ISSN 20700504, Catalysis in Industry, 2011, Vol. 3, No. 1, pp. 41–46. © Pleiades Publishing, Ltd., 2011.

Original Russian Text © E.N. Efremenko, N.A. Stepanov, A.B. Nikolskaya, O.V. Senko, O.V. Spiricheva, S.D. Varfolomeev, 2011, published in Kataliz v Promyshlennosti.

BIOCATALYSIS

Biocatalysts Based on Immobilized Cells of Microorganisms

in the Production of Bioethanol and Biobutanol
E. N. Efremenkoa, b, N. A. Stepanova, b, A. B. Nikolskayab, O. V. Senkoa,
O. V. Spirichevaa, and S. D. Varfolomeeva, b
a
Faculty of Chemistry, Moscow State University, Moscow, 119991 Russia
bEmanuel Institute of Biochemical Physics, Russian Academy of Sciences, Moscow, 119334 Russia

Received May 11, 2010

Abstract—In this work, we discuss the processes for the production of bioethanol and biobutanol, which are
promising alternative fuels, using biocatalysts based on cells of various microorganisms immobilized in
poly(vinyl alcohol) cryogel. Biocatalysts based on immobilized cells reliably allow ethanol production from
a variety of industrial and agricultural wastes (wheat straw, beet and sugarcane bagasse, parchment, corn cobs,
soybean processing waste) with a high degree of conversion of consumed substrates to the target product. Eth
anol concentrations are appreciably higher in media with biocatalysts than in free cells of the same microor
ganisms. It is found that immobilized cells of filamentous fungi can convert a wider range of the sugars con
tained in processed media to ethanol than commonly used yeasts. It is shown that the immobilization of the
genus Clostridium cells that produce butanol enables us to reliably change the ratio of solvents that accumulate
in the medium during acetone–butanol–ethanol fermentation in the direction of a greater amount of
butanol, thereby improving the process’s characteristics relative to presentday technologies based on free
bacterial cells.

Keywords: biocatalyst, immobilized cells, yeasts, filamentous fungi, bioethanol, biobutanol.

DOI: 10.1134/S207005041101003X

INTRODUCTION losecontaining feedstock (CCF) leads to the resulting

The immobilization of cells of microorganisms
refers to any restriction on the freedom of their move
ment in space, and is achieved by using different cell
carriers. Immobilization allows us to generate and
maintain high concentrations of cells in reactionary
media during biotechnological processes, to increase
their rates and the stability of cell operation, to use
them repeatedly and consistently, and to move on to
the organization of continuous process procedures [1].
The use of biocatalysts of immobilized forms of cells of
microorganisms in various processes of biofuel pro
duction is an innovative approach to solving the prob
lems of intensifying and improving the economic and
environmental attractiveness of existing facilities [2].
It is known that the stability of heterogeneous biocat
alysts based on immobilized cells of microorganisms
(bacteria, yeasts, and filamentous fungi) remains
unchanged after their longterm storage [3].
Our vast natural reserves of renewable organic feed
stock containing cellulose, along with our large vol
umes of agricultural and industrial wastes, are one
basis for the development and use of technologies for
producing biofuels from them. It is known that the
mandatory physicochemical pretreatment of cellu
media containing furfurol, phenol derivatives, tan
nins, acetic acid, terpenes, and other substances [4]
that inhibit the catalytic activity of suspension yeast
cells and reduce the efficiency of ethanol production
in general. In this regard, the interest in using biocata
lysts based on immobilized yeast cells, which exhibit
high resistance to negative factors, is quite high [5–8].
It is well known that yeasts, which are commonly
used for the conversion of the monosaccharides con
tained in hydrolysates of various CCFs into ethanol,
cannot convert pentoses (xylose and arabinose),
which are often contained in high concentrations (up
to 20 g/l) in media cultivated by cells, to the target
product. It has been shown that it is possible to use fil
amentous fungi of the genera Aspergillus, Mucor, and
Rhizopus for the conversion of pentoses and monosac
charides into ethanol, but it is also known that they are
extremely sensitive to the ethanol that accumulates in
the medium [9, 10], and a concentration of 45 g/l
results in almost complete inhibition of the fermenta
tion process.
At present, butanol is produced from petroleum via
the hydrolysis of halogenoalkanes or the hydrolysis
and hydration of alkenes. Due to the growing interest

41
42 EFREMENKO et al.
Table 1. Concentration, g/l, (numerator) and yield, %, (denominator) of ethanol from the theoretically possible level as a
result of the conversion of sugars contained in fermentolysates of agricultural and industrial wastes by (A) cells immobilized
in PVA cryogel and (B) free cells of S. bayanus yeast
init
Substrate C substr , g dry wt–wt/l
Soybean processing wastes 265
Sugarcane bagasse 50
Parchment 50
Wheat straw 50
Beet bagasse 50
Corn cobs 50
in biofuels from renewable energy sources, however,
the production of butanol by the biotechnological
method is of particular interest. The fermentation of
sugars by genus Clostridium cells with the formation of
a mixture of acetone–butanol–ethanol (ABE) sol
vents has long been known, but its industrial use is lim
ited, since a 1–2% concentration of butanol accumu
lating in the medium substantially inhibits the metab
olism of cells [11, 12]. In this context, it is better to use
butanolproducing cells in the immobilized form,
which contributes to their high stability relative to the
target product [13, 14].
In this work, we studied the possibility of using a
new biocatalyst based on Saccharomyces cerevisiae
ph.v. bayanus yeast cells immobilized into a cryogel of
poly(vinyl alcohol) (PVA) [15] to produce ethanol
from agricultural and industrial wastes subjected to
enzymatic treatment. For the sake of comparison, the
target metabolite was produced under similar condi
tions using free cells of the same culture. We also stud
ied the effectiveness of using a new biocatalyst devel
oped on the basis of filamentous fungi cells immobi
lized in PVA cryogel [16] to produce ethanol from
CCF fermentolysates, as opposed to free cells of the
same cultures. In addition, we studied the effect of
immobilizing Clostridium acetobutylicum cells in PVA
cryogel on their efficiency for biobutanol.
The choice of PVA cryogel in a work on designing
biocatalysts was not random; it was dictated by this
particular carrier being one of the best for the immo
bilization of microorganism cells used effectively in
various biotechnological processes [17–21].
MATERIALS AND METHODS
In this work, we used filamentous fungi Mucor cir
cinelloides VP 426 un 20, Rhizopus oryzae NNRL
395, Fusarium oxysporum 11 dn1, and Aspergillus ter
reus yu4a1, which were maintained on an agarized
medium with a composition of 20 g/l glucose, 0.2 g/l
Ethanol
τprocess, h
A B
24 53.7/90.3 50.5/85.0
90 9.0/95.1 8.7/92.0
90 10.8/94.9 10.3/91.9
90 11.7/92.6 11.1/87.8
96 15.0/82.9 8.2/45.3
24 8.6/98.3 7.6/87.4
MgSO4, 0.2 g/l CaCO3, 200 g potatoes, and 20g/l agar
(pH = 6.8). To grow spores, the fungal culture was
plated in Petri dishes and on pads with the agarized
medium. After spore formation, the Petri dishes and
pads were stored at +4°C.
Cells of Saccharomyces cerevisiae ph.v. bayanus yeasts
(Zymasil Killer) (AEBgroup) were stored at 4–8°C on a
agarized semisynthetic medium. For the accumulation of
yeast biomass, we used a liquid culture medium with a
composition, g/l, of 10 glucose, 2.0 yeast extract,
0.5NaCl, 2.5KH2PO4, 0.5MgSO4 ⋅ 7H2O, and
2.0(NH4)2SO4.
The yeast cells were cultivated at 26°C under strict
aerobic conditions to the end of the logarithmic
growth phase under constant stirring (180 rpm) using
an IRC1U thermostated shaker (Adolf Kunner G
Apparaebau, Switzerland). The resulting yeast biom
ass was separated at 10000 rpm for 10 min on a Beck
man 2–21 centrifuge (United States) and subse
quently used for immobilization in PVA cryogel.
To obtain biobutanol, we used strictly anaerobic
cells of the Clostridium acetobutylicum B1787 strain.
The accumulation of biomass of C. acetobutylicum
cells for their immobilization in PVA cryogel and the
cultivation of immobilized cells were performed under
anaerobic conditions at 37°C in a medium with a
composition, g/l, of 10 peptone (tryptone), 5 yeast
extract, and 25 glucose.
The immobilization of cells of filamentous fungi,
yeasts, and bacteria in PVA cryogel was performed
according to previously developed methods correspond
ing to different cell types [15, 16, 21]; pH in the experi
ments was controlled by potentiometric measurements
using a PBL modem pH meter (Switzerland).
The concentration of sugars in the different media
was determined by liquid chromatography under high
pressure using an 1100 Agilent chromatograph with an
amperometric detector (United States) and a Dionex
Carbopak PA20 anion exchange column. A solution
of 7.5 mM NaOH was used as eluent. The concentra
CATALYSIS IN INDUSTRY Vol. 3 No. 1 2011
 BIOCATALYSTS BASED ON IMMOBILIZED CELLS OF MICROORGANISMS 43

tion of glucose in the medium was determined by the Table 2. Ethanol concentrations accumulating during the con
glucosidase method using a standard reagent (Impakt, version of various sugars (45 g/l) into ethanol for 48 h under the
Russia). action of free (numerator) and immobilized (denominator)
The concentrations of ethanol and butanol were cells of the Mucor circinelloides fungus
determined by gas chromatography using a Kristaly Ethanol concen Ethanol con
uks 4000 M chromatograph with a flame ionization Sugars Sugars
tration, g/l centration, g/l
detector (FID). Nitrogen was used as the carrier gas.
The temperature of thermostat columns was 190°C, Arabinose 1.9/7.2 Raffinose 2.3/4.0
while those of the detector and evaporator were 240 Galactose 2.2/3.4 Ribose 3.4/16.5
and 260°C, respectively. Glucose 4.0/16.0 Saccharose 2.8/4.1
Xylose 1.6/2.3 Fructose 6.4/14.3
RESULTS AND DISCUSSION Maltose 3.7/5.7 Cellobiose 3.8/12.7
Biocatalyst based on immobilized yeast cells for bio
ethanol production. We studied the possibility of using
the conversion of sugars contained in industrial and Table 3. Concentration of major monosaccharides in the fer
agricultural wastes after their enzymatic pretreatment mentolysates of CCF samples (g/l)
for ethanol production (Table 1). In the fermentation of CCF Glucose Arabinose Xylose
similar substrates (e.g., sugarcane bagasse, wheat straw,
and waste paper), foreign researchers observed a 50– Wheat straw 24.0 – 5.2
80% yield of ethanol for free yeast cells [22–24], which Parchment 23.0 – 4.2
is much lower than the value obtained in this work using
Beet bagasse 13.0 10.4 0.2
both free and immobilized cells. There is no similar
information on immobilized yeast cells in the literature Sugarcane bagasse 18.5 2.9 6.1
describing the conversion of the studied substrates,
which means that these types of feedstock were treated
with this biocatalyst for the first time. cells. In this regard, our biocatalyst developed on the
It was shown that in all of the tested samples of basis of immobilized yeast cells can be of great practi
feedstock fermentolysates, the degree of conversion of cal interest.
sugars to ethanol under the action of immobilized Biocatalyst based on immobilized cells of filamen
yeast cells was higher than in the case of free cells. In tous fungi for ethanol production. We initially studied
addition, our biocatalyst in the form of immobilized the possibility of using cells of filamentous fungi
cells acted effectively in various media and provided a immobilized in PVA cryogel in the processes of con
yield of the target product above 90% (see Table 1). verting sugars to ethanol. A comparative analysis of the
Note that soybean wastes are mostly used for the effectiveness of the action of free cells and biocatalyst
production of biodiesel fuel [25]. There are no litera based on immobilized cells of Mucor circinelloides fil
ture data on the use of soybean processing waste for amentous fungus in the conversion of sugars to ethanol
the production of ethanol fuel. It was thus shown for showed that the concentration of accumulating target
the first time that our biocatalyst based on yeast cells product was 1.4–4.8 times higher when using immobi
immobilized in PVA cryogel can be effectively used to lized cells than it was for free cells over the same time
produce ethanol from soy hydrolysates, which are (Table 2). It was found that the same immobilized cells
found as production waste at plants producing edible of fungi can be multiply used for the fermentation of
soy protein; in this case, the resulting concentration of various individual sugars in a batch mode; in addition,
ethanol can be 5–10 times higher than that from other the efficiency of biocatalysts did not changed for at
sources of raw materials (Table 1). least five operation cycles (24 h each).
Despite intensive research conducted all over the We studied the possibility of using biocatalysts
world to find ways of raising the production of ethanol based on immobilized cells of filamentous fungi for
and reducing its cost, there is an extremely short list of the conversion of sugars in fermentolysates of agricul
works [26–29] in which the industrial use of immobi tural wastes whose composition is given in Table 3. It
lized cells in these processes is discussed; this can be was found that under the action of free and immobi
attributed to the absence of practically meaningful lized cells of filamentous fungi, the conversion of sug
samples of biocatalysts in the present day world mar ars contained in the media resulted in the accumula
ket. Moreover, today the possibility of using immobi tion of ethanol. When using biocatalysts based on
lized yeast cells for ethanol production is being dis immobilized cells, the concentration of ethanol was
cussed more actively with respect to starchcontaining 22.5 times higher (depending on the strain and the ini
feedstock [26–8, 30] and lactosecontaining wastes tial feedstock) than the results obtained in the same
[29, 31], rather than CCFs, since it is assumed that the media using free cells of filamentous fungi (Table 4).
last source of sugars is hard to process and contains a The yields of ethanol in media that are fermentoly
large number of substances that can adversely affect sates of beet and sugarcane bagasse, calculated

CATALYSIS IN INDUSTRY Vol. 3 No. 1 2011

44 EFREMENKO et al.
Table 4. Use of free cells of filamentous fungi (numerator) and cells immobilized in PVA cryogel (denominator) to produce
ethanol from enzymatically pretreated CCF samples
CCF, filamentous fungus Glucose*, g/l
Sugarcane bagasse Mucor circinelloides 18.5
Wheat straw Mucor circinelloides 24
Beet bagasse Mucor circinelloides 13
Parchment Rhyzopus oryzae 23
* In the medium after enzymatic processing of CCF.
according to the concentration of converted glucose,
exceeded the 100% theoretically possible level (see
Table 4). It is obvious that these values, which exceed
the expected concentrations of ethanol, were found in
these particular media due to the conversion of pen
toses (mostly arabinose) into ethanol, which is consis
tent with the data of Table 3.
As in the case of immobilized yeast cells (see Table 1),
for biocatalysts based on different immobilized cells of
filamentous fungi, we studied the conversion of soy
bean processing wastes into ethanol (Table 5). The ini
tial feedstock sample contained saccharose, stachy
ose, raffinose, fructose, and galactose. Due to the high
viscosity of the initial solutions for the conversion of
sugars to ethanol, before introducing the immobilized
cells of filamentous fungi, they were diluted with water
to a final total concentration of sugars in the medium
of 50 g/l.
As in other experiments, the best parameters for the
conversion of sugars to ethanol were found for the bio
catalyst prepared on the basis of Mucor circinelloides
cells, which provides an almost 90% yield of the target
product in a given medium. In comparing immobi
lized cells of filamentous fungi and yeasts, the latter
must be regarded the best producers of ethanol; they
Table 5. Conversion of sugars contained in soybean processing
wastes into ethanol under the action of immobilized cells of fil
amentous fungi for 96 h
Immobilized cells of filamentous fungi Ethanol, g/l
Fusarium oxysporum 9.3 ± 0.5
Mucor circinelloides 23.4 ± 1.0
Aspergillus terreus 10.9 ± 0.3
Ethanol
C, g/l Yield, % of theoretically possible
3.9 41.5
 
11.0 117.0
0.4 3.2
 
9.0 73.5
6.9 104.1
 
9.1 137.3
1.4 11.9
 
9.1 77.6
exhibit higher rates of ethanol production, due appar
ently to their high tolerance to negative factors accom
panying the process, particularly the accumulation of
the target product and declining pH of the medium in
the fermentation process. However, filamentous fungi
demonstrated the possibility of using a much wider
range of substrates that can be converted to ethanol.
Biocatalyst based on immobilized cells of Clostrid
ium acetobutylicum bacteria for biobutanol production.
We developed a biocatalyst based on hydrogenpro
ducing anaerobic cells of Clostridium acetobutylicum
bacteria immobilized in PVA cryogel. The concentra
tion of humid cell biomass in the composition of this
biocatalyst was 10%, and the concentration of PVA
cryogel was 15%.
The ABE process with the participation of the biocat
alyst was performed in a minireactor in which the ratio
of medium volume to the gas phase volume was 2.0.
Analysis of the culture liquid after 72 h of the action
of the biocatalyst based on immobilized Clostridium
acetobutylicum cells showed that the medium con
tained, g/l, 0.95 acetone, 2.90 butanol, 0.23 ethanol,
0.66 acetate, and 3.00 butyric acid. A comparison of
these data with the same characteristics of a medium
prepared under similar conditions yet using suspen
sion cells allowed us to conclude that the immobiliza
tion of cells and their use in the ABE process under the
chosen conditions lead to changes in the composition
of the culture liquid. The concentration of butanol
that accumulates in the culture medium of immobi
lized cells for three days was thus 2.5 times higher than
that in the medium with free cells (figure). This change
affected the ratio of solvents (acetone : butanol : etha
nol) that accumulate in the medium: almost 4 : 12 : 1
instead of the 3 : 6 : 1 found for free cells of the same
culture. Determination of the residual concentration
of glucose in the medium with immobilized and free
cells showed that its consumption was higher by 35%
in the case of immobilized cells.
CATALYSIS IN INDUSTRY Vol. 3 No. 1 2011
 BIOCATALYSTS BASED ON IMMOBILIZED CELLS OF MICROORGANISMS
Alcohol concentration, g/l
3.0
Ethanol
Butanol
2.5
2.0
1.5
1.0
0.5
0 in the amount of butanol. This makes the industrial
a b
Accumulation of alcohols by (a) free and (b) immobilized
cells of Clostridium acetobutylicum bacteria under similar
conditions of their cultivation for 72 h (cell concentration
in the medium, 43 g/l; 37°C).
Earlier, in works concerned with studying immobi
lized cells of the genus Clostridium [13–14], the
authors observed no similar changes in the ratio of
produced solvents. This result was apparently due to
the selected carrier for cells as well as conditions of
their immobilization and use in the ABE process.
It was shown that the biocatalyst based on Clostrid
ium acetobutylicum cells immobilized in PVA cryogel
can be repeatedly used to produce butanol in the batch
mode of minireactors. In addition, the above ratio of
solvents in the medium remained almost unchanged
for at least five operation cycles of 72 h each.
The presentday development of butanol produc
tion involves the preparation of new genetically modi
fied strains of the genus Clostridium cells and their use
in the ABE process to derive media with a changed
ratio of accumulating solvents with an increased
amount of butanol. In this work, the immobilization of
cells for the production of butanol allowed us to obtain
a similar result using a thoroughly studied bacterial
culture and to increase the effectiveness of its use, i.e.,
to increase the yield of the target product without
searching for new strains, recombinant or nonrecom
binant, and introducing them into the process.
CONCLUSIONS
We showed that the production of bioethanol from
a variety of industrial and agricultural wastes (wheat
straw, beet and sugarcane bagasse, parchment, corn
cobs, and soybean processing waste) using biocatalysts
based on yeast and filamentous fungi cells immobi
lized in PVA cryogel is highly efficient. It was found
that immobilized cells of filamentous fungi can con
vert a wider range of sugars contained in cultivated
media to ethanol than the yeasts commonly used in
ethanol fermentation.
CATALYSIS IN INDUSTRY Vol. 3 No. 1 2011
45
Biocatalysts based on immobilized cells of both
yeasts and filamentous fungi exhibited higher degrees
of conversion of consumed substrates into the target
product in the processes of ethanol production, and
thus the accumulation of higher concentrations of
ethanol in comparison to free cells of the same micro
organisms. Analysis of the results from studies of yeasts
and filamentous fungi showed that the described ten
dencies in the change of the characteristics of the pro
cesses using immobilized cells are general.
It was found that the immobilization of butanol
producing cells enables us to reliably change the ratio
of solvents accumulating in a medium with an increase
production of this target product by the biotechnolog
ical method using our new biocatalyst more attractive
than the process that traditionally employs free bacte
rial cells.
ACKNOWLEDGMENTS
This work was supported by the Program for Basic
Research no. 19 of the Presidium of the Russian Acad
emy of Sciences, “Chemical Aspects of Power Engi
neering,” and the Moscow State University Program
for Promising Lines of Development in 2010–2013,
PNR5 “Energy Efficiency, Materials, and Bionano
systems.”
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