Pretreatment of fibre sludge in ionic liquids followed by enzyme and acid catalysed hydrolysis

Article abstract of DOI:10.1016/j.cattod.2012.04.001

Pretreatment of fibre sludge in ionic liquids and enzyme or acid catalysed hydrolysis of fibre sludge is studied. Ionic liquids, i.e. 1-allyl-3- methylimidazolium chloride ([AMIM]Cl) and 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), are used in the pretreatment step. Effect of ionic liquid (IL) pretreatment on the acid and enzyme catalysed hydrolysis of fibre sludge is considered. Cellulose content of fibre sludge is more than 80 wt%, and therefore, reducing sugars obtained as a result of hydrolysis contain mostly glucose. To maximize the yield of reducing sugars during the enzymatic hydrolysis, the combinations of selective enzymes are required. Results also show that the use of ionic liquids in the pretreatment step before acid and enzyme hydrolysis increases significantly the yield of total reducing sugars.

Full text of DOI:10.1016/j.cattod.2012.04.001

Catalysis Today 196 (2012) 11–15
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Catalysis Today
journal homepage: www.elsevier.com/locate/cattod
Pretreatment of fibre sludge in ionic liquids followed by enzyme and acid
catalysed hydrolysis
Jana Holma,b,∗, Ulla Lassia,b, Henrik Romara,b, Riikka Lahtia,b, Johanna Kärkkäinenb, Marja Lajunenb
a Kokkola University Consortium Chydenius, Talonpojankatu 2 B, FI-67100 Kokkola, Finland
b University of Oulu, Department of Chemistry, P.O. Box 3000, FI-90014 Oulu, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Pretreatment of fibre sludge in ionic liquids and enzyme or acid catalysed hydrolysis of fibre
sludge is studied. Ionic liquids, i.e. 1-allyl-3-methylimidazolium chloride ([AMIM]Cl) and 1-butyl-3-
methylimidazolium chloride ([BMIM]Cl), are used in the pretreatment step. Effect of ionic liquid (IL)
pretreatment on the acid and enzyme catalysed hydrolysis of fibre sludge is considered.
Cellulose content of fibre sludge is more than 80 wt%, and therefore, reducing sugars obtained as a result
of hydrolysis contain mostly glucose. To maximize the yield of reducing sugars during the enzymatic
hydrolysis, the combinations of selective enzymes are required. Results also show that the use of ionic
liquids in the pretreatment step before acid and enzyme hydrolysis increases significantly the yield of
total reducing sugars.
Received 31 October 2011
Received in revised form 31 January 2012
Accepted 2 April 2012
Available online 5 May 2012
Keywords:
Biomass
Acid hydrolysis
Enzymatic hydrolysis
Glucose
© 2012 Elsevier B.V. All rights reserved.
Ionic liquid
1. Introduction
In this study, fibre sludge was pretreated with ionic liquids
followed by hydrolysis into fermentable sugars. In our previous
Over the years, a significant amount of research has been
conducted worldwide to convert lignocellulosic biomass (forestry
waste, agricultural residues and energy crops) through a sugar
platform towards other useful materials [1]. Lignocellulose, for
example, can work as a sustainable feedstock for future valuable
products such as biofuels and chemicals.
studies [5], we proved that ionic liquid [BMIM]Cl is efficient sol-
vent for fibre sludge that contains cellulose. This is consistent with
several previous studies where lignocellulose is pretreated in ionic
liquids [6,7]. Recently, the use of ionic liquids in the pretreatment
of paper sludge [8] has also been published, but only a few publi-
cations related to fibre sludge are available.
Research has shown that cellulose forms intramolecular hydro-
gen bonds, which makes it hardly soluble in conventional solvents
[2]. Furthermore, some ionic liquids (ILs) also possess the necessary
properties to be potential chemicals for the pretreatment of bioma-
terials. Effective hydrolysis of cellulose to fermentable sugars is a
fundamental and necessary step in cellulosic biofuel production
[3]. For example, biobutanol can be produced from forest waste
materials through a sugar platform. Biobutanol is a suitable fuel
for combustion engines (gasoline engines) due to its high energy
density and its sufficient air-to-fuel ratio which can be adjusted in
existing engines. Biobutanol as a fuel is also suitable for the distribu-
tion via existing pipelines due to its low vapour pressure. Compared
with ethanol and methanol fuels, biobutanol is less corrosive, more
energy efficient and less dissolvable in water [4].
Therefore, in this study ionic liquids are used as a pretreatment
step for the acid and enzyme hydrolysis of fibre sludge. If the fibre
sludge, a by-product from the pulping process, can be dissolved and
hydrolysed to reducing sugars with high activity and selectivity this
will open up new possibilities for biofuel production.
2. Experimental
2.1. Materials and characterization
Fibre sludge (provided by a Finnish pulp mill), which is also
known as a primary sludge, is a by-product from the pulping pro-
cess. It contains mostly cellulose (approximately 80–90 vol%) and
water. In laboratory experiments filter paper (Whatman 1) was
used as a reference for pure cellulose.
Ionic liquids [BMIM]Cl and [AMIM]Cl were prepared and used
as presented in Section 2.2. Butyl chloride (Reagent Plus®, 99%),
allyl chloride (Reagent Plus®, 99%) and N-methylimidazole (≥99%)
as well as anolyte and catolyte chemicals, Hydranal-Coulomat AG
∗
Corresponding author at: University of Oulu, Department of Chemistry, P.O. Box
3000, FI-90014 Oulu, Finland. Tel.: +358 408086623; fax: +358 68253100.
E-mail address: jana.holm@cou.fi (J. Holm).
0920-5861/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cattod.2012.04.001

12
J. Holm et al. / Catalysis Today 196 (2012) 11–15
and CG, respectively were purchased from Sigma Aldrich (Schnell-
dorf, Germany). Ethyl acetate (99%, Rathburn) and acetonitrile (98%,
Labscan) were used as obtained from the supplier. Electrospray ion-
ization mass spectra (ESI MS) were recorded with a Micromass, LCT
mass spectrometer and 1H NMR spectra with a Bruker DBX 200
instrument (200.13 MHz) in CDCl3.
Hydrolysis of cellulose was performed by acids or enzymes. Both
maleic and sulphuric acid were used in acid hydrolysis whilst in the
enzymatic hydrolysis, ACCELLERASE enzyme mixture was used, as
described in Section 2.3.
␣, ␤- and ␥-cellulose content of fibre samples were determi-
nated with the T 203cm-99 Official Standard method [9]. This
standard method can be applied to bleached or delignified pulps
and therefore, it is a suitable method in this study since fibre sludge
is a delignified pulp. ␣-Cellulose indicates undegraded, higher-
molecular-weight cellulose content in pulp, ␤-cellulose indicates
a degraded cellulose, whereas ␥-cellulose consists mainly of hemi-
cellulose [9].
Total carbon content (TC) of fibre sludge was determined by
elementary analysis using a Perkin Elmer CHNS analyser in which
elements of the sample are combusted to simple gases (CO2, H2O,
N2 and SO2) in a pure oxygen atmosphere. Gases are homogenized
and detected by controlling the exact conditions of pressure, tem-
perature and volume. The homogenized gases are de-pressurized
through a column where they are separated and thus are identified
as a function of their thermal conductivities.
Fig. 1. Ground fibre sample (left), [BMIM]Cl (middle) and a mixed solution of the
ground fibre sample and [BMIM]Cl (right).
2.3. Enzymes
Various combinations of selective enzymes are required to
maximize the yield of reducing sugars during enzymatic hydrol-
ysis. Optimal enzyme mixture in the hydrolysis depends upon the
composition of the various biomass fractions (i.e. cellulose, hemi-
cellulose, and lignin) in the biomass substrate [12]. The required
enzyme dosage may vary significantly based on the specific compo-
sition of the biomass feedstock and the particular physical and/or
chemical pretreatment method used. The pretreatment can cre-
ate inhibiting products that can reduce the activity of the enzyme,
resulting in lower cellulose conversion and/or increased enzyme
dosage [13].
Chloride (Cl−) and sulphate (SO42−) concentration in the sugar
solution after the hydrolysis were analysed by ion chromatogra-
phy (761 Compact IC, Metrohm). Metal analysis of fibre sludge
was also performed by Inductively Coupled Plasma (ICP-AES and
ICP-SFMS).
A cellulase enzyme mixture is used to catalyse the breakdown of
cellulosic material into glucose, cellobiose, and higher glucose poly-
mers. The cellulase enzyme mixture can contain various enzymes
mainly exo- and endoglucanase, hemi-cellulase and ␤-glucosidase.
␤-Glucosidase, also known as cellobiase is the main compound that
hydrolyses cellobiose to glucose [14].
2.2. Ionic liquids
Two chloride-based ionic liquids, [BMIM]Cl and [AMIM]Cl, were
synthesized according to the literature methods [10,11], respec-
tively. Reactions were carried out under nitrogen atmosphere and
followed by ESI-MS and 1H NMR. Produced ILs were dried overnight
in a high vacuum at 50–70 ◦C and stored in a desiccator. Water con-
tent of the IL determined by the Mettler Toledo DL36 Karl Fischer
coulometer was <0.1% (w/w).
In this study, ACCELLERASE® 1500 enzyme complex (provided
by Genencor) was used. It contains a combination of enzymes
which effectively modify and digest non-starch carbohydrates.
It is a lignocellulosic material which is composed mainly of
cellulose, hemicellulose, and ␤-glucans. ACCELLERASE® 1500 is
capable of efficiently and synergistically hydrolysing lignocellu-
losic biomass into fermentable monosaccharides and contains
higher levels of ␤-glucosidase activity, to ensure almost com-
plete conversion of cellobiose to glucose. ACCELLERASE® 1500
is produced with a genetically modified strain of Trichoderma
reesei [15] which results in a cellulase activity of 2283 CMCU/g
(CMCU = carboxymethyl cellulose unit) and ␤-glucosidase activity
of 715 pNPG U/g. One pNPG unit denotes 1 ␮mol of nitrophenol
liberated from paranitrophenyl-B-d-glucopyranoside per minute
at 50 ◦C [15]. The operational stability of the ACCELLERASE® 1500
enzyme complex is at its best between the temperature range of
50–65 ◦C and at pH values from 4.0 to 5.0. In addition, the enzyme
complex can be inactivated at temperatures above 70 ◦C or at pH
levels above 7.0 or below 4.0.
[BMIM]Cl was prepared from butyl chloride (159.5 g, 1.72 mol)
and N-methylimidazole (103.0 g, 1.25 mol) in
a 500 ml flask
by mixing and refluxing until all methylimidazole had reacted
(24–48 h). The crude product was then recrystallized from an ethyl
acetate–acetonitrile mixture (55:45). The yield of white [BMIM]Cl
was 174.8 g (80%). 1H NMR (200 MHz, CDCl3): ı 0.96 (3H, t,
JHH = 7.3 Hz), 1.41 (2H, m), 1.89 (2H, m), 4.13 (3H, s), 4.34 (2H, t,
JHH = 7.3 Hz), 7.47 (1H, t, JHH = 1.8 Hz), 7.62 (1H, t, JHH = 1.8 Hz), 10.67
(1H, s). MS(ESI+) [m/z (rel. int. (%))]: 139 (100, [BMIM]). MS(ESI−)
[m/z (rel. int. (%))]: 210 (100, Cl[BMIM]Cl).Preparation of [AMIM]Cl
was carried out by placing N-methylimidazole (125.0 g, 1.53 mol)
in a 500 ml round-bottom flask and cooling the reaction vessel in an
ice bath. Allyl chloride (155.0 g, 2.03 mol) was added slowly into the
cooled flask. Reaction was mixed for a while at 0 ◦C and allowed to
warm up to the room temperature. Then the reaction mixture was
gently heated up with an oil bath and refluxed for 24 h until all N-
methylimidazole had reacted. A crude product was washed with
ethyl acetate (3 × 40 ml) and dried overnight under high vacuum
at 50 ◦C. The yield was 236.1 g (98%). 1H NMR (200 MHz, CDCl3) ı:
4.09 (3H, s), 4.99 (2H, d, JHH = 6.2 Hz), 5.44 (2H, m), 5.99 (1H, m), 7.39
(1H, t, JHH = 1.8 Hz), 7.59 (1H, t, JHH = 1.8 Hz), 10.72 (1H, s). MS(ESI+)
[m/z (rel. int. (%))]: 123 (100, [AMIM]). MS(ESI−) [m/z (rel. int. (%))]:
193 (100, Cl[AMIM] Cl).
2.4. Sample pre-treatment in ionic liquids
Fibre sample from the pulp mill was ground in a ball mill for
more than 1 h with 55 larger balls (Ø200 mm) and 30 smaller
balls (Ø90 mm) (Fig. 1). Due to a hygroscopic nature of chloride-
based ILs they were dried prior use (water content below 0.5%)
to achieve efficient dissolution of cellulosic material. It is known
that lignocellulosic material dissolves better in dry IL than in water
contaminated IL [2].

J. Holm et al. / Catalysis Today 196 (2012) 11–15
13
Table 1
Metal analysis of fibre sludge.
Oxides
Value
Metals
Value
SiO2
Al2O3
CaO
Fe2O3
K2O
MgO
Na2O
4.92% DS
1.20% DS
1.07% DS
0.3% DS
0.147% DS
0.5% DS
Ba
Cr
Cu
Ni
Pb
S
59.8 mg/kg DS
43.8 mg/kg DS
2.86 mg/kg DS
4.78 mg/kg DS
1.46 mg/kg DS
1370 mg/kg DS
34.5 mg/kg DS
0.381% DS
Zn
DS, dry substance.
Glucose (GO) assay kit (Sigma) is an enzymatic analysing
method for glucose concentration. Enzymatic methods are
specific, sensitive and rapid. In this method d-glucose is oxidized to
d-gluconic acid and hydrogen peroxide by glucose oxidase. Hydro-
gen in the presence of peroxide reacts with reduced o-dianisidine
(colourless) to form brown oxidized o-dianisidene. Oxidized o-
dianisidene reacts further with sulphuric acid to form a more stable
coloured product namely pink oxidized o-dianisidine. This com-
pound can be detected by UV–vis at 540 nm and its concentration
is proportional to the original glucose concentration [17] UV–vis
(UV-1800 SHIMADZU) was used in this study to detect the colour
differences in solutions.
2.6. Hydrolysis of samples
Pretreated, regenerated cellulose samples (0.1 g) were hydrol-
ysed in sulphuric acid or maleic acid at different acid concen-
trations. Based on the preliminary experiments, optimal acid
concentrations of 2–3 M were used. Acid hydrolysis was per-
formed with different reaction times (0.5–3 h) at temperatures
of 23–100 ◦C whilst gently stirred (150 rpm). Based on this, the
optimized time for hydrolysis was 90 min (dependent on acid con-
centration which was 2–3 M). The reaction was followed by taking
the samples from the solution and by measuring the amount of total
reducing sugars (TRS%) by the DNS assay.
Pretreated, regenerated cellulose samples (0.1 g) were also
hydrolysed by using ACCELLERASE enzyme mixture. Conditions for
enzymatic hydrolysis are presented in detailed in Section 2.3. Buffer
solution of citrate (1 M) was used. The enzymatic hydrolysis was
followed by taking the samples from the solution in every 10 min
and by measuring the amount of total reducing sugars (TRS%) by the
DNS assay. The enzymatic hydrolysis was performed at pH of 5.0
and at temperature of 50 ◦C whilst being gently stirred for 5–24 h.
Fig. 2. Fractionation scheme in the pretreatment and hydrolysis of dried fibre
sludge.
100 mg of dry fibre sludge was combined with 5.0 g of IL
([BMIM]Cl or [AMIM]Cl) to create a solution which was then heated
on an oil bath at 100 ◦C for 30 min (optimized time for ionic liquid
pretreatment). The sample was gently stirred at a rate of 150 rpm.
Filter paper (Whatman 1) was used as a reference sample in the
experiments. 20 ml of hot deionized water was then added to the
solution of fibre sludge and IL after dissolution to act as an anti-
solvent for regenerating cellulose from ILs. The solution was left
to set for 15 min during which a solid precipitate was formed. The
supernatant containing the IL was removed with a Bühner funnel
and the precipitate was washed with deionized water. The ionic
liquid has to be removed carefully from the precipitate because it
is harmful for enzyme activity. The pretreated precipitate was then
collected and stored to be used in acid or enzymatic hydrolysis.
Fractionation scheme is presented in Fig. 2.
3. Results and discussion
3.1. Fibre sludge characterization
Dried fibre sludge contained 80 wt% of ␣-cellulose, 13 wt% of
␤-cellulose and 7 wt% of ␥-cellulose based on analysis by the T
203cm-99 Official Standard method [9]. Based on the elemental
analysis (as described in Section 2.1), dry fibre sludge contained
carbon (33.9 wt%), hydrogen (3.7 wt%) and nitrogen (<0.5 wt%), and
also sulphur (<0.5 wt%) originating from the pulping process. Chlo-
ride (Cl−) and sulphate (SO42−) in the sugar solution were analysed
by ion chromatography. As a result, chloride and sulphate concen-
trations after the hydrolysis were measured to be 2.7 g/l and 30 g/l
respectively. Results of metal analysis of dry fibre sludge are shown
in Table 1. Metal concentrations are on the ppm level, and there-
fore, have no direct effect on the pretreatment or hydrolysis step
of fibre sludge.
2.5. Analysis of TRS and glucose
Total reducing sugars (TRS) which can form some aldehyde or
ketone (including disaccharide, monosaccharide) can be measured
by the DNS method. 3,5-Dinitrosalicylic acid (DNS) reacts with
reducing sugars and other reducing molecules to form 3-amino-
5-nitrosalicylic acid, which absorbs light strongly at 540 nm [16].
Yield of total reducing sugars (TRS%) from fibre sludge was calcu-
lated as follows:
reducing sugars weight
fibre sludge weight
total reducing sugar yield (TRS%) =
× 100

14
J. Holm et al. / Catalysis Today 196 (2012) 11–15
120
100
80
60
40
20
0
[BMIM]Cl pretreated
reference sample
[AMIM]Cl pretreated
reference sample
[BMIM]Cl pretreated
fiber sludge
[AMIM]Cl pretreated
fiber sludge
no pretreated fiber
sludge
0
2
4
6
8
Hydrolysis ꢀme (h)
Fig. 3. Acid hydrolysis over fibre sludge (after ionic liquid pretreatment). Com-
parison between maleic and sulphuric acid under optimized conditions (acid
concentration 2–3 M, hydrolysis time 90 min).
Fig. 5. Relative yield of total reducing sugars after enzymatic hydrolysis. Enzymatic
hydrolysis of fibre sludge was done by ACCELLERASE 1500 enzyme mix (enzyme
loading 0.2 ml in 10 ml sample volume).
3.2. Hydrolysis results
results of enzymatic hydrolysis without any IL pretreatment, a 50%
increase in relative amount of measured sugars was obtained. There
was no significant difference between the use of ILs [BMIM]Cl and
[AMIM]Cl.
Acid hydrolysis was performed with sulphuric acid and maleic
acid in the concentration range of 2–3 M. Results of acid hydrolysis
are presented in Fig. 3. Hydrolysis with 3 M maleic acid provided the
best TRS yield of 1.4%, with a hydrolysis period of 90 min at 100 ◦C,
compared to 3 M sulphuric acid which resulted in a TRS yield of
7.4%. There is no significant concentration dependency using 2 M or
3 M maleic acid and sulphuric acid respectively. On the other hand,
the use of sulphuric acid results in much higher yields compared
with maleic acid.
Enzymatic hydrolysis gave a TRS yield of over 30% in 90 min at
50 ◦C and maximum yield of TRS 34.4% at 120 min. These results are
presented in Fig. 4. Based on these results, enzymatic hydrolysis
provides the highest yields in milder conditions and is the most
suitable hydrolysis method for fibre samples.
4. Conclusions
Using ILs in the pretreatment process of fibre sludge increased
the yield of total reducing sugars. TRS yields both in acid and enzy-
matic hydrolysis were higher compared to the relative TRS yield
of untreated fibre sludge samples. It was also shown that there
was no significant difference in using [BMIM]Cl or [AMIM]Cl in the
pretreatment step of fibre sludge. In both cases ionic liquids made
cellulose structure more accessible to acid or enzymatic degrada-
tion. However, enzymatic hydrolysis was much more efficient than
acid hydrolysis when fibre sludge was used as a feedstock for the
conversion of cellulose to reducing sugars. Most of these sugars in
the final solution were supposed to be glucose.
3.3. Effect of ionic liquid pretreatment
Ionic liquid pretreatment procedure is illustrated in Fig. 2. Yield
of solid material, mostly cellulose, after pretreatment was approx-
imately 50% (calculated from mass balance) of the dry fibre sludge.
Enzymatic hydrolysis results over fibre sludge are presented in Fig.
5, which shows the relative yield of total reducing sugars (%) after
ionic liquid pretreatments. As can be seen in Fig. 5, the use of ionic
liquid pretreatment gave a significant benefit. Compared to the
Acknowledgements
This work has been carried out with the financial support from
the Academy of Finland (project SusFuFlex and SusProc) within the
research program for Sustainable Energy. Authors acknowledge Mr.
Mark Jackson for linguistic corrections.
TRS% as a funcꢀon of hydrolysis ꢀme (min)
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