Cellulose Biorefinery Based on a Combined Catalytic and Biotechnological Approach for Production of 5-HMF and Ethanol

Article abstract of DOI:10.1002/cssc.201601244

In this study, a combination of catalytic and biotechnological processes was proposed for the first time for application in a cellulose biorefinery for the production of 5-hydroxymethylfurfural (5-HMF) and bioethanol. Hydrolytic dehydration of the mechanically activated microcrystalline cellulose over a carbon-based mesoporous Sibunt-4 catalyst resulted in moderate yields of glucose and 5-HMF (21.1–25.1 and 6.6–9.4 %). 5-HMF was extracted from the resulting mixture with isobutanol and subjected to ethanol fermentation. A number of yeast strains were isolated that also revealed high thermotolerance (up to 50 °C) and resistance to inhibitors found in the hydrolysates. The strains Kluyveromyces marxianus C1 and Ogataea polymorpha CBS4732 were capable of producing ethanol from processed catalytic hydrolysates of cellulose at 42 °C, with yields of 72.0±5.7 and 75.2±4.3 % from the maximum theoretical yield of ethanol, respectively.

Full text of DOI:10.1002/cssc.201601244

Accepted Article
Title: Cellulose biorefinery based on combined catalytic and
biotechnological approach for production of 5-HMF and ethanol
Authors: Ksenia N. Sorokina, Oxana P. Taran, Tatiana B. Medvedeva,
Yuliya V. Samoylova, Alexandr V. Piligaev, and Valentin N.
Parmon
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ChemSusChem
10.1002/cssc.201601244
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Cellulose biorefinery based on combined catalytic and
biotechnological approach for production of 5-HMF and
ethanol
Ksenia N. Sorokina*[a,b], Oxana P. Taran [a,c], Tatiana B. Medvedeva , Yuliya V. Samoylova ,
[a]
[a]
[
a]
[a,b]
Alexandr V.Piligaev , Valentin N. Parmon
renewable feedstocks for the production of motor fuels
and valuable chemicals. The main advantages of this
approach are its sustainability[1] accompanied by the low
power consumption[2] as well as the reduced emission of
Abstract
In this study,
a combination of catalytic and
biotechnological processes was proposed for the first
time for application in a cellulose biorefinery for the
production of 5-HMF and bioethanol. Hydrolytic
CO
2
.[3] For this purpose, modern biorefineries have been
developed for the conversion of lignocellulosic biomass
into a spectrum of marketable products and energy.[4]
The lignocellulosic feedstock sources for the
biorefineries include energy crops,[5] forestry[6] and
dehydration
of
the
mechanically
activated
microcrystalline cellulose over
a
carbon-based
mesoporous Sibunt-4 catalyst resulted in moderate yields
of glucose and 5-HMF (21.1–25.1% and 6.6–9.4%). 5-
HMF was extracted from the resulting mixture with
isobutanol and subjected to ethanol fermentation. A
number of yeast strains were isolated that also revealed
high thermotolerance (up to 50°C) and resistance to
inhibitors found in the hydrolysates. The strains
Kluyveromyces marxianus C1 and Ogataea polymorpha
CBS4732 are capable of producing ethanol on processed
catalytic hydrolysates of cellulose at 42°C, with yields of
[7]
agricultural waste and residues . Biomass itself consists
of complex biopolymers comprised of cellulose (40-60%),
hemicellulose (30%), and lignin (10–30%).[8] The main
products of its processing of are holocellulose and
cellulose. They can be separated from lignin by various
pretreatment methods,[9] including hydrolysis with dilute
and concentrated sulfuric acid,[10] alkaline hydrolysis,[11]
the organosolv process,[12] steam explosion,[13] ammonia
fiber explosion (AFEX),[14] etc. The relative simplicity of
cellulose processing allows a wide variety of products
that are so-called “platform molecules” for industrial
applications to be obtained.[15] Sugar monomers can be
derived from biomass by hydrolysis with homogenous
soluble catalysts such as mineral acids,[10] alkalis[16] and
enzymes,[17] and in many cases, a relatively high yield
can be observed (> 90%).[18] However, the main
disadvantage of this process is that the catalyst needs to
be separated from the products and regenerated after
the reaction. To avoid this problem, carbon-based solid
acid catalysts may be used for the hydrolysis of cellulose.
Moreover, this allows its conversion into the valuable
products via a one-pot process. The combination of
cellulose hydrolysis with acid into glucose[19,20] and its
72.0±5.7% and 75.2±4.3% from the maximum theoretical
yield of ethanol, respectively.
Introduction
Due to instability on the global energy and
hydrocarbon feedstock markets, there is an increasing
trend toward the development of sustainable
technologies that employ locally available sources of
[
a]
K.N. Sorokina, Prof. O.P. Taran, T.B. Medvedeva, Y.V. Samoilova,
A.V.Piligaev, Prof. V.N. Parmon
Boreskov Institute of Catalysis (BIC)
630090, Novosibirsk, Lavrentieva ave. 5 (Russian Federation)
E-mail: sorokina@catalysis.ru
K.N. Sorokina, Prof. V.N. Parmon
Novosibirsk State University (NSU)
[
[
b]
c]
630090, Novosibirsk, Pirogova str. 2 (Russian Federation)
Prof. O.P. Taran
Novosibirsk State Technical University (NSTU)
subsequent dehydration into 5-hydroxymethylfurfural (5-
HMF)[21-23] will produce the key platform molecules as a
630037, Novosibirsk, Prosp. Karla Marksa, 20 (Russian Federation)
Supporting information for this article is given via a link at the end of
the document.
1
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ChemSusChem
10.1002/cssc.201601244
FULL PAPER
feedstock for the manufacturing of a wide variety of
products (diesel and jet fuels, plastics, etc.).[24]
catalyst that was preliminarily oxidized with wet air.[22,23]
5-HMF is recovered by isobutanol extraction from the
mixture, and the remaining sugar mixture is neutralized
and concentrated for subsequent fermentation. The most
effective ethanol producers were selected by screening
of the isolated thermotolerant yeast strains that were able
to grow at 40°C. The parameters studied were the
efficiency of their biomass production at temperatures up
to 50°C as well as tolerance to fermentation inhibitors.
Strains that revealed superior properties after screening
(all belonging to K. marxianus and O. polymorpha
species) were used for the ethanol fermentation of the
processed hydrolysate.
5-HMF can be separated from the sugars remaining
in the mixture by organic solvent extraction.[25] This
approach will not only allow the recovery of 5-HMF but
also allow the fermentation substrate to be obtained with
a low content of furan derivatives that have an inhibitory
effect on culture growth during the fermentation
process.[26] Glucose, as one of the major reaction
products, can serve itself as the primary substrate for
fermentation
into
various
chemicals,
including
bioethanol,[27] butanol,[28] isobutanol,[29] lipids[30] and
succinic[31] or lactic[32] acids. Among them, ethanol is the
most common product of glucose fermentation and
Results and Discussion
constitutes over 90% of the global bio-fuel production [33]
It is also recognized as a “platform molecule” serving as
precursor for hydrocarbon fuels, chemicals and
.
Preparation and analysis of catalytic
hydrolysates
of
the
mechanically
activated
a
microcrystalline cellulose
aromatic compounds in integrated biorefineries.[34,35] The
fermentation of sugars to ethanol is usually performed
using the yeast Saccharomyces cerevisiae[36] or bacteria
Cellulose is a chemically resistant solid compound
whose structure is stabilized by numerous hydrogen
bonds. This results in the formation of a crystalline
[37]
and Escherichia coli.[38] To
Zymomonas mobilis
structure that significantly complicates the process of the
conversion of cellulose into chemicals.[44,45] Therefore,
reduce the energy consumption of this process, the use
of thermotolerant yeasts such as Kluveromyces
marxianus[39] and Ogataea polymorpha[40] has been
proposed recently. The major advantage of ethanol
production by K. marxianus[41] and O. polymorpha[40] is
their ability to ferment sugars at temperatures up to 45°C,
as opposed to the mesophilic yeast S. cerevisiae that
predominantly grows at temperatures less than 37°C. It
has been shown that the high temperature fermentation
significantly reduces the cost of cooling the fermentation
broth[42] and thereby improves the process economics. In
addition, the high temperature process allows reduction
before processing cellulose by hydrolysis, one has to
pre-activate the cellulose by destroying its crystalline
structure. Among the variety of activation methods,
mechanical activation seems to be a quite effective and
environmentally safe method of increasing the reactivity
and capacity of cellulose by reducing its particle size[46]
and crystalline structure.[47] Moreover, mechanical
activation allows the reduction of the reaction
temperature as well as shortening the time of the
cellulose hydrolysis process.[9]
Before the mechanical activation of the
microcrystalline cellulose, its average particle size,
according microscopic examination, was 114±35 m
of
the
contaminating
microorganism
growth.
Thermotolerant yeasts are able to grow on media
containing glucose, xylose, cellobiose and some other
sugars[40,43] that can be found in biomass hydrolysates.
Thus, these yeasts are promising for application for
cellulosic ethanol production.
(
Figure 1a), while the degree of crystallinity studied by X-
ray diffraction was 80-92%. After mechanical activation,
the particle size decreased to 22.3±8.0 m (Figure 1b).
The X-ray pattern of the mechanically activated cellulose
demonstrates an expansion and a decrease in the
intensities of the 10-1, 101, 021, 002 and 040 peaks of
the sample (Figure 2b), which confirms destruction of the
cellulose crystal structure. The amorphous halo has the
appearance of the X-ray pattern of the mechanically
The aim of this study is to examine the combination
of a one-pot catalytic process of cellulose conversion into
5-HMF and glucose with a solid acid catalyst and a
biotechnological process of glucose fermentation into
ethanol with thermotolerant yeasts. First, the
mechanically activated microcrystalline cellulose is
hydrolyzed in the presence of a solid carbon-based
activated cellulose at 2 equal to 21.5 ° (Figure 2b).
However, in this study the same efficiency of activation
2
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ChemSusChem
10.1002/cssc.201601244
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was not observed compared to results of a previous
study.[22,23] The degree of crystallinity of the sample
estimated by the method[45] was approximately 36%.
For the preparation of the solid acid catalyst for
The oxidation of Sibunit-4 (Sibunit-4-ox) by wet air
has been proven to be efficient for increasing the amount
of acidic groups on the catalyst surface by a factor of six
(0.28 mmol per gram of carbon).[23,48] The acidic group
content was determined by an acid-base titration with
microcrystalline cellulose hydrolysis,
a carbon-based
material Sibunit-4 was used. This material has
a
sodium hydroxide. The specific surface area (SBET)
graphite-like nature, which makes it more durable and
resistant to chemical attack in comparison with activated
carbons. Another advantage of Sibunit is its mesoporous
morphology, which reduces the diffusion limitations
during the catalytic reactions in liquid media and provides
determined by a low-temperature nitrogen adsorption
method for Sibunit-4 was 379 m2 g , while the
mesoporous volume was 0.21 cm3 g . Nitrogen
adsorption isotherms on the surfaces of Sibunit-4 and
Sibunit-4-ox are shown in Figure S1 (supporting
information).
-1
-1
Figure 1.
Microphotographs and size distributions of the
microcrystalline cellulose particles before and after the
mechanical activation: (a) before pretreatment; and (b)
after grinding in the planetary-type mill for 40 min.
Figure 2.
Diffraction patterns of microcrystalline cellulose samples:
(a) before mechanical pretreatment; and (b) after
grinding in the planetary-type mill for 40 min. Dashed
line: amorphous cellulose.
an efficient transport of substrates to the catalyst surface.
The unmodified Sibunit-4 has an extremely low content
of acidic groups on its surface (0.042 mmol per gram of
carbon), which limits its application as a catalyst for the
hydrolysis of chemically stable substrates such as
cellulose.
After the oxidation pretreatment, only
a small
decrease in SBET was observed for the Sibinit-4-ox
catalyst (348 m2 g ), which indicates the slight
destruction of its structure by oxidation. However, the
volume of mesopores increased up to 0.33 cm3 g ,
which can improve the mass-transfer inside the catalyst
-1
-1 [49]
3
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10.1002/cssc.201601244
FULL PAPER
particles. The solid acid catalyst Sibunit-4-ox[23,48] allows
the production of glucose and 5-HMF from the
mechanically activated microcrystalline cellulose with
relatively high yields of up to 45 mol.% and 23 mol.%,
respectively, at a temperature of 180°C. It should be
noted that 5-HMF, as one of the most valuable products
formed during the reaction, appears to be more stable in
the presence of solid acid catalysts, unlike with
homogenous soluble acid catalysts. In the latter case, 5-
HMF is transformed rapidly into a mixture of levulinic and
formic acids.[22,23]
HMF and glucose. Other compounds that were also
found in the mixture included cellobiose, mannose,
fructose, furfural, glycolic, lactic, formic acid and tartaric
acid. Kinetic curves of the reaction are shown on Figure
3. In this study, the maximum glucose yield (23.1±1.5
mol.%) from the cellulose hydrolysate was observed after
5 h. For this reason, in the subsequent experiments, the
reaction was terminated at this time point. The yields of
the main reaction products during activated cellulose
hydrolytic dehydration over Sibunit-4-ox were as follows:
glucose (21.1–25.1 mol.%) and 5-HMF (6.6–9.4 mol.%).
The average yields of the products with their standard
deviations after 5 h of the reaction are presented in Table
1. In this study, after 5 h, the glucose and 5-HMF yields
did not exceed 23.1±2.0 mol.% and 8.0±1.4 mol.%,
respectively. The conversion of unreacted cellulose was
estimated on a TOC analyzer as 54.3%. One may expect
that the reason was a lower efficiency of cellulose
grinding than in recent studies.[23]
Table 1. Product composition of the activated
cellulose hydrolytic dehydration over the solid carbon
catalyst Sibunit-4-ox (after 5 h of reaction).[
a]
-
1
Product
cellobiose
glucose
mannose
fructose
Yield, % [mol%]
0.5 ± 0.0
23.1 ± 2.0
1.4 ± 0.2
1.1 ± 0.1
8.0 ± 1.4
0.8 ± 0.1
0.6 ± 0.1
0.9 ± 0.2
1.2 ± 0.2
0.7 ± 0.1
[g L ]
0.11 ± 0.00
2.48 ± 0.16
0.15 ± 0.02
0.12 ± 0.01
0.60 ± 0.11
0.05 ± 0.01
0.02 ± 0.00
0.04 ± 0.01
0.06 ± 0.01
0.06 ± 0.01
5-HMF
furfural
formic acid
glycolic acid
lactic acid
tartaric aid
[
a] Reaction conditions were same as in Figure 1.
Extraction of 5-HMF with organic solvents
The isolation of 5-HMF, as one of the main products
of the hydrolysis of cellulose, is well-known as an
important step in biomass processing. 5-HMF is poorly
extracted from the reaction mixtures; however, a number
of solvents, including MIBK, ethyl acetate, THF, diethyl
Figure 3.
Kinetic curves of the accumulation of the reaction
products for the hydrolytic dehydration of the activated
cellulose over the solid carbon acid Sibunit-4-ox.
acetone,[50]
ether,
n-butanol, and 2-butanol in
Reaction
conditions:
mechanically
activated
combination with each other and in the presence of ionic
liquids[51] were successfully applied for this purpose. The
−
1
−1
microcrystalline cellulose, 10 g L ; catalyst, 10 g L ;
180°С; 1 MPa Ar; and 1500 rpm. (a) Yields of glucose
extraction of 5-HMF from solutions is often performed at
and 5-HMF. (b) Yields of all other compounds. The data
are obtained from the average of 6 experiments.
concentrations.[51]
high NaCl
However, for the
subsequent fermentation of the extracted mixture, a high
salt concentration is not desirable due to the sensitivity of
some yeasts to osmotic stress.[52] For this reason, for the
extraction of 5-HMF from the aqueous phase, we tested
4
HPLC analysis of the hydrolytic dehydration of
cellulose showed that the main reaction products were 5-
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ChemSusChem
10.1002/cssc.201601244
FULL PAPER
various solvents with different boiling points and
polarities, such as diethyl ether, MIBK, isobutanol and n-
butanol (note that the latter alcohols are the products of
microbial biosynthesis).[53] The HPLC analysis of the
extracted hydrolysates is shown in Table S1 (supporting
information). The most efficient solvents for the extraction
of furan derivatives (5-HMF and furfural) from the
reaction mixture appeared to be n-butanol and isobutanol.
Extraction with diethyl ether showed the highest
was confirmed by quantitative 13C NMR (Figure S2,
supporting information) and revealed that isobutanol is
an admixture.
Isolation of yeasts and screening of their
thermotolerances
Recent studies showed that thermotolerant yeasts
belonging to the species K. marxianus[54] and O.
polymorpha[55] are promising ethanol producers.
Moreover, these yeasts are able to co-ferment glucose
and xylose, thus allowing their use for the fermentation of
the biomass hydrolysates.[56] In contrast to thermotolerant
species, S. cerevisiae, a commonly used yeast for
ethanol fermentation, has no natural ability to consume
xylose. It also needs to be cultured at temperatures of
25-37°C, which increases the cost of ethanol
production.[57] In our study, a number of thermotolerant
yeast strains were isolated from various dairy products at
40°C. Identification of these isolates according to their
18s rRNA sequences revealed that most of them belong
to the thermotolerant yeast species K. marxianus (9
strains) as well as to Clavispora lusitaniae and Pichia
kudriavzevii. In addition to the isolated strains, the
thermotolerant yeast strain O. polymorpha CBS4732 was
also used in this study. A list of strains isolated in this
study is provided in Table 3.
-
1
concentration of 5-HMF (0.2±0.0 g L ) remaining in the
aqueous phase. In all of the samples, after the extraction,
the concentrations of all sugars remained at the same
level, while the concentrations of organic acids varied
slightly. In general, most of the extracts had a low
concentration of 5-HMF, and furfural was not even
detected in all of the samples. Only minor amounts of
-
1
butanol and isobutanol (0.1±0.0 g L ) were found in the
mixtures after extraction. Low concentrations of inhibitors
make these extracted hydrolysates suitable for
subsequent fermentation. According to these findings, we
selected isobutanol as the solvent for extraction to obtain
the final fermentation mixture, whose final composition is
presented in Table 2.
Table 2. HPLC analysis of the processed
catalytic hydrolysate when cellulose was used as the
fermentation medium.[a]
Table 3. Thermotolerant yeast strains isolated in
this study.
-
1
Compound
glucose
[g L ]
Yeast strain
Source of strain
cheese
cheese
kefir
kefir
15.0 ± 1.0
2.0 ± 0.1
1.3 ± 0.1
3.0 ± 0.1
0.3 ± 0.1
0.4 ± 0.0
1.4 ± 0.2
0.6 ± 0.0
0.3 ± 0.1
0.1 ± 0.0
Kluyveromyces marxianus C1
Kluyveromyces marxianus D5
Kluyveromyces marxianus B5
Kluyveromyces marxianus B3
mannose
fructose
cellobiose
fermented
milk
goat
Clavispora lusitaniae С3
5-HMF
Kluyveromyces marxianus А1
Kluyveromyces marxianus A5
Kluyveromyces marxianus D4
Pichia kudriavzevii B4
kumiss
kumiss
cheese
quark
formic acid
glycolic acid
lactic acid
Kluyveromyces marxianus A4
Kluyveromyces marxianus B1
Kluyveromyces marxianus 4C
quark
quark
quark
tartaric acid
isobutanol
[
a] Hydrolysate was obtained under the same
conditions as in Table 1, concentrated and extracted
with isobutanol, and further supplemented with YNB
salts and (1.0 g L ) yeast extract.
Complete utilization of sugars comprising the
hydrolysate mixture is required to obtain the maximal
yield of ethanol from the processed biomass. Screening
of the isolated thermotolerant yeast strains for their sugar
utilization efficiencies was performed at 42-48°C in
medium supplemented with sugars that were found in the
catalytic hydrolysate of cellulose (glucose, mannose,
fructose or cellobiose). Yeast cultivation was carried out
-1
After the extraction of 5-HMF with isobutanol, it was
separated from the solvent by distillation. A yield of 1.2 g
of this product was obtained, which corresponds to a
yield of 7.2 wt.%. The structure of the isolated 5-HMF
was confirmed by 1H and 13C NMR (Figures S2-S4,
supporting information). The purity of isolated 5-HMF
5
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ChemSusChem
10.1002/cssc.201601244
FULL PAPER
in sealed 96-well plates. The results are shown in Figure
a-c.
The data presented demonstrate that all of the
phase of growth) of the four chosen yeast strains,
screening in 96-well deep well plates on the YNB
4
-1
medium supplemented with glucose (20.0 g L ) was
strains studied are able to grow at 42°C on medium
containing all four of the sugars, except the medium with
-1
cellobiose (20.0 g L ), which significantly reduced the
growth. With a further increase in temperature up to 45-
48°C (Figure 4b, c), considerable growth was maintained
only by some of the K. marxianus strains and by the
strain O. polymorpha CBS4732. At 45-48°C, a high
consumption of fructose for most of the strains and
differences in the thermotolerances for K. marxianus
strains were observed. These findings are consistent with
those of other studies, which revealed the significant
growth for this yeast species at temperatures above 40°C
and up to 48-50°C.[58] Furthermore, previously it was
shown that these yeast species can consume various
sugars, including glucose, mannose, cellobiose, xylose,
etc.[41] with high efficiency. The O. polymorpha CBS4732
strain was previously used at 37°C for the fermentation of
glucose, mannose, galactose, maltose and xylose (with
the exception of cellobiose)[40] into ethanol. In our study,
the strain O. polymorpha CBS4732 has shown the
highest ability for growth at 42°C (Figure 4a) on the
-1
medium with fructose (20.0 g L ). For another yeast, C.
lusitaniae, the possibility of the aerobic fermentation of
xylose[59] and cellobiose[60] was demonstrated previously,
but at lower temperatures (25°C and 28°C, respectively).
Yeast species P. kudriavzevii is known to be able to
assimilate glucose, sucrose, galactose, fructose, and
mannose, but it could not assimilate xylose, arabinose,
cellobiose, raffinose, or trehalose[61] at 40°C. This
species can also produce ethanol with a productivity 28 g
L-1 h-1 and yield of 85.4% on cassava starch
hydrolysate.[62] In our study, it was shown that the growth
in the presence of all sugars for C. lusitaniae C3 and P.
kudriavzevii B4 strains was limited at 45°C, while the
optimal temperature for the production of biomass for
both of them was 42°C. According to the data obtained,
the strains K. marxianus C1, O. polymorpha CBS4732, K.
marxianus D5 and K. marxianus B5 were selected for the
further studies as they all had the ability to grow at
temperatures up to 48°C.
Figure 4.
Growth of thermotolerant yeast strains on YNB medium
containing sugars (glucose, mannose, fructose or
For the detailed elucidation of the growth
parameters (specific growth rate, biomass yield, biomass
volumetric productivity and dry cell weight at the mid-log
-1
cellobiose, 20.0 g L ) at various temperatures in 96-well
microtiter plates: (a) 42°C; (b) 45°C; and (c) 48°C.
6
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FULL PAPER
Table 4. IC50 values for inhibitors of biomass production of the thermotolerant yeast strains.
IC50 of Compound
O. polymorpha
K. marxianus C1
K. marxianus D5
K. marxianus B5
-
1
[
g L ]
CBS4732
5
-HMF
1.96 ± 0.01
0.67 ± 0.02
1.11 ± 0.03
1.30 ± 0.03
15.33 ± 1.06
15.18 ± 1.71
30.71 ± 2.24
51.39 ± 0.61
6.78 ± 0.54
2.85 ± 0.19
2.44 ± 0.18
1.25 ± 0.10
0.39 ± 0.03
0.99 ± 0.02
20.65 ± 1.94
9.52 ± 0.30
30.56 ± 1.77
62.48 ± 1.18
2.81 ± 0.14
1.75 ±0.08
1.84 ± 0.02
1.59 ± 0.20
0.79 ± 0.01
1.36 ± 0.09
18.70 ± 1.10
20.38 ± 1.76
34.36 ± 1.96
49.37 ± 0.85
3.84 ± 0.21
1.53±0.09
2.03 ± 0.06
1.24 ± 0.12
0.79 ± 0.01
1.21 ± 0.06
16.67 ± 0.47
20.05 ± 3.42
39.38 ± 1.34
46.50 ± 1.32
4.63 ± 0.34
3.20 ± 0.19
furfural
acetic acid
formic acid
lactic acid
glycolic acid
tartaric acid
ethanol
isobutanol
n-butanol
performed at temperatures of 37–50°C (Table S2,
supporting information). The optimum temperature range
for biomass production for the strains being studied
appeared to be 37-42°C. At 37°C, the strains K.
marxianus D5, K. marxianus B5 and O. polymorpha
CBS4732 all have high specific growth rates, while the
strain K. marxianus C1 has the highest  of 0.40±0.05 g
L-1 h-1 at 42°C, which correlates with the data reported
previously.[63] At 37°C, the highest biomass volumetric
productivities and dry cell weights were observed for all
of the strains, with the exception of O. polymorpha
CBS4732 that has its optimal growth rate at 42°C with a
Isobutanol and n-butanol tolerances were also examined
for yeast strains, as minor amounts of them remained in
the aqueous phase after extraction (Table S1, supporting
information). The results are shown in Table 4.
It can be seen that 5-HMF, furfural, acetic acid and
formic acid have the highest inhibitory effects on the
growth of yeast strains. The strain O. polymorpha
CBS4732 shows the highest tolerance to 5-HMF
(IC50=2.44±0.18
g
L-1) compared to other strains
belonging to the K. marxianus species. The strain K.
marxianus C1 has the highest sensitivity to furfural
-1
(IC50=0.67±0.02 g L ). It is consistent with the result of a
previous study,[64] in which the inhibition of yeast growth
by furfural was reported at a concentration of 40 mM
-1
DCW of 2.05±0.14 g L . The highest biomass yields
were achieved for all of the strains belonging to K.
marxianus at 42°C, and thus, this temperature was used
for subsequent studies. It should be noted that a feature
of the O. polymorpha CBS4732 strain is its significant
biomass growth at 50°C (Yb/s=0.10±0.01 g g-1 and
-1
(3.84 g L ). Furthermore, 5-HMF in concentrations above
40 mM exhibited the lag phase of the tested strain K.
marxianus TISTR5925. The strain K. marxianus C1 in
our study has also shown high tolerance to acetic acid
-
1
-1
DCW=1.59±0.07 g L ). Thus, despite O. polymorpha
CBS4732 having a lower ability for the production of
biomass, it has a higher thermotolerance.
(IC50=1.11±0.03 g L ), but it has a lower IC50 value
compared to the strain K. marxianus TISTR5925.[64]
However, the yeast strain O. polymorpha CBS4732
revealed the highest sensitivity to acetic acid
Inhibitor tolerance of the yeast strains
-
1
Their tolerance to inhibitors of fermentation is one of
the most important properties of the ethanol-producing
yeast strains. As 5-HMF is the main product of the
catalytic hydrolysis of the activated microcrystalline
cellulose, the high tolerance of yeasts to this compound
is highly desirable as well as to other products of
hydrolysis. In this study, the IC50 values for substances
produced during the catalytic hydrolysis of cellulose and
released during the further fermentation were determined.
(IC50=0.39±0.03 g L ) among all of the tested strains. It is
also has moderate sensitivities to formic (IC50=0.99±0.02
-1
L-1) acids.
CBS4732
g
L ) and glycolic (IC50=9.52±0.30
g
Nevertheless, strain O. polymorpha
demonstrates relativity high levels of biomass production
-1
in the presence of lactic acid (IC50=20.65±1.94 g L ).
Furthermore, it was shown that tartaric acid has no
significant effect on the biomass growth of all the strains
studied. O. polymorpha CBS4732 demonstrates the
7
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ChemSusChem
10.1002/cssc.201601244
FULL PAPER
highest tolerance to ethanol, which is the main product of
It was shown that biomass production decreases for
all of the tested strains after NaOH neutralization up to
pH>4.74. The highest biomass growth was observed at a
pH of 3.75 for the strain O. polymorpha CBS4732.
However, cultivation at a pH of 4.75 resulted in a
significant increase in the biomass growth for the strain K.
marxianus C1 (up to 79.5±4.4%) and a slightly lower
growth for the rest of the strains including K. marxianus
B5 (59.1±13.8%), O. polymorpha CBS4732 (50.9±5.9%)
and K. marxianus D5 (31.9±3.9%). Thus, the optimal pH
for the fermentation of all of the strains studies was 4.75.
This is consistent with the data obtained in other studies,
where the optimal value for the fermentation of K.
marxianus on various biomass hydrolysates was found to
be a pH of 5.0.[65,66]
-1
fermentation, with an IC50 value of 62.48±1.18 g L , while
the lowest value was observed for K. marxianus B5 with
-
1
an IC50 value of 46.50±1.32 g L . For all of the strains, n-
butanol is considerably more toxic solvent than
a
isobutanol. The IC50 value for isobutanol was relatively
low only for the O. polymorpha CBS4732 strain
-1
(
2.81±0.14 g L ), but according to its minor content in the
hydrolysate after extraction, it would not significantly
affect the fermentation parameters. Thus, according to
the analysis of the inhibitor tolerance for all of the tested
substances, it can be concluded that the O. polymorpha
CBS4732 strain has the highest resistance to the furan
derivatives (furfural and 5-HMF). However, it is worth
noting that this strain proved to be sensitive to high
concentrations of organic acids, as opposed to the strain
K. marxianus C1.
Cultivation of thermotolerant yeasts on the extracted
and neutralized catalytic hydrolysates of activated
microcrystalline cellulose
To determine the optimal conditions for yeast
biomass growth on the extracted hydrolysate, it was
neutralized to pH values of 3.75-7.00 by various methods.
The results were compared to model mixtures with the
same pH value to estimate the relative biomass
production (Figure 5).
Figure 6.
Influence of the hydrolysate neutralization on the growth
of biomass of the thermotolerant yeast strains at 42°C for
16 h. The biomass growth corresponding to 100% was
accepted as biomass growth during cultivation on a
model mixture of sugars at a pH of 4.75.
Previously, it was shown that the neutralization of
the hydrolysate can significantly affect the fermentation
parameters. In the previous study, [67] it was found that
the neutralization of spruce hydrolysate with NH
allows better results to be achieved for ethanol
fermentation with the yeast S. cerevisiae (Q is 86% from
one of the model mixtures) rather than with Ca(OH)
=41%). Another effective method of neutralization that
proved to be efficient is an overliming of the hydrolysate
with Ca(OH) . It consists of an initial increase in the pH of
4
OH
Figure 5.
e
Biomass growth of the thermotolerant yeast strains at
2
42°C for 16 h at different pH values on the extracted
(Q
e
cellulose hydrolysate mixture after neutralization with
NaOH. The value of 100% of biomass production was
taken as the biomass production on a model sugar
mixture at the same pH.
2
the mixture up to 10.0-11.0, with subsequent adjustment
to a pH of 4.5-5.0. This type of neutralization can
8
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ChemSusChem
10.1002/cssc.201601244
FULL PAPER
significantly increase the effectiveness of yeast and
bacterial fermentation.[68] Therefore, to choose the
effective methods of neutralization for all of the strains
were NH
4
OH treatment and overliming with Ca(OH)
2
. For
optimal method of neutralization, we performed
comparative analysis of different methods, including
treatment with NaOH, treatment with NH OH and
. The results are presented in
a
example, in the case of the NH
4
OH-neutralized
hydrolysate, the relative biomass productions for K.
marxianus C1 and O. polymorpha CBS4732 were
comparable (78.5±8.2% and 74.6±9.1%, respectively).
In the case of K. marxianus C1, application of
overliming allowed the maximum biomass production of
97.0±3.1% to be achieved. However, the use of
overliming requires the application of an additional step
4
overliming with Ca(OH)
Figure 6.
2
The thermotolerant yeast strains studied revealed
different sensitivities to the hydrolysate treatment, except
for the strains K. marxianus C1 and K. marxianus B5,
which showed comparable levels of biomass production
4
in the case of neutralization with NH OH. The most
Figure 7.
Fermentation of the yeast strains K. marxianus C1 and O. polymorpha CBS4732 on the extracted and neutralized catalytic
hydrolysates of cellulose and a model sugar mixture: (a) fermentation of K. marxianus C1 on the model sugar mixture; (b)
fermentation of K. marxianus C1 on processed catalytic cellulose hydrolysate; (c) fermentation of O. polymorpha CBS4732
on the model sugar mixture; and (d) fermentation of O. polymorpha CBS4732 on the processed catalytic cellulose
hydrolysate.
9
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FULL PAPER
Table 5. Analysis of the composition of the fermentation liquid for yeast strains K. marxianus C1 and O. polymorpha
CBS4732 cultivated on processed catalytic cellulose hydrolysates and model sugar mixes.
Mannose+
Fructose [g L ]
-1
[e]
Ethanol [g L-1] [f]
DCW [g L-1] [f]
Fermentation
Glucose [g L ]
-1 [e]
S1 [
S2 [
a]
16.1±0.6
14.7±0.7
14.0±0.3
15.7±0.6
3.1±0.2
3.3±0.2
2.7±0.2
3.6±0.2
8.4±0.2
6.8±0.3
7.4±0.3
7.6±0.5
2.7±0.2
1.9±0.2
1.9±0.2
1.3±0.2
b]
[
c]
S3
S4 [
d]
[
a] Fermentation of K. marxianus C1 on model sugar mixture for 7 h. [b] Fermentation of K. marxianus C1 on processed
cellulose hydrolysate for 14 h. [c] Fermentation of O. polymorpha CBS4732 on model sugar mixture for 10 h. [d]
Fermentation of O. polymorpha CBS4732 on processed cellulose hydrolysate for 16 h. [e] Fermentation broth at 0 h. [f]
At the end of fermentation.
-1
in biomass processing, which is accompanied by the
necessity to separate the liquid phase of the mixture from
the resulting precipitate. For this reason, in this study,
0.12±0.01 h ), with relatively similar other
characteristics to those of K. marxianus C1 during
fermentation under similar conditions. The fermentation
of K. marxianus C1 on the processed hydrolysate
4
neutralization with NH OH was used as a more
-
1
appropriate technique to achieve the desired level of
biomass production. For further fermentation studies, the
strains K. marxianus C1 and O. polymorpha CBS4732
were selected as they had the highest biomass
productions during growth on extracted and neutralized
cellulose hydrolysate at a pH of 4.75.
resulted in a higher biomass yield (0.11±0.01 g g )
compared to that of O. polymorpha CBS4732 (0.06±0.00
-1
g g ). The volumetric productivity of ethanol as well as
the specific growth rate on the hydrolysate were lower
than those for the model sugar mixture for both strains,
but in the case of K. marxianus C1, all of the observed
parameters were markedly decreased. During the
fermentation, the value of Ye/s of 0.39±0.03 g g-1 for O.
polymorpha CBS4732 was higher than that for the strain
K. marxianus C1, which indicates the strong efficiency of
the former strain for the production of ethanol. The strain
K. marxianus C1 has a higher biomass yield, which did
not result in an increased yield of ethanol. Nevertheless,
both strains showed relatively high theoretical yields of
ethanol during cultivation on the processed hydrolysate
with respect to the model mixture (72.0±5.7% for K.
marxianus C1 and 75.2±4.3% for O. polymorpha
CBS4732) and are able to metabolize 5-HMF.[69] The
concentrations of other acids present in the hydrolysate
(e.g., glycolic and tartaric) remained constant throughout
the fermentation for both strains and corresponded to the
initial amounts found in the initial processed hydrolysate.
As opposed to thermotolerant yeasts, the ethanol
fermentation of biomass hydrolysates with common S.
cerevisiae strains has been extensively studied[70, 71].
Goswami et al.[72] studied the process of S. cerevisiae
fermentation on the catalytic hydrolysate of alkali-
pretreated rice straw preliminarily purified with an ion
4
Fermentation on the extracted and NH OH-
neutralized hydrolysates in shake flaks in anaerobic
conditions was carried out for the strains K. marxianus
C1 and O. polymorpha CBS4732 at a starting biomass
DCW of 1.0 g L-1 for up to 16 h (Figure 5). The
concentrations of the main products (Table 5) and
fermentation parameters were analyzed (Table 6).
The data presented indicate that both yeast strains
revealed the production of ethanol during fermentation on
the processed cellulose hydrolysates. The fermentable
sugars include glucose, mannose and fructose.
Cellobiose was consumed during the fermentation in
minor amounts. K. marxianus C1 has the ability to use all
three sugars during the fermentation simultaneously,
while O. polymorpha CBS4732 preferably uses glucose,
leaving the remaining sugars to be consumed after the
preferential depletion of glucose. The strain K. marxianus
C1 has both
a
high level of biomass production
-1
(
Y
b/s=0.14±0.02 g g-1 and =0.29±0.01 h ) and a high
volumetric productivity of ethanol (1.69±0.10 g L-1 h-1)
during fermentation on the model sugar mixture.
In contrast, the strain O. polymorpha CBS4732 has
lower production of biomass (Yb/s=0.11±0.01 g g-1 and
exchange
resin.
1
0
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FULL PAPER
Table 6. Fermentation parameters for yeast strains K. marxianus C1 and O. polymorpha CBS4732 cultivated on
processed catalytic cellulose hydrolysates and model sugar mixes.
[
e]
[e]
-1
Q
g
1
Q
e
Y
e/s
Y
b/s
[g g ]
[f]
[h-1]
Fermentation
Ye/s [%]
-
-1
-1 -1
-1
[
g L h ]
[g L h ]
[g g ]
S1 [
S2 [
a]
-3.78±0.15
-1.40±0.05
-1.80±0.05
-1.24±0.04
1.69±0.10
0.41±0.02
1.10±0.05
0.53±0.02
0.44±0.02
0.34±0.02
0.44±0.02
0.39±0.03
0.14±0.02
0.11±0.1
0.11±0.01
0.06±0.00
84.0±4.2
72.0±5.7
84.5±5.0
75.2±4.3
0.29±0.01
0.06±0.0
0.12±0.01
0.07±0.0
b]
[
c]
S3
S4 [
d]
[
a] – [d] are the same as for Table 5. [e] Yield from fermentable sugars (glucose, mannose, fructose). [e] Degree of
-1
ethanol yield to the maximal theoretical yield from fermented sugars (that was accepted as 0.521 g g ).
It was shown that the ethanol production achieved
2.29% of the maximal theoretical yield but was also
with overliming was fermented with the K. marxianus
5
ATCC 36907 strain at 30°C. It was shown that the
volumetric productivity of ethanol is reduced ten-fold
compared with model mixtures (0.86 g L-1 h-1 and 0.08 g
L-1 h-1, respectively), which was associated with the
presence of furan derivatives in the hydrolysate. In our
study, the ethanol yield for the yeast strain K. marxianus
C1 from the catalytic hydrolysate of cellulose was 1.3
times smaller than with the model sugar mixture. For the
strain O. polymorpha CBS4732, this ratio was 1.1, which
indicates the high tolerance of this strain to fermentation
inhibitors. Thus, strains belonging to the species K.
marxianus are capable of a relatively high efficiency of
the conversion of sugars derived from cellulose into
ethanol using a solid catalyst but concede to the
fermentation parameters of S. cerevisiae at 30°C.
accompanied by the formation of high levels of acidic
compounds. In addition to the yeast S. cerevisiae, some
of the thermotolerant yeasts were also previously used
for the fermentation of biomass hydrolysates. Dasgupta
et al.[73] showed that the fermentation of the enzymatic
hydrolysate of bagasse pith at 45°C with the yeast strain
Kluyveromyces sp. IIPE45 resulted in an ethanol
production of 0.36 g L-1 h-1 and 88% of the theoretical
yield on a mixture of hexoses, which is comparable with
the data obtained for K. marxianus C1 in our study.
Among all of the hydrolysis methods, the process of
biomass hydrolysis with inorganic acids and its
subsequent fermentation into ethanol is well-studied. Its
main drawback is the formation of a large variety of
byproducts, including organic acids and furan derivatives,
which must be separated from the sugars before
fermentation due to their cell toxicities. Hydrolysate
purification before fermentation with the yeast K.
marxianus was previously performed with overliming[69]
and ion exchange chromatography[65]. These approaches
allow a significant reduction of the inhibitor content and
thus improved the fermentation parameters, but they
result in waste accumulation. In the previous study,[74] the
fermentation of K. marxianus CCA 510 on sulfuric acid
hydrolysates of apple bagasse was studied at 30°C. It
was shown that at the end of the fermentation of the
hydrolysate containing glucose, xylose and arabinose,
the latter was not completely assimilated by the yeast,
and the final yield of ethanol was 11.89±0.34 g L-1 at 96 h.
Under similar conditions during fermentation with S.
cerevisiae at 30°C, the ethanol concentration was 10.0 g
L-1 after 4 h of fermentation with a yield of 0.49 g g-1 and
The use of the AFEX method is a promising
approach for bioethanol production and an alternative to
acid hydrolysis pretreatment. For example, the AFEX-
treated corn stover after enzymatic hydrolysis and
fermentation of complex media and hydrolysates at a
temperature of 30°C with the strain S. cerevisiae 424A
(LNH-ST), which was engineered to express genes of
xylose metabolism,[75] has shown an insignificant
difference in the volumetric productivities of ethanol
(0.9±0.0 g L-1 h-1 and 0.8 g L-1 h-1, respectively). The
theoretical yield of ethanol from fermentable sugars
(glucose and xylose) was 83.6±0.9% and 88.5%,
respectively, thus exceeding the values obtained for the
strains K. marxianus C1 and O. polymorpha CBS4732 in
this study. However, after 24 h of fermentation with S.
cerevisiae 424A (LNH-ST), the reduction of biomass in
the fermentation broth was approximately 30% (9.5±0.2 g
L-1 for the hydrolysate and 6.7 g L-1 on a complex
medium),[75] which is comparable to the results observed
for both strains being studied. The higher efficiency of
-
-1
[69]
productivity 1.43 g L 1 h . In another study,
the
cellulosic hydrolysate of sugarcane bagasse neutralized
1
1
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ChemSusChem
10.1002/cssc.201601244
FULL PAPER
ethanol production of the strain S. cerevisiae 424A (LNH-
ST) may be related to its high metabolic efficiency due to
its genetic modification. However, both K. marxianus C1
and O. polymorpha CBS4732 strains have higher ethanol
volumetric productivities at elevated temperature (42°C)
and are capable of the production of a high amount of
biomass during fermentation on catalytic hydrolysates of
cellulose. This study also showed that fermentation
parameters of the examined strains are comparable to
analogues strains with respect to lab-scale fermentation.
However, industrial processes for ethanol production
from biomass require an ethanol concentration of at least
hydrolysates of cellulose.
The application of
thermotolerant yeasts for fermentation at 42 °C will also
reduce production costs. Compared with other methods
of producing ethanol from hydrolysates (for example, the
acid hydrolysis treatment and AFEX), the catalytic
process is recognized as an environmentally safe
process. It is shown that the use of the strains K.
marxianus C1 and O. polymorpha CBS4732 for the
fermentation of processed catalytic cellulose hydrolysate
has a relatively high efficiency since the strains are
resistant to fermentation inhibitors. Thus, the proposed
combination of the catalytic processing of mechanically
activated cellulose for the production of 5-HMF and
subsequent fermentation of the extracted hydrolysate
-
1 [76]
4
0 g L .
The scale-up of this biorefinery process will
result in the application of a more concentrated sugar
solution after 5-HMF and furfural extraction and possibly
require the application of additional purification stages.
Conclusions
with thermotolerant yeasts may be
a
promising
alternative method for ethanol production by fermentation
and can be used to produce other valuable substances,
such as bioacids or bioalcohols.
In this study, we have explored the potential of
combining catalytic and biotechnological methods in a
cellulose biorefinery with the goal of producing 5-HMF
and ethanol. Moreover, the one-pot hydrolytic
Experimental Section
Materials
NaOH (Panreac) was used for the titration of the
catalyst. Microcrystalline cellulose with a particle size <
dehydration
of
the
mechanically
activated
microcrystalline cellulose into glucose simultaneously
produces 5-HMF and can be conducted in water in the
presence of a solid acid catalyst. As such a catalyst, we
used a mesoporous graphitic carbon material Sibunit-4,
which was oxidized in an environmentally friendly
manner by wet air. The recorded yields of glucose and 5-
HMF were 21.1–25.1% and 6.6–9.4%, respectively, while
fructose, mannose and some organic acids were found in
minor concentrations. Additionally, we revealed that the
extraction of the catalytic hydrolysate of cellulose with
organic solvents (n-butanol, isobutanol and MIBK) can
effectively recover the 5-HMF from aqueous solutions of
sugars. Diethyl ether was found to be a poor extractant,
and isobutanol was selected for the 5-HMF extraction in
this study due to its effectiveness. 5-HMF was separated
from the solvent by distillation. The structure and purity of
the isolated 5-HMF was confirmed by H and 13C NMR.
A relatively low content of fermentation inhibitors
0
.1 mm (Vekton, Russia) was used for mechanical
activation. 5-Hydoxymethylfurfural (Sigma-Aldrich), D-
fructose (Sigma-Aldrich), D-mannose (Sigma-Aldrich), D-
glucose (Reahim, Russia), D-cellobiose (Alfa Aesar),
levulinic acid (Acros Organics), malic acid (Sigma-
Aldrich), glycolic acid (Sigma-Aldrich), formic acid
(
(
Panreac), lactic acid (Panreac), and tartaric acid
Reahim, Russia) were purchased for use as HPLC
standards. n-Butanol, diethyl ether, methylisobutylketone
NevaReactive, Russia) and isobutanol (99.3%, Reaktiv,
(
Russia) were used for 5-HMF extraction. All other
reagents were of analytical grade. Argon (99.998% purity,
Sibtehgaz, Russia) and synthetic air (20% O
) were used as an inert gas in the reactor and for the
synthesis of the catalyst, respectively. Milli-Q water
Millipore, France) was used for the preparation of all of
2
and 80%
N
2
(
1
the solutions.
Mechanical activation of microcrystalline cellulose
Mechanical activation of microcrystalline cellulose
was performed in a Pulverizette 5 planetary mill (Fristch,
Germany) for 40 min with a cellulose loading of 15 g and
an acceleration of 22 g. The size of the cellulose particles
was measured by optical microscopy on a Biomed-5
(
including 5-HMF) and compete absence of furfural in the
mixture obtained after the extraction of 5-HMF allowed
the production of ethanol via fermentation with
a
relatively high efficiency. It should be noted that in this
case, ethanol is a byproduct of the 5-HMF production,
which allows the complete recycling of catalytic
1
2
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10.1002/cssc.201601244
FULL PAPER
(
Biomed Ltd.-M, Moscow, Russia) microscope equipped
Isolation and identification of thermotolerant yeasts
Samples of various dairy products were placed in
YPD medium supplemented with streptomycin (20 g
with a digital camera.[49]
Catalyst preparation
-
1
The carbon-based material Sibunit-4 (Institute of
Hydrocarbon Processing, Omsk)[77,78] was used as a
catalyst in this study. The Sibunit-4 was preliminarily
oxidized with wet air at 450°C and then ground in a
mortar. The 56-94 m fraction was separated and used
mL ) and grown at 30°C for 48 h. Cultures were then
plated on YPD agar and incubated at 40°C for 48 h. The
individual colonies were isolated and twice reseeded in
the same conditions for pure culture preparation.
Genomic DNA was isolated with
a genomic DNA
for
cellulose
hydrolysis
(Sibunit-4-ox).[23]
The
GeneJET Genomic DNA Purification Kit (Thermo). PCR
amplification of the partial 18s rRNA sequence was
performed using the universal primers 18SUnivFwd (5'-
ACCTGGTTGATCCTGCCAGT-3') and BMB-B (5'-
concentration of the strongly acidic groups, such as
phenolic, lactone, carboxylic and sulfo-groups, on the
surface of the carbon catalyst was determined by titration
of the acid with sodium hydroxide by Boehm’s
method.[79,80]
CCGTCAATTCVTTTPAGTTT-3'),
followed
by
sequencing with BigDye Terminator v3.1 Cycle
a
One-pot process for the catalytic hydrolysis-
dehydration of cellulose
Sequencing Kit (Thermo). All isolates were stored in 96-
well plates at -80°C in 15% glycerol.
The one-pot hydrolysis-dehydration of the activated
cellulose was performed at a temperature of 180°C and a
pressure of 1 MPa of argon in the 150 mL Mini-Reactor
autoclave (Autoclave Engineers, USA) under vigorous
mechanical stirring (1500 rpm) with a propeller-type
Screening of yeast biomass growth in the 96-well
microtiter plates
To determine the yeast biomass growth on various
substrates, sealed 96-well microtiter plates[81] were used.
Overnight yeast culture was grown up to an OD of 0.8-
1.0 at 595 nm in an YNB salt medium supplemented with
-1
magnetic drive. The activated cellulose (10 g L ) and
-
1
-1
-1
catalyst (10 g L ) in water (45 mL) were incubated for 5 h.
During the reaction, the composition of the reaction
mixture was analyzed by HPLC.
peptone (5.0 g L ), yeast extract (3.0 g L ), and glucose
-1
(20.0 g L ). Yeast culture (20 L) at a final concentration
of 5×106 cells mL-1 per well was added to the YNB
medium with a pH of 4.75 (180 L) supplemented with
Extraction of 5-HMF from the catalytic hydrolysate
5-HMF was isolated from the mixture obtained from
-1
the yeast extract (1.0 g L ) and required sugar (glucose,
the catalytically processed activated cellulose by
extraction with organic solvents. Before extraction, the
hydrolysate was filtered and evaporated up to 10 times in
a rotary evaporator at 70°C. The evaporated mixture was
then placed in a separatory funnel, an equal volume of
an organic solvent was added, the solution was
thoroughly mixed, and the aqueous portion was
withdrawn. The extraction was repeated three times. The
organic fractions were combined, and the resultant
solution was further evaporated on a rotary vacuum
evaporator (2 mm Hg) for 5-HMF isolation. The
remaining aqueous solution was re-evaporated to a final
glucose concentration of 20 g L-1 and combined with the
mannose, sucrose or cellobiose), followed by culturing at
350 rpm for 16 h at 42-48°C. If the processed
hydrolysate was used as a culture medium, a model
mixture comprised of a mixture of sugars in the same
concentrations and at the desired pH was used as a
reference. All experiments were carried out in triplicate,
and the data are expressed as a mean±SD.
Determination of the dry cell weight
The relationship between the OD and dry cell weight
(
DCW) was determined for each strain. During the
exponential phase of growth on YNB medium
-1
supplemented with peptone (5.0 g L ), yeast extract (3.0
-1
-1
g L ), and glucose (20.0 g L ), the OD was measured at
95 nm. An aliquot of culture liquid was transferred onto
-1
following YNB medium components: KH
2
PO
4
(1.0 g L ),
5
-
1
-1
MgSO
4
(0.5 g L ), NaCl (0.1 g L ), CaCl
2
(0.1 g L-1),
a glass fiber filter, filtered, dried at 105°C overnight and
weighed. The relationship between the OD and DW was
determined through calculation of linear regression and
-1
-1
(
NH
4
)
2
SO
4
(5.0 g L ), and yeast extract (1.0 g L ). The
mixture was then neutralized to the desired pH with an
alkaline reagent (NaOH or NH OH) or by overliming with
Ca(OH)
,[68] and then, it was fermented by yeasts.
-1
2
4
converted to g L with R > 0.98.
2
1
3
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Quantitative screening of thermotolerant yeast
neutralized catalytic hydrolysate of cellulose at a pH of
4.75 (10 mL) supplemented with YNB salts and yeast
growth in the deep 96-well plates
-
1
Quantitative analysis of the effect of temperature on
the productivity of the yeast biomass was performed by
aerobic cultivation in deep 96-well plates in the
Microflask (Applikon Biotechnology) system. Yeast
strains were pre-grown overnight in YNB medium
extract (1.0 g L ) in sealed flasks (25 mL). Fermentation
was carried out in microaerobic conditions at 100 rpm
and 42°C. A model mixture of sugars (in concentrations
corresponding to those in the hydrolysate processed for
fermentation) in YNB medium at
a pH of 4.75
-
1
-
-1
supplemented with peptone (5 g L ), yeast extract (3 g L
supplemented with yeast extract (1.0 g L ) was used as
a reference. During the experiment, the biomass growth
was monitored at 595 nm, and the composition of the
culture broth was analyzed by HPLC. All experiments
were carried out in triplicate, and data are expressed as
a mean±SD.
1)
, and glucose (20.0 g L ), followed by separation by
-1
centrifugation at 500 × g for 10 min. Then, they were
twice washed with PBS and dissolved in 1 ml of YNB
-1
medium containing glucose (20.0 g L ) and yeast extract
-1
5
(
1.0 g L ) at a final concentration of 5×10 cells mL-1.
Yeasts were cultured at 400 rpm for up to 10 h at
temperatures of 37-50°C until the log phase was reached.
The cell growth was monitored at 595 nm, and the
amount of glucose in the culture was quantified using the
GOD-PAP assay. All experiments were carried out in
triplicate, and data are expressed as a mean±SD.
Calculation of the fermentation parameters
During the fermentation, the following parameters
were evaluated. The specific biomass growth rate ()
was calculated in the exponential growth phase by (h-
1)
=(lnX2-lnX1)×(t2-t1)-1 where X1 and X2 are the
-1
biomass dry weight concentrations (mg L ) on days t1
Screening of yeast tolerance to inhibitors
(
start of exponential growth) and t2 (end of exponential
To determine the effects of various compounds on
biomass production during fermentation, the cultivation of
yeasts was performed in 96-well sealed microtiter plates
as described above in the presence of inhibitors at 42°C
for 8 h, and biomass growth was assessed at 595 nm.
The inhibitory effects of the substances were evaluated
as IC50 values, corresponding to the concentration of the
substance that induced an inhibition of 50% of the growth
of biomass. IC50 values were determined from the semi-
log plots of the relative values of biomass yield at the
growth), respectively. The ethanol (Ye/s) and biomass
-1
yields (Yb/s) (g g ) were calculated as the ratios between
-1
ethanol and biomass production (g L ) and fermentable
-1
sugar consumption (g L ), respectively. Ethanol (Q
e
) and
) (g L-1 h-1) was the
biomass volumetric productivity (Q
b
-1
ratio between ethanol (biomass) production (g L ) and
the fermentation time (h). All data are expressed as a
mean±SD.
Experimental techniques
XRD: The XRD study of cellulose particles was
performed on a Bruker D8 (Germany) diffractometer with
the CuKα radiation (=1.5418 Ǻ). Scanning was
performed in the 2 range of 10-40 °. The degree of
crystallinity of cellulose was calculated according to XRD
stationary phase of growth as
a function of the
concentration of the compound. All experiments were
carried out in triplicate, and data are expressed as a
mean±SD.
Shake flask fermentation of the extracted catalytic
hydrolysates of activated microcrystalline cellulose
A single colony of overnight yeast culture on a YPD
plate was inoculated into 10 ml of YNB medium a pH of
study data as the ratio of the peak area of crystalline
cellulose to the total area of all peaks.[82]
BET: The textural characteristics of the catalyst’s surface
2
were determined by N adsorption isotherms at 77 K on
-1
4
.75 supplemented with peptone (5.0 g L ), yeast extract
an ASAP-2400 (Micromeritics, USA) system. The surface
-1
(
3.0 g L ) and a mixture of sugars (corresponding to the
area was calculated by the BET model.
sugar composition of the neutralized and prepared
hydrolysate). The yeasts were cultured aerobically at
NMR: 1H and 13C NMR spectra have been recorded on a
Bruker AVANCE-400 spectrometer at 400.13 and 100.61
42°C at 250 rpm until the mid-log phase of the growth
MHz, respectively, using 5 mm NMR tubes in CDCl
3
phase corresponding to an OD of 1.0 at 565 nm. The
cells were collected by centrifugation at 500 × g for 10
min, washed with PBS, and combined with the
solutions at 298 K. Standard DEPT90 and DEPT135
experiments have been used for spectral editing.
Quantitative 13C NMR spectra were recorded using
1
4
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ChemSusChem
10.1002/cssc.201601244
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inverse gated decoupling. 1JCH coupling constants were
estimated by the fitting of normal gated decoupled
spectra.
Keywords: biomass conversion
•
heterogeneous
catalysis • 5-HMF • ethanol • biosynthesis
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FULL PAPER
Ksenia N. Sorokina*, Oxana P. Taran,
Tatiana B. Medvedeva, Yuliya V.
Samoylova, Alexandr V. Piligaev,
Valentin N. Parmon
Page No. – Page No.
Cellulose biorefinery based on
Clean and effective: A cellulosic biorefinery process based on a catalytic and
biotechnological approach was developed. Glucose (yield: 21.1-25.1%) and 5-HMF
combined
biotechnological
catalytic
approach
and
for
(
yield: 6.6–9.4%) were produced over a carbon-based catalyst. After 5-HMF
production of 5-HMF and ethanol
extraction, the latter was then fermented into ethanol by the thermotolerant yeast
strains K. marxianus C1 and O. polymorpha CBS4732 at 42°C, with yields of
72.0±5.7% and 75.2±4.3%, respectively.
1
8
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