence, what is already carried out, is a
pre-treatment withsteamat relativelylowtemperatures andpres-
sures, which does not lead to an optimumuse of the enzymes for
saccharification. For the combination, the best conditions must
be found.
6.6. Enzymatic hydrolysis
Preliminary experiments have been carried out and have been
reported by Baig et al. [20]. Further work is going on involv-
ing experts from microbiology, who involve hyper-thermopile
enzymes for saccharification.
The table in Fig. 29 confirms also the fact that the
isolated hydrolysis is efficient for xylose conversion from hemi-
cellulose, but glucose yield is still quite poor. The pre-treatment
gives favorable conditions for the subsequent enzyme-catalyzed
conversion of cellulose (Figs. 28–32).
The addition of enzymes (commercial, Depol 692, max.
cellulase-activity of 800 U/g at 68

C and pH 5; at a pH-value of
4 or 6, activity is reduced to 10%of the maximumvalue) to a non
treated rice bran suspension resulted in a glucose concentration
of 730 mg/L. In pre-treated suspensions of rice bran the enzyme
treatment resulted in glucose concentrations up to 2100 mg/L
(Fig. 30).
Glucose concentration after combined pre-treatment and
enzyme-catalyzed hydrolysis decreases with increasing temper-
ature during pre-treatment, as has been reported by other groups
[27]. This may be due to reduced enzyme activity caused by
enhanced lignin concentrations in solution. The amount of dis-
solved lignin increases with temperature and residence time,
whichleads toreducedyieldof the enzymatic hydrolysis [53,57].
The maximum concentration of 2100 mg/L is obtained at log
(R
0
) of 3.5 to 4, which corresponds to a temperature for the
pre-treatment of 200

C and 5 min residence time, or 230

C at
Fig. 28. (a) Hot water rice bran liquefaction in a piston reactor at a pressure of
200 bar and (b) production of side products in function of time.
1 to 2 min residence time. This is about the same values as for
xylose and arabinose. If the feed suspension is saturated with
CO
2
, glucose concentrations remain constant in the range of
1000 mg/L and 1500 mg/L. The reason could be found in is the
reduced temperature dependence, when CO
2
is used, at higher
values for the temperature.
Fig. 29. Influence of hot water hydrolysis on the liquefaction of rice bran and
yield of xylose and glucose.
316 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Fig. 30. Glucose: concentration after enzymatic hydrolysis.
7. Fermentation to ethanol and separation with CO
2
Fermentation of the resulting mono-sugars to ethanol is a
technique which is known for a long time and usually carried out
with strains of bakers yeast (Saccharomyces cerevisiae). In order
to obtain ethanol from the sugar solution of about 100–200 g/L,
containing C5- and C6-sugars, still a satisfying consortium of
micro-organisms has to be developed. This is outside of the
topic covered here, therefore, we assume that an aqueous ethanol
solution of 7 to 10% is available after fermentation, which now
has to be concentrated to ≥99.8 wt.% ethanol. Sakaki et al. [76]
contributed to the description of the fermentability of cellulose
decomposition products resulting froma near-critical hydrolysis
treatment.
SFE-technology can provide an alternative for the separation
of CO
2
solution, reducing the number of columns and applying
only separating agents inherent to the process [77,78].
In the past decade, numerous studies have been performed
to describe the separation efficiency of the system carbon diox-
ide/ethanol/water at elevated pressures and temperature [79].
Vapor–liquid equilibrium (VLE) data of CO
2
+ethanol +water
and its binary mixtures have been published in a wide range
of temperature and pressure. From an economic point of
view, gas extraction of ethanol +water mixtures should yield
ethanol of high purity to compete with conventional pro-
cesses. However, in order to get a high solubility of ethanol
in the vapor phase, many studies were carried out at condi-
tions of complete miscibility of ethanol and CO
2
[80–82]. At
these conditions, the phase behavior of the ternary mixture
CO
2
+ethanol +water is of type I and anhydrous ethanol cannot
be produced.
One of the first studies reporting the possibility to pro-
duce anhydrous ethanol by means of CO
2
without adding any
entrainer was by Nagahama et al. [83]. Experiments were car-
ried out at conditions of type II phase behavior. Further VLE
measurements of the mixture CO
2
+ethanol +water were per-
formed at Kobe Steel Ltd. in Japan (e.g. Furuta et al., [84,85])
at 10.1 MPa and 313 K, 323 K and 333 K. The separation factor
at 100 bar, 60

C, decreased from around 30 at infinite dilution
Fig. 31. The system EtOH–H
2
O–CO
2
at (a) 100 bar, 60

C (type II) and (b) 140 bar, 60

C (type I) .
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 317
of ethanol in water to approximately 1.25 at infinite dilution of
water in ethanol.
Data have been concluded and transformed to Fig. 31.
The Ponchon-Savarit diagrams Fig. 31 can be interpreted
as follows: at a pressure of 100 bar and a temperature of
60

C, the phase equilibrium between the light phase and the
heavy liquid phase exists up to absolute purity. The con-
odes which are connecting the referring liquid and gaseous
phase show a high slope. This indicates immediately that an
important number of equilibrium plates in a counter-current
supercritical fluid extraction column is necessary when the pure
ethanol extract shall be produced from low concentrated feed
input.
At a pressure of 140 bar, the phase equilibrium line closes at
a maximum ethanol concentration of about 90%, which means
that this pressure is not adapted for the absolute ethanol produc-
tion. However, the difference in extract and raffinate density
allows to establish a stripping system, which separates pure
water from the feed solution with a low number of equilibrium
plates.
Consequently, process design may be optimized when car-
rying out the supercritical extraction in two pressure steps: the
enrichment section (7%→100% EtOH) at 100 bar, 60

C, and
a stripping section (7%→0% EtOH) at 160 bar and 60

C.
7.1. Estimation of the necessary number of theoretical
equilibrium plates
Numerical modeling tools or graphical methods allow to cal-
culate the number of equilibriumplates for a required product or
raffinate purity, provided a reflux ratio and the number minimum
equilibrium plates has been defined.
The number of theoretical stages were calculated by the
Ponchon-Savarit method [88]. Fig. 32 illustrates calculated
results that are based on a feed mixture of 10 wt.% ethanol that
Fig. 32. Calculation of the number of theoretical plates for separation of
EtOH–H
2
O: CC-SFE: 100 bar, 60

C, EtOH concentrations: feed 7%, extract
99.9%, raffinate 0.1%.
is separated into an ethanol-rich extract (99.9 wt.% ethanol) and
a water-rich raffinate (0.1 wt.% ethanol). The minimum reflux
ratio for this separation task was calculated to be 10, equal
to a minimum solvent-to-feed ratio of around 2.5, while the
minimum number of stages is 35–40.
Ethanol separation froma feed mixture with 10 wt.%ethanol
should be carried out at solvent-to-feed ratios between 20 and
30.
Solvent-to-feed ratios are relatively small compared to other
counter-current gas extraction processes. This is due to large
separation factors and a solubility of pure ethanol in CO
2
of
5 wt.% at the conditions investigated. Instead of the solvent-to-
feed ratio, the ratio of solvent flow rate to product flow rate
should be used as an indicator for operating costs.
If the extract is the only product, as in ethanol purification,
the solvent-to-extract ratio becomes 300 kg/kg at a solvent-to-
feed ratio of just 30 kg/kg. The influence of feed composition
and the number of theoretical stages on the solvent-to-extract
ratio reveals that the production of pure ethanol by means of
supercritical CO
2
needs the highest solvent-to-extract ratiowhen
using a feed with less than 20 wt.% ethanol [74].
Ethanol solubility below 0.2 wt.% can only be achieved at
pressures close to the vapor pressure of liquid CO
2
. Large
amounts of CO
2
dissolve in the ethanol-rich phase. Therefore,
separation of ethanol and CO
2
has to be optimized by solvent
distillation.
7.2. Experimental reliability tests
7.2.1. Counter current SFE
Lab scale experiments on ethanol/water purification have
been performed to confirmthe numerical estimation and to prove
the feasibility of enriching ethanol above the azeotrope point. In
our work, the experimental unit and the procedure is given in
detail by Budich et al. [77] (Fig. 33).
Fig. 33. Draft of the lab scale CC-SFE unit.
318 C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321
Table 4
Experimental results for purification of ethanol from aqueous solutions by countercurrent extraction. Extractor: temperature 60

C; pressure: 100 bar; separator:
temperature: 50–60

C; pressure: 50–60 bar
Expt. no. Wt.% ethanol in feed Feed flow rate (kg/h) CO
2
flow rate (kg/h) Solvent to feed ratio Wt.% ethanol in extract Wt.% ethanol in raff.
1. 70 0.36 2 5.5 99.18 69.82
2. 80 0.36 3.6 10 99.62 74.04
3. 80 0.36 4.32 12 98.65 70.22
4. 80 0.36 5.76 16 99.68 59.75
Counter-current multistage extraction was carried out in lab-
oratory extraction columns of 6 m total height (17.5 mm and
25 mm i.d., equipped with 6 m and 2 m×2 m, respectively of
Sulzer EX packing) [75].
Loaded solvent was withdrawn from the top. Solvent and
extract were separated by pressure reduction down to 5 MPa. In
addition, solvent distillation was established as proposed by De
Filippi and Vivian [86] and reported by Ikawa et al. [87].
Experimental results for a purification of ethanol with a
counter-current extraction column are summarized in the fol-
lowing table (Table 4).
7.2.2. Hydrodynamics
Mass transfer is of some concern in the process of ethanol
purification. During experiments with feed mixtures of low
ethanol content, the height equivalent to one theoretical stage
(HETS) was found to be in the range of 1 m. When a feed with
94 wt.% ethanol was used, HETS was in the range of 0.33 m.
HETS values are reported in the literature [77]. From these data
can be concluded that there are two different regimes of mass
transfer, the one in the mostly aqueous region with high HETS,
and the one in the ethanol region with low HETS.
Determination of the capacity of a counter-currently operated
gravity driven column requires information about the flooding
point. Budich et al. [77] have measured fluid dynamic capacities
for the system ethanol–water–CO
2
in a counter-current column
with Sulzer EX packing. Data of flooding point measurements
are shown in the Fig. 34. Ageneral correlation has been reported
by Stockfleth and Brunner [89] (Fig. 34).
Fig. 34. Flooding point diagram of the system EtOH–water–CO
2
for Sulzer EX
Packings, parameter: concentration of EtOH in water.
Maximum cross-section capacity was found to be a func-
tion of the ethanol content of the solvent-free liquid phase.
Changes in flooding behavior at high ethanol concentrations
between100 wt.%and70 wt.%are mainlydue tothe influence of
phase composition on the density of the phases. With a further
decrease in ethanol content of the liquid phase, the influence
of viscosity and surface tension of the liquid phase becomes
significant. Therefore, for the removal of ethanol from the fer-
mentation broth down to lowconcentrations, the counter-current
gravity column seems not to be sufficient. Instead, a mixer-
settler was experimentally applied for that task, with excellent
results.
7.2.3. Mixer settler SFE
The mixer settler unit (Fig. 35) has been described by
Schaffner et al. [90]. Experiments in the high-pressure pilot
plant have been performed with a feed input of 5% EtOH.
Process conditions were 140 bar and 60

C. Although simula-
tion suggested the necessity of only 2 mixing plates, due to
constructive reasons, the apparatus was operating with 5 cells.
A pure water raffinate output (99.6% water) was achieved at
S/F=74, mixer-settler extract contains consequently 20–30%
ethanol.
7.3. Design
With the information given above, it may directly be con-
cluded that a “conventional” counter-current SFE, operating at
100 bar and 60

C may lead to unrealistic diameter design in
industrial scale (Table 5). Even a CC-SFE which operates with
two pressure steps will still require considerable investment in
autoclave volume:
Table 5
Scale up of the ethanol purification process with a single countercurrent SFE
unit
Feed 1 t/h
Solvent 3 t/h
Extract 70 kg/h 99.95% wt EtOH
Reflux 700 kg/h
Raffinate 930 kg/h 0.5% wt EtOH
Process conditions stripping section 160 bar, 60

C
Min diameter stripping section D=1.6 m, h =1 m,
vol =2 m
3
Process conditions enriching section 100 bar, 60

C
Min diameter enriching section D=0.35 mh =22 m,
vol =2.2 m
3
Packing: Sulzer EX
C. Schacht et al. / J. of Supercritical Fluids 46 (2008) 299–321 319
Fig. 35. Draft of the lab scale mixer-settler unit.
Alternatively the production of high purity ethanol (≥99.5%)
from fermentation broth (7–10% ethanol) by using supercritical
CO
2
is suggested in the following steps:
(1) Feed of 7–10% ethanol is fed to a counter current mixer set-
tler unit with 2–3 stages, operating at 150 bar, 60

C. Ethanol
is enriched to about 75–80%.
(2) The extract from the mixer-settler unit is fed to counter cur-
rent packed column operating at 100 bar and 60

C. In this
column, ethanol is concentrated to ≥99.5% purity.
(3) The raffinate from this packed column contains 60–70%
ethanol. It is fed to the mixer settler unit as reflux.
The design of the process plant was performed for the treat-
ment of 4000 t/year (resp. 1 t/h) of a feed input of a 7% ethanol
solution. Operating time is 4000 h/year, since for this design a
typical sugar cane processing plant in India with a capacity of
25,000 t/year was used as basis. Hence the distilleries operate at
comparable capacities.
Investment cost for the CC-SFE and mixer settler can be
estimated according delValle et al. [91]. Extraction column has
an inner diameter of 0.3 m, the autoclave volume of the mixer
settler is negligible.
Side product carbon dioxide from fermentation (approxi-
mately 34 kg/h) is equivalent to about 0.5% of the CO
2
cycle,
it may be used to replace the mass losses of the solvent at the
extract and raffinate outlet.
8. Conclusion
Fuel ethanol production from ligno-cellulosic material is not
commercial so far. There is still a wide range of improvements
to be achieved before such a process is competitive to mineral oil
based fuels. SFE technology can contribute at several process
steps advantageously. Several aspects could not be discussed,
e.g. the role of SFE in lignin processing or the role of SCWO in
handling the residues. But it became obvious that only a com-
bination of technologies will be able to achieve the goal. In this
case it is biotechnology and high-pressure technology. There-
fore, the cooperation of experts in these fields seems to be the
most efficient way to overcome certain difficulties within this
field.
Acknowledgements
Numerous co-workers and students contributed to the data
base of this work, as can be seen from the list of references.
In addition, several organisations and companies contributed
over the years: Deutsche Forschungsgemeinschaft (DFG),
Deutsche Bundesstiftung Umwelt (DBU), DSM Vitamins Ltd.
(former F. Hoffmann-La Roche AG).
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