Technical Note

Received: 1 August 2011 Revised: 1 November 2011 Accepted: 1 November 2011 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.3695

Alkali pretreatment of softwood spruce and

hardwood birch by NaOH/thiourea, NaOH/urea,
NaOH/urea/thiourea, and NaOH/PEG to
improve ethanol and biogas production
Abas Mohsenzadeh,a Azam Jeihanipour,a,b∗ Keikhosro Karimia,c
and Mohammad J. Taherzadeha

Abstract
Alkali-dissolution pretreatment of softwood spruce and hardwood birch to improve ethanol and biogas production was
investigated. The pretreatments were carried out at different temperatures between −15 and 80 ◦ C with NaOH/thiourea
(7/5.5 wt%), NaOH/urea (7/12 wt%), NaOH/urea/thiourea (7/8/6.5 wt%), and NaOH/PEG (7/1 wt%) aqueous solutions. The
pretreated materials were then subjected to enzymatic hydrolysis for 72 h. The pretreatments by NaOH/thiourea at −15 ◦ C
improved the hydrolysis yields of spruce from 11.7% to 57% of theoretical yield, and for birch from 23.1% to 83% of theoretical
yield. The enzymatic hydrolysis and fermentation of these pretreated materials by NaOH/thiourea with baker’s yeast resulted
in 54.0% of theoretical yield compared with 10.9% for untreated spruce and 80.9% of theoretical yield compared with 12.9% for
untreated birch. Furthermore, anaerobic digestion of pretreated materials resulted in 0.36 L g−1 VS methane compared with
0.23 L g−1 VS for untreated birch, and 0.21 L g−1 VS compared with 0.03 L g−1 VS for untreated spruce.

c 2012 Society of Chemical Industry

Keywords: alkaline pretreatment; spruce; birch; enzymatic hydrolysis; bioethanol; biogas

INTRODUCTION re-association of cellulose hydroxyl groups. Thus, it can positively

Production of biofuels such as ethanol and biogas from ligno- affect the dissolving process.7,9 Based on the cellulose dissolution
celluloses demands a pretreatment to make the recalcitrant and capability, these chemicals can be used for pretreatment of
crystalline structure of lignocelluloses available to enzymatic or lignocelluloses.10 Alkaline pretreatment by NaOH was previously
bacterial attack.1 In addition, this structure is covered by lignin, used successfully to improve ethanol and biogas production from
which contributes to reducing the access of enzymes. Differ- wood under mild operating conditions.11 No investigation of
ent physical, chemical and biological methods are used as the hardwood and softwood pretreatment prior to enzymatic or
pretreatment step such as milling, steam explosion, ammonia bacterial hydrolysis for ethanol or biogas production by these
fiber explosion, alkaline, dilute- and concentrated-acid processes.2 solvents was found in the literature.
Among these methods, chemical methods are more efficient and The overall goal of this work was to investigate the effects
economically feasible. of different alkali dissolution systems including NaOH/thiourea,
One of the most effective chemical pretreatment methods is NaOH/thiourea/urea, NaOH/urea, and NaOH/PEG aqueous solu-
dissolution of lignocellulosic materials. Dissolution can efficiently tions at different temperatures on hard- and softwoods, to develop
disintegrate the structure of lignocellulose and thus improve the an efficient pretreatment method for bioconversion of lignocel-
biological conversion of lignocelluloses. Sodium hydroxide is a luloses. Two common wood species including a softwood spruce
simple chemical that can swell or even dissolve cellulose at high and a hardwood birch were pretreated and then subjected to
concentrations. The dissolution mechanism is that soda hydrates
can penetrate the amorphous area of cellulose, and destroy
the neighboring crystalline regions.1 Furthermore, addition of ∗
Correspondence to: Azam Jeihanipour, School of Engineering, University of
chemicals such as thiourea,3 urea,4 or a combination of urea Borås, Borås, Sweden. E-mail: Azam.Jeihanipour@hb.se
and thiourea5 have been shown to improve the dissolution
behavior of lignocelluloses in the NaOH solution. The proposed a School of Engineering, University of Borås, Borås, Sweden
mechanism is that these components trap the free water and
b Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan,
prevent the interaction between cellulose chains via hydrogen
81746-73441, Iran
bonds.6 Polyethylene glycol (PEG)/NaOH7 aqueous solution has
recently been used as a potential ‘green’ solvent for cellulose.8 PEG c Department of Chemical Engineering, Isfahan University of Technology,
molecules act as hydrogen-bonding acceptors and prevent the Isfahan, 84156-83111, Iran

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c 2012 Society of Chemical Industry
 www.soci.org A Mohsenzadeh et al.

enzymatic hydrolysis for 72 h, and the best results were applied Digestion
for ethanol and biogas production. Using thermophilic bacteria at 55 ◦ C, anaerobic digestion was
performed in serum glass bottles which were closed with butyl
rubber seals and aluminum caps.18 The working volume of the
MATERIALS AND METHODS bottles was 118 mL. The inoculum was delivered from a 3000 m3
Raw materials and their analyses municipal solid waste digester operating at thermophilic (55 ◦ C)
Spruce (Picea abies) and birch (Betula pendula) wood species were conditions (Borås Energi och Miljö AB, Borås, Sweden). Pretreated
collected from the forests around the city of Borås in Sweden. They or untreated wood (0.25 g), 20 mL inoculum, and 5 mL deionized
were debarked and milled to particle size between 75 and 500 µm, water were added to each flask. In order to estimate the amount
and their dry weights were determined by drying at 105 ◦ C for 24 h. of biogas produced by just the inoculum, deionized water and
The carbohydrate and lignin fractions were analyzed according to inoculum were used as blank. Furthermore, each flask was flushed
NREL procedure.12 In this method, acid hydrolysis of cellulose and with a mixture of 80% nitrogen and 20% carbon dioxide to achieve
hemicelluloses is carried out to depolymerize them to sugars, and anaerobic conditions. In order to calculate the amount of methane
the lignin is measured as acid-insoluble and acid-soluble lignins. produced during digestion, samples of gas were taken from the
The acid-soluble lignin was measured by UV–vis spectroscopy at headspace of flasks on a regular basis and analyzed by a gas
205 nm with absorptivity of 30 (L g−1 cm−1 ). chromatograph (GC).

Pretreatments
Analytical methods
Aqueous alkali solutions were prepared by mixing 7 wt% NaOH
The methane and carbon dioxide in biogas were analyzed
with 12 wt% urea, 5.5 wt% thiourea, 1 wt% PEG, or 8 wt%
using a GC (Auto System Perkin Elmer, Waltham, MA) supplied
urea/6.5 wt% thiourea in deionized water. For the pretreatments,
with a packed column (Perkin Elmer, 6 × 1.8 OD, 80/100,
7 g of wood species was added to 93 g alkaline solution and stirred
Mesh) and a thermal conductivity detector (Perkin Elmer)
for 30 min at room temperature. The mixtures were stored at −15,
with inject temperature of 150 ◦ C. The carrier gas used was
0, 22, and 80 ◦ C for 16 h then, 100 mL of deionized water was
added to each solution and stirred for 1 h at ambient temperature. nitrogen with a flow rate of 23 mL min−1 at 60 ◦ C. Sugars and
The pretreated wood samples were then filtered and washed with fermentation products were analyzed by HPLC (Waters, Milford,
deionized water to remove the chemicals, and neutralize to pH 7. MA) supplied with RI detector (Waters 2414) and an ion-exchange
These pretreated materials were then freeze-dried to maintain column (Aminex HPX-87H, Bio-Rad, Hercules, CA) at 60 ◦ C using
the water swelling state and to prevent structural collapse before 5 mmol L−1 H2 SO4 as eluent at flow rate 0.6 mL min−1 .
further use.13 The crystallinity of pretreated lignocelluloses was measured
by a Fourier transform infrared (FTIR) spectrometer (Impact 410,
Nicolet Instrument Corp., Madison, WI). The spectra were acquired
Enzymatic hydrolysis
with an average of 64 scans and a resolution of 4 cm−1 , in the
The pretreated and untreated lignocelluloses were enzymatically
range from 600 to 4000 cm−119 monitored by ‘Nicolet OMNIC 4.1’
hydrolyzed using 5% w/v dry matter in 50 mmol L−1 sodium citrate
software.
buffer at pH 4.8. The suspensions were autoclaved, and then
All experiments and analyses were performed in duplicate,
supplemented with 20 FPU cellulase (celluclast 1.5L, Novozyme,
except the digestions, which were performed in triplicate, and
Denmark) and 50 IU β-glucosidase (Novozyme 188, Novozyme,
results are presented as mean values. The average standard
Denmark) per gram of dry wood. The enzymatic hydrolyses were
deviation was 4.3%.
carried out at 45 ◦ C in 100 mL flasks in a water bath shaker (OLS
200, Grant Instruments, UK) at 120 rpm for 72 h. The activities of the
cellulase and β-glucosidase were 80 FPU mL−1 and 240 IU mL−1
measured according to Adney and Baker14 and Ximenes et al.,15
RESULTS
respectively. Spruce and birch wood species were pretreated by four different
alkaline solutions including [7 wt% NaOH, 5.5 wt% thiourea],
[7 wt% NaOH, 8 wt% urea, 6.5 wt% thiourea], [7 wt% NaOH, 12 wt%
Fermentation
urea], or [7 wt% NaOH, 1 wt% PEG] at −15, 0, 22 and 80 ◦ C for 16 h.
Fermentations were performed in 118 mL anaerobic bottles. The
The effects of all pretreatments on the composition and enzymatic
fermenting microorganism was a flocculation strain of Saccha-
hydrolysis of the wood species were investigated. Then, the best
romyces cerevisiae (CCUG 53 310, Culture Collection, University of
pretreatment conditions were applied to further study of the
Gothenburg, Sweden). The strain was preserved on agar plates
crystallinity changes of the lignocelluloses, and yields of ethanol
containing (g L−1 ): yeast extract, 10; soy peptone, 10; glucose, 20;
and biogas in fermentation and anaerobic digestion, respectively.
agar, 20. It was grown at 30 ◦ C and maintained at 4 ◦ C. A volume
of 20 mL of each hydrolyzate was supplemented with 5 mL of all
other nutrients16 and were inoculated with 1.0 ± 0.1 g L−1 yeast Effects of the pretreatments on the composition of birch
and were incubated at 30 ◦ C in a shaking bath for 30 h anaero- and spruce
bically. Samples were analyzed for ethanol and sugar by HPLC. The effects of pretreatments of birch and spruce on their cellulose
All experiments were performed in duplicate and average values and lignin compositions are summarized in Table 1. Acid-insoluble
were reported. The inocula were prepared by aerobic cultivation of lignin (AIL) was dominant in these wood species. The AIL content
the yeast in 1 L cotton-plugged Erlenmeyer flasks in a water-bath of birch was 21.9%, which was increased to 23.1–27.3% after
shaker at 120 rpm and 30 ◦ C for 24 h. A 500 mL growth medium various pretreatments, while the AIL in spruce which was 28.3%
was prepared as previously reported.17 The yeast was separated was not significantly changed (Table 1). On the other hand, the
after 5 min settlement followed by 5000 rpm centrifugation for effect of alkaline pretreatment on the acid-soluble lignin (ASL)
10 min under aseptic conditions. content was more significant. By decreasing the temperature of

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Table 1. Cellulose and lignin content of treated and untreated birch and spruce

Acid insoluble lignin (AIL) Birch (wt%) Acid insoluble lignin (AIL) Spruce (wt%)

Reagent Temp.( ◦ C) −15 0 22 80 −15 0 22 80

NaOH/Thiourea 27.3 25.1 24.6 23.1 28.4 27.9 28.1 27.4

NaOH/Thiourea/Urea 26.8 25.3 24.6 22.1 28.5 28.4 28.2 27.3
NaOH/Urea 26.1 24.7 24.6 25.3 28.2 27.9 28.1 29.4
NaOH/PEG 26.1 24.3 24.3 25.5 28.0 27.4 28.1 30.3
Untreated 21.9 21.9 21.9 21.9 28.3 28.3 28.3 28.3

Acid soluble lignin (ASL) Birch (wt%) Acid soluble lignin (ASL) Spruce (wt%)

NaOH/Thiourea 5.8 3.8 4.9 4.4 0.63 0.63 0.68 0.68

NaOH/Thiourea/Urea 5.6 5.6 4.6 4.4 0.60 0.60 0.69 0.69
NaOH/Urea 5.7 3.8 4.6 4.6 0.61 0.60 0.60 0.53
NaOH/PEG 5.8 5.8 4.6 4.4 0.48 0.48 0.57 0.49
Untreated 2.7 2.7 2.7 2.7 0.50 0.50 0.50 0.50

Cellulose Birch (wt%) Cellulose Spruce (wt%)

NaOH/Thiourea 37.5 53.4 56.1 58.4 32.3 45.8 45.7 48.5

NaOH/Thiourea/Urea 38.8 55.8 57.3 58.1 33.5 46.8 46.3 48.7
NaOH/Urea 38.8 55.7 56.8 57.8 32.8 45.7 45.3 48.6
NaOH/PEG 37.6 53.6 55.7 58.4 32.2 44.6 45.9 48.3
Untreated 40.0 40.0 40.0 40.0 43.0 43.0 43.0 43.0

the pretreatment, the ASL content of birch increased between 41 The cellulose contents of the pretreated and untreated
and 115% compared with an untreated sample. However, it did lignocelluloses are presented in Table 1. For all reagents, increasing
not change more than 38% for spruce (Table 1). the temperature of pretreatment generally resulted in higher
Although it seems that the pretreatment has increased the cellulose content of the pretreated materials (Table 1). The effect
lignin content, the calculated lignin removal of pretreatments of the pretreatments on the cellulose is clearer when calculating
showed dissimilar results (Table 2). A considerable part of the cellulose recovery (Table 2). The recoveries of cellulose were
lignin, i.e. 11.1–38.9%, was removed by the pretreatments. defined as the ratio of the difference between the amount of
The lignin removal by NaOH/thiourea and NaOH/thiourea/urea cellulose before and after the pretreatments to the amount of
was more successful at higher temperatures, for both hard- cellulose in untreated lignocellulose. Cellulose recovery for spruce
and softwoods – whereas at 80 ◦ C, the NaOH/thiourea solution was more than that for the birch species, and in both cases
removed 34.2 and 18.7%, and NaOH/thiourea/urea removed 38.9 it increased with the temperature due to better solubility of
and 19.4% of the lignin of birch and spruce, respectively. However, cellulose at the lower temperature (Table 2). It is apparent from
the best temperatures for lignin removal by NaOH/urea and the data in Table 2 that the cellulose recovery is more dependent
NaOH/PEG from both hard- and softwoods were 0 and 22 ◦ C, on the temperature than on the type of mixed reagent with
respectively (Table 2). NaOH. However, for all the temperatures, the recovery of cellulose

Table 2. Lignin removal and cellulose recovery after alkali pretreatment of birch and spruce species under different conditions

Birch Spruce

Reagent Temp.( ◦ C) −15 0 22 80 −15 0 22 80

Lignin removal (wt%)

NaOH/Thiourea 17.4 30.8 29.9 34.2 15.1 17.4 17.6 18.7

NaOH/Thiourea/Urea 19.8 28.8 33.2 38.9 14.8 16.4 18.1 19.4
NaOH/Urea 22.2 32.8 28.8 29.5 16.6 17.9 16.9 13.8
NaOH/PEG 13.6 28.1 28.5 28.1 16.8 19.4 23.4 11.1

Cellulose recovery (wt%)

NaOH/Thiourea 63.8 80.7 83.9 88.3 63.3 89.0 87.7 94.1

NaOH/Thiourea/Urea 62.2 81.1 82.8 85.6 65.8 90.3 88.0 94.2
NaOH/Urea 61.6 82.8 87.3 86.1 63.6 88.3 88.0 94.2
NaOH/PEG 64.2 81.3 84.0 83.4 62.9 86.3 82.3 93.6

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when treating spruce with NaOH/thiourea/urea was a little higher

Table 3. Yield of ethanol production of untreated spruce and birch
compared with the treatment with other solutions (Table 2). and samples pretreated by NaOH/thiourea aqueous solution at −15 ◦ C
Spruce Birch
Sample (% theoretical yield∗ ) (% theoretical yield∗ )
Enzymatic hydrolysis
Pretreated and untreated lignocelluloses were subjected to Untreated 10.9 12.9
enzymatic hydrolysis in order to investigate the effect of the Pretreated with 54.9 80.9
pretreatment. The results are presented in Figs 1 and 2. The NaOH/thiourea
enzymatic hydrolysis of untreated birch and spruce for 72 h ∗ Percentage of theoretical ethanol yield = [ethanol produced
resulted in 23.1% and 11.7% hydrolysis compared with the (g L−1 )]/[0.51×1.111×initial weight of biomass (g L−1 ) × F]×100,
theoretical yield. A large improvement was observed after where F is cellulose fraction in biomass.
pretreatment of hardwood birch, and the hydrolysis yields were
improved to 48–83% of the theoretical yield (Fig. 1). The effect
of temperature on the improvement of the saccharification of The materials pretreated by NaOH/thiourea at −15 ◦ C were
birch is more significant for NaOH/thiourea than for the other selected for more investigation of fermentation, crystallinity and
reagents (Fig. 1). In other words, the final cellulose conversion of biogas production.
birch, at 72 h, pretreated with NaOH/urea/thiourea, NaOH/urea,
or NaOH/PEG was not significantly changed by changing the
temperature (Fig. 1(B), 1(C), 1(D)). The best saccharification yield Fermentation
of birch, i.e. 83% of theoretical yield, was obtained using The fermentation experiments with S. cerevisiae for 30 h were
NaOH/thiourea at −15 ◦ C. carried out for untreated birch and spruce species and samples
The pretreatment of spruce improved its hydrolysis yields from pretreated with NaOH/thiourea at −15 ◦ C, which had the best
11% to 27–57% of the theoretical yield (Fig. 2). The effect of yield of enzymatic hydrolysis after 72 h for both lignocelluloses. A
temperature on the yield of enzymatic hydrolysis of pretreated significant improvement in ethanol yield was obtained, from 10.9%
spruce with NaOH/urea was not significantly affected by the of theoretical yield for untreated spruce to 54.9% for the pretreated
pretreatment temperature (Fig. 2(C)). However, temperatures of one, and also from 12.9% of theoretical yield for untreated birch
−15 and 0 ◦ C were the most effective ones in pretreating spruce to 80.9% for the pretreated one (Table 3). Hence, NaOH/thiourea
with NaOH/thiourea and NaOH/PEG (Fig. 2(A) and 2(D)). In contrast, pretreatment at −15 ◦ C of hardwood birch for ethanol production
was more efficient than that of the softwood spruce.
the efficiency of pretreatment of spruce with NaOH/urea/thiourea
at 80 ◦ C on the yield of enzymatic hydrolysis was as effective as
pretreatment at −15 ◦ C (Fig. 2(B)). The highest saccharification Biogas production
yield of spruce after 72 h, which was 57% of the theoretical Selected pretreated and untreated birch and spruce species
yield, was obtained by pretreatment with NaOH/thiourea at were also subjected to anaerobic digestion for 30 days under
−15 ◦ C. thermophilic conditions (55 ◦ C). The pretreatment greatly affected

60 60
(A) NaOH/thiourea (B) NaOH/thiourea/urea
50 50
(% of theoretical yield)

(% of theoretical yield)
Glucose yield

Glucose yield

40 40

30 30

20 20

10 10

0 0
0 24 48 72 0 24 48 72

60 60
(C) NaOH/urea (D) NaOH/PEG
50 50
(% of theoretical yield)

(% of theoretical yield)
Glucose yield

Glucose yield

40 40

30 30

20 20

10 10

0 0
0 24 48 72 0 24 48 72
Time (h) Time (h)

Figure 1. Enzymatic hydrolysis of untreated and pretreated birch by aqueous solutions of NaOH/thiourea, NaOH/thiourea/urea, NaOH/urea, and
NaOH/PEG at different temperatures: (•) untreated wood, and pretreatment at (
) −15 ◦ C, () 0 ◦ C, () 22 ◦ C, and (×) 80 ◦ C. The glucose yield is
calculated as the percentage of theoretical yield [glucose produced (g L−1 ) (1.111× biomass (g L−1 ) ×F), where F is the cellulose fraction in biomass].

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100 100
(A) NaOH/thiourea (B) NaOH/thiourea/urea
90 90

(% of theoretical yield)
(% of theoretical yield)
80 80
70 70

Glucose yield
Glucose yield
60 60
50 50
40 40
30 30
20 20
10 10
0 0
0 24 48 72 0 24 48 72
100 100
(C) NaOH/urea
90 90 (D) NaOH/PEG
(% of theoretical yield)

(% of theoretical yield)
80 80
70 70
Glucose yield

Glucose yield
60 60
50 50
40 40
30 30
20 20
10 10
0 0
0 24 48 72 0 24 48 72
Time (hr) Time (hr)

Figure 2. Enzymatic hydrolysis of untreated and pretreated spruce by aqueous solutions of NaOH/thiourea, NaOH/thiourea/urea, NaOH/urea, and
NaOH/PEG at different temperatures: (•) untreated wood, and pretreatment at (
) −15 ◦ C, () 0 ◦ C, () 22 ◦ C, and (×) 80 ◦ C. The glucose yield is
calculated as the percentage of theoretical yield [glucose produced (g L−1 ) (1.111× biomass (g L−1 ) ×F), where F is the cellulose fraction in biomass].

0.4 0.8
Untreated
Methane yield (l/g VS)

0.3 Treated

0.2
Crystallinity Index

0.7

0.1

0
0 5 10 15 20 25 30 35 0.6
Time (days)

Figure 3. Effect of pretreatment of spruce and birch by NaOH/thiourea

aqueous solution at −15 ◦ C on the yield of methane in biogas production:
() untreated birch, () treated birch, () untreated spruce, and () treated
spruce. 0.5
Birch Spruce

Figure 4. Total crystallinity index of untreated spruce and birch and

the methane production for both birch and spruce species (Fig. 3). pretreated by NaOH/thiourea aqueous solution at −15 ◦ C.
The biogas yields of untreated birch and spruce were 0.23 and
0.03 L g−1 VS, respectively, which were improved by 56% and
600% after pretreatment. DISCUSSION
The hydrolyses of both hard- and softwood species was signifi-
Change in structure of the wood by pretreatment cantly improved by the selected pretreatments under all condi-
Using FTIR spectroscopy, changes in the crystallinity of the tions tested. Dissolving in NaOH/thiourea, NaOH/thiourea/urea,
treated and untreated birch and spruce were studied. The NaOH/urea, and NaOH/PEG aqueous solutions significantly mod-
1427 and 898 cm−1 absorption bands assigned to the crystalline ified the structure of wood and increased the yield of its
cellulose I and cellulose II, respectively, were used to investigate biodegradation products. The hydrolysis of softwoods is usu-
the crystallinity changes. The crystallinity index, which is the ally more difficult than that of hardwoods,20,21 and the presence
absorbance ratio A1427 /A898 is presented in Fig. 4. The spruce was of lignin is an important factor for the hydrolysis.22 This claim is
more crystalline than the birch species. However, the pretreatment in line with our results in which the lignin content of hardwood
reduced the crystallinity of both materials. The crystallinity index birch was less than that of softwood spruce, and consequently,
was decreased by pretreatment from 0.56 for untreated birch to the pretreatment process was more efficient for birch. However,
0.54 for pretreated birch, and from 0.71 for untreated spruce to lignin removal is not at the highest level for pretreatment by
0.68 for pretreated spruce. NaOH/thiourea at −15 ◦ C, which gave the best yield of enzymatic

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 www.soci.org A Mohsenzadeh et al.

hydrolysis. Therefore, the parity of lignin removal with improving 2 Taherzadeh M and Karimi K, Pretreatment of lignocellulosic wastes
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this process, which affects the composition of wood. dilute-acid pretreated rice straw by simultaneous saccharification
and fermentation with Mucor indicus, Rhizopus oryzae, and
Saccharomyces cerevisiae. Enzyme Microbiol Technol 40:138–144
(2006).
CONCLUSION 18 Hansen T, Schmidt J, Angelidaki I, Marca E, Jansen J, Mosbæk H, et al,
Among the different alkali dissolution systems, pretreatment by Method for determination of methane potentials of solid organic
NaOH/thiourea aqueous solution at −15 ◦ C and atmospheric pres-
waste. Waste Manage 24:393–400 (2004).
19 Carrillo F, Colom X, Suñol JJ and Saurina J, Structural FTIR analysis
sure is an effective alternative for pretreating hard- and softwoods and thermal characterisation of lyocell and viscose-type fibres. Eur
to enhance biogas and bioethanol production. However, the pro- Polym J 40:2229–2234 (2004).
cess is more effective for hardwood birch than for softwood spruce. 20 Gregg D and Saddler J, Bioconversion of lignocellulosic residue
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21 Palonen H, Thomsen A, Tenkanen M, Schmidt A and Viikari L, Evaluation
ACKNOWLEDGEMENT of wet oxidation pretreatment for enzymatic hydrolysis of softwood.
This work was supported financially by Sparbanken Sjuhärad Appl Biochem Biotechnol 117:1–17 (2004).
(Sweden). 22 Isogai A and Atalla R, Dissolution of cellulose in aqueous NaOH
solutions. Cellulose 5:309–319 (1998).

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c 2012 Society of Chemical Industry J Chem Technol Biotechnol (2012)