Bioresource Technology 102 (2011) 9105–9110

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Investigation into the structural, morphological, mechanical and thermal

behaviour of bacterial cellulose after a two-step purification process
Saharman Gea a,b, Christopher T. Reynolds c, Nima Roohpour a, Basuki Wirjosentono b,
Nattakan Soykeabkaew a,d, Emiliano Bilotti a,c, Ton Peijs a,c,e,⇑
a
School of Engineering and Material Science and Centre for Materials Research, Queen Mary University of London, London E1 4NS, UK
b
Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of Sumatera, Utara Medan, 20155, Indonesia
c
Nanoforce Technology Ltd., Queen Mary University of London, London E1 4NS, UK
d
Mae Fah Luang University, 333 Moo 1, School of Science, Thasud Muang, Chiang Rai 57100, Thailand
e
Eindhoven University of Technology, Eindhoven Polymer Laboratories, P.O. Box 513, 5600 MB Eindhoven, The Netherlands

a r t i c l e i n f o a b s t r a c t

Article history: Bacterial cellulose (BC) is a natural hydrogel, which is produced by Acetobacter xylinum (recently renamed
Received 18 July 2010 Gluconacetobacter xylinum) in culture and constitutes of a three-dimensional network of ribbon-shaped
Received in revised form 23 April 2011 bundles of cellulose microfibrils. Here, a two-step purification process is presented that significantly
Accepted 23 April 2011
improves the structural, mechanical, thermal and morphological behaviour of BC sheet processed from
Available online 29 April 2011
these hydrogels produced in static culture. Alkalisation of BC using a single-step treatment of 2.5 wt.%
NaOH solution produced a twofold increase in Young’s modulus of processed BC sheet over untreated
Keywords:
BC sheet. Further enhancements are achieved after a second treatment with 2.5 wt.% NaOCl (bleaching).
Bacterial cellulose
Alkalisation
These treatments were carefully designed in order to prevent any polymorphic crystal transformation
Bleaching from cellulose I to cellulose II, which can be detrimental for the mechanical properties. Scanning electron
Crystallinity microscopy and thermogravimetric analysis reveals that with increasing chemical treatment, morpholog-
Mechanical properties ical and thermal stability of the processed films are also improved.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction and Gram-negative Acetobacter xylinum is the only species known

to be capable of producing cellulose in commercial quantities.
Bacterial cellulose (BC) has attracted a great deal of academic Therefore, methods of producing BC have been developed with
and commercial interests, with numerous applications in biotech- the aims of improving yield, structure, and other desired physical
nology, pharmacy, biobased packaging and other fields of biomed- properties (Yamanaka et al., 2000). Besides using different produc-
ical science (Iguchi et al., 2000). The reason of such interests tion methods, the medium used for culture, the pH level, and the
resides in the ease in producing a fully biobased cellulosic material source of nitrogen and phosphate as the main food source for A.
which has good mechanical properties such as high tensile xylinum, as well as several additives, have also all been studied
strength and modulus, high purity, high water-binding capacity, (Huang et al., 2010).
and an ultra-fine network structure. The relative high mechanical A crucial step in the production of any cellulose product is its
properties of BC microfibrils have also led to their use as reinforc- purification, which is traditionally, known as a chemical pulping
ing agents in composite materials (Soykeabkaew et al., 2009; Gea process in the paper-making industry. This process is intended to
et al., 2010). The Young’s modulus of a single filament or microfi- remove essentially all of the undesired residual insoluble lignin
bril of BC as estimated using a Raman spectroscopy technique is and other chemicals bound to the cellulose fibre as well as impuri-
130–140 GPa (Hsieh et al., 2008; Eichhorn et al., 2010). ties occurring during processing and converting them into com-
Many strains of bacteria possess the ability to produce cellulose pounds which are soluble in alkali water. If lignin is not
at the surface of a medium containing carbon and nitrogen as food removed, photo-oxidation, which causes discolouration of paper
source. These include Sarcina, Agrobacterium, Rhizobium, and Aceto- with age, is likely to occur, and also the final paper product is brit-
bacter (Deinema and Zevenhuizen, 1971). However, the rod-shaped tle (Robert, 1996). Therefore, various methods have been devel-
oped and different chemicals have been used to produce a good
quality paper as well as to overcome low pulp yields (Bajpai, 2005).
⇑ Corresponding author at: School of Engineering and Material Science and
Centre for Materials Research, Queen Mary University of London, London E1 4NS,
Alkaline is one of the most commonly used chemicals in the
UK. mercerization process since it is able to hydrolyse and remove
E-mail address: [email protected] (T. Peijs). impurities present in cellulose. Unfortunately, this mercerization

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.04.077
9106 S. Gea et al. / Bioresource Technology 102 (2011) 9105–9110

process is often accompanied by an unwanted transformation of these sheets is envisaged by retaining the more high-performance
the crystal structure from cellulose I to cellulose II. The concentra- cellulose I crystal phase in the BC microfibrils.
tion of NaOH used for this transformation varies, depending on the
type and the origin of cellulose being treated. Previous researchers 2. Methods
have reported that the transformation of cellulose I to cellulose II
occurs at concentrations of NaOH above 6% (Borysiak and Gar- 2.1. Materials
barczyk, 2003; Moigne and Navard, 2010). Many researchers have
investigated the effect of immersion time on the purification of cel- In this study, strains of A. xylinum for the production of BC were
lulose, from few minutes to 48 h (Moharram and Mahmoud, 2008; supplied by the Microbiology Laboratory of the Institute of Agricul-
Mansikkamäki et al., 2005; Nakagaito and Yano, 2008; Oh et al., ture, Bogor, Indonesia. The chemicals used for the culture medium
2005; Shibazaki et al., 1997; Aziz and Ansell, 2004). were glucose, ammonium sulphate, potassium hydrogen ortho-
Research into the change of cellulose I to cellulose II as a result phosphate, magnesium sulphate and yeast extract. In the purifica-
of alkalisation is still the subject of debate (Zhou et al., 2002; Oh tion process, sodium hydroxide, sodium hypochlorite and
et al., 2005). Dinand et al. (2002) has hypothesised and tested that additional vitamins such as B1, B3 and B5 used to assist the growth
samples immersed in NaOH solutions for 2–24 h, with a given con- of the bacteria were used as received from VWR International (UK).
centration, yielded the same results.
There are three steps in the transformation process, which be-
2.2. Sample preparation
gins with fibre swelling due to water absorption, leading to a sig-
nificant increase in mobility of the cellulose chains. Then the
2.2.1. Preparation of BC pellicle
alkaline solution penetrates the amorphous areas of the cellulose,
The culture medium for the production of BC was prepared by
before saturating the cellulose and disrupting the crystalline re-
the procedure reported by Gea et al. (2007). For every litre of dis-
gions, finally forming new crystalline lattices after the mercerising
tilled water, 50 g glucose, 5 g (NH3)3PO4, 4 g KH2PO4, 5 g yeast ex-
solution is washed off (Lee et al., 2004). Usually, the amorphous
tract, and 0.1 g MgSO47H2O were added. This culture medium was
and crystalline regions which cover the upper surface of the cellu-
autoclaved at 121 °C for 2 h, and, once cooled to ambient temper-
lose structure react with the alkali solution and are removed when
ature, vitamins B1, B2 and B5 were added. The acidity of the med-
the solution is finally washed off (Gassan and Bledzki, 1999; Liu
ium was adjusted to pH = 4 using glacial acetic acid. A strain of
and Hu, 2008).
A. xylinum was inoculated into the static culture medium for
During the polymorphic transformation from cellulose I to cel-
21 days at 28 °C.
lulose II, a change in conformation from parallel to anti-parallel oc-
curs, which is accompanied by the breaking of numerous primary
inter- and intra-molecular hydrogen bonds in cellulose I. This 2.2.2. Treatment of BC pellicle
transformation is a secondary phenomenon, which could not occur After harvesting, some of the pellicles of BC were left on the
without first breaking the primary hydrogen bonds in cellulose I sieve to allow the liquid from the pellicles to escape through the
(Laszkiewicz, 1997). The result of this transformation is a signifi- sieve without any treatment. These samples are called untreated
cant decrease in mechanical properties. Ishikawa and Okano BC. The remaining pellicles were washed under running tap water,
(1997) have reported a drop in Young’s modulus of a single ramie and then these pellicles were immersed overnight in 2.5 wt.%
fibre from 27 GPa (cellulose I) to 21 GPa (cellulose II). The appear- NaOH. These are hereafter referred to as NaOH-treated BC (sin-
ance of cellulose polymorphs is easily detected by conventional gle-step treated BC). A third sample was prepared in the same
wide angle X-ray diffraction (Mansikkamäki et al., 2005). The mea- way as the second sample, i.e. NaOH-treated BC, and successively
surement of single cellulose fibres/fibrils has been performed by treated also with 2.5 wt.% NaOCl (two-step treated BC). The pelli-
Matsuo et al. (1990) using an X-ray diffraction method with cellu- cles were then rinsed under running tap water to remove any sol-
lose I having a modulus of 120–135 GPa and cellulose II 106– vent until neutral pH conditions.
112 GPa. Kroon-Batenburg and Kroon (1997) have also reported
the moduli of both polymorphs and found them to be 130– 2.2.3. Processing of BC sheet
137 GPa and 71–90 GPa for cellulose I and II, respectively. For the preparation of specimens for morphological analysis,
Bleaching is another very common process in the paper-making samples were left in the form of a gel, while, for other tests, such
industry. This stage is aimed at enhancing the brightness of the as mechanical, thermal, and structural analysis, the samples were
pulp to provide a bright white paper for applications such as print- processed into sheet form. For the preparation of BC sheet, the pel-
ing and tissue paper. It also eliminates the problem of yellowing of licles (90–95% water) was sandwiched between two stainless steel
the paper exposed to light due mainly to residual lignin in the pulp plates of 20 mm thickness along with a wire mesh, and compres-
(3–6 wt.%) and removes resin and other extractives present in sion moulded for 5 min at 115 °C at 70 MPa pressure to remove
unbleached pulp (Bajpai, 2005). Bleaching agents are often used any remaining water.
based on their reduction and oxidation behaviour in nature. Chem-
icals which are usually employed include bisulphite, dithionite and 2.3. Characterisation
borohydride, while also peroxide, hypochlorite, paracetic acid and
ozone have been used (Robert, 1996). 2.3.1. Morphological analysis of BC gel
Within this research, both treatments (pulping/mercerization The morphology of untreated BC, single-step treated BC and
and bleaching) have been performed on BC, in analogy with tradi- two-step treated BC was investigated using a cryo-field emission
tional paper-making practice, in order to remove cellulose by-prod- scanning electron microscope (FE-SEM) Quanta 3D ESEM from
ucts and other organic compounds (like nucleic acids and proteins FEI (Eindhoven, The Netherlands). Each gel was cut with a sharp
remaining from the culture medium) but, at the same time, prevent- knife, securely mounted on a sample holder under ambient condi-
ing the polymorphic transformation from cellulose I to cellulose II. tions, and then quickly frozen in liquid nitrogen. The sample was
Although the effect of purification on the improvement of mechan- subsequently transferred under vacuum into the cryo-SEM sample
ical properties of processed BC sheet has already being reported ear- preparation chamber, with the sample temperature constantly
lier, a further enhancement in mechanical and thermal properties of maintained at 150 °C via a cold stage. The temperature of the
 S. Gea et al. / Bioresource Technology 102 (2011) 9105–9110 9107

anti-contaminator in the sample preparation chamber was main-

tained at 190 °C. The sample was then fractured using a cold
knife kept at 150 °C to reveal a clean surface of frozen BC gel.
The sample was sublimed for more than 5 min at 100 °C to etch
away water on the surface of the gel following fracturing. Next,
the sample temperature was lowered to 135 °C and once the
temperature remained constant, the sample was sputter-coated
at 11 mA for at least 60 s to deposit a thin layer of conductive plat-
inum on top of the sample. The sample was then transferred under
the protection of high vacuum into the cryo-FE-SEM microscopy
chamber and imaged at an accelerating voltage of 2 kV and at a
working distance of 6–8 mm.

2.3.2. Tensile testing of BC sheet

Tensile tests were performed on a tensile test machine Instron
5566 equipped with a 1 kN static load cell. The 0.2–0.3 mm thick
sheets were cut into rectangular strips (30  5 mm). Samples were
stored in desiccators for at least 24 h before testing. Tensile testing
was undertaken at room temperature and at a speed of
1 mm min1. All measurements were performed for at least five
samples, and the average value recorded.

2.3.3. Thermogravimetric analysis

Thermogravimetric analysis (TGA) and differential thermal
analysis (DTGA) data of all samples were carried out using a TA
Instrument Q500 thermal analyser. The average weight of the sam-
ples was approximately 15 mg. Scan rates of 20 °C min1 over a
temperature range of 20–1000 °C were applied. All specimens
were in sheet form and tested under an inert (N2) atmosphere.

2.3.4. X-ray diffraction analysis

Fig. 1. Pellicles of (a) untreated BC (b) and two-step treated B.
The crystalline structure of the BC sheets was analysed using a
Siemens D5000 X-ray diffractometer (XRD), where the X-ray beam
was Ni-filtered Cu Ka (k = 0.154 nm) and radiation operated at
NaOCl overnight consecutively. The two samples, single-step trea-
40 kV with a filament of 40 mA. The sheet was directly mounted
ted BC and two-step treated BC, were compared with native un-
on the sample holder. The aperture slits were set at 0.1° and the
treated BC. A fixed concentration of 2.5 wt.% NaOH solution was
scanning step was 0.02° with a scan time of 2.5 s per step.
used in this study based on the intent to maintain the cellulose I
polymorph of BC and prevent the formation of cellulose II which
2.3.5. Infrared analysis is characterised by lower mechanical properties. It has been widely
Fourier transfer infrared (FTIR) spectra of the BC sheets were reported that concentrations of over 6% NaOH can potentially
obtained using a Nicolet 8700 FTIR spectrometer (Thermo Electron change the crystal structure of BC from cellulose I to cellulose II
Corporation, UK) in conjunction with an MTEC Photoacoustic Spec- (Shibazaki et al., 1997; Dinand et al., 2002; Mansikkamäki et al.,
trum (PAS) cell. Spectra were obtained in the mid infrared region 2005; Oh et al., 2005; Gomes et al., 2007). From the work of Las-
(4000–500 cm1) at 8 cm1 resolution and averaging of 128 scans. zkiewicz (1997), it can be seen that the change in structure from
cellulose I to cellulose II as a result of the alkalisation process is
3. Results and discussion accompanied by the breaking of many inter- and intra-molecular
hydrogen bonds which are naturally present in cellulose.
3.1. Morphology of the BC gels By using a two-step purification process, i.e. an alkaline solution
followed by a NaOCl solution, non-cellulose materials such as pro-
Fig. 1 shows the contrast between BC gel before (a) and after (b) tein and nucleic acids derived from bacterial cells and the culture
two-stage purification. Unlike plant cellulose, which is bound with broth are removed from the pellicle, allowing the formation of
lignin, hemicellulose and other chemicals which occur naturally strong inter- and intra-fibrillar hydrogen bonds. Besides being free
with cellulose, BC is not bound to other chemicals and is of relative from impurities, this two-step treated BC gel shown in Fig. 1b was
high purity (Klemm et al., 2006). Therefore the purification process odourless with a neutral pH. Within a few weeks, mould starts to
applied in this work differs from plant cellulose purification as it is grow on untreated BC sheet, resulting in a colour change with
aimed at removing only the remaining organic material, which is a the untreated BC becoming darker. Two-step treated BC sheet
source of food for the microbes in the medium. Proteins and nu- can instead be stored for a long time without experiencing a
cleic acids, either from yeast extract or produced by microbes dur- change in colour and/or quality.
ing incubation, cause impurities as well as give the gel a yellowish The reason for choosing this method is to maintain the three-
colour as shown in Fig. 1a. Vitamin B also contributed to the col- dimensional structure of BC gel, allowing the inside of the gel to
ouration. The remaining rod-shape bacteria of A. xylinum, as shown be observed easily. The surface of untreated BC is still opaque
on the surface of untreated BC gel, are the other main target of meaning that it is impossible to study the internal structure of
alkalisation (Fig. 2a). the untreated BC network directly, as shown in Fig. 2a. The organic
There are two stages in the purification process. First, BC gel impurities coming from the medium which are used in the culture
was washed in 2.5 wt.% NaOH solution and then with 2.5 wt.% process, as well as the rod-shaped A. xylinum, cover the pores of the
9108 S. Gea et al. / Bioresource Technology 102 (2011) 9105–9110

a moderately strong base (OCl1) and a weak acid (HOCl). When

dissolved in water it forms hypochlorous acid, which is known as
an active bleaching agent as shown in this equation below,

NaOCl þ H2 O ! Naþ þ OCl ð1Þ


OCl þ H2 O ! HOCl þ OH ð2Þ
For this reason, NaOCl has traditionally been used as a bleaching
agent to remove stains on clothing such as fats, waxes, pectin
impurities including dyestuffs which naturally contain colourant
in the pulp and other chemicals in the textiles which contain con-
jugated double bonds.

3.2. Mechanical properties of BC sheets

The purification of the pellicle is one of the most important

steps in improving the mechanical properties of hot-pressed BC
sheets (Yamanaka et al., 1989). Fig. 3 shows the stress–strain curve
of untreated BC, single-step treated BC and two-step treated BC
sheet. The Young’s modulus, tensile strength, and strain at break
of all samples are listed in Table 1. The Young’s modulus of a BC
sample after a single-step purification process is double that of un-
treated BC. However, subsequent purification with 2.5 wt.% NaOCl
for 12 h (two-step purification process) improves properties even
further as shown in Table 1. It is believed that the mechanical
properties are increased due to the removal of impurities from
within the gel network allowing the BC fibrils to better interact
with one another. Therefore purification increases the intrinsic
hydrogen bonding found within BC sheet by removing residual
impurities that remained from the growing process while main-
taining the cellulose I polymorph.

3.3. Thermal behaviour and purification of BC sheets

Thermogravimetric analysis (TGA) and differential thermo-

gravimetric (DTG) curves of the BC sheets are shown in Fig. 4a
and b, respectively. The corresponding data are listed in Table 2.
TGA can be used to characterise any material that exhibits a weight
change upon heating and to detect the phase change due to a
decomposition processes. The two-step treated BC sample under-
goes one main degradation step at about 360 °C, which is typical
of cellulose breakdown (Roma and Winter, 2004), apart from a
minor mass loss at about 100 °C, which can be attributed to water
evaporation. The less pure single-step treated BC sample with-

Fig. 2. SEM image of (a) untreated BC, (b) single-step purified BC and (c) two-step
purified BC gel. 200 Two-step treated BC
Single-step treated BC
Untreated BC
Stress [MPa]

gel surface preventing contact between fibrils within the network. 150
As a result, the number of hydrogen bonds that can form is dramat-
ically reduced, so reducing the strength of the dried sheet material
(Yamanaka et al., 1989). This is confirmed by the value of Young’s 100
modulus of untreated BC sheet as shown in Fig. 3 and Table 2. This
can be compared with the single-step treated BC in Fig. 2b, where
the rod-like shape of A. xylinum and other impurities can no longer 50
be seen on the surface of the gel, hence revealing the internal
structure of the gel. Cloudy aggregations of BC are beginning to
be formed. This is in sharp contrast to the pellicle which was fur-
ther treated with 2.5 wt.% NaOCl, as shown in Fig. 2c, where nano- 0.004 0.008 0.012 0.016
fibres of BC can be clearly seen. This also shows the effectiveness of
Strain [-]
NaOCl as a bleaching agent in removing impurities which are not
removed with NaOH alone. The cloudy aggregation found in sin- Fig. 3. Stress–strain curves of untreated BC, single-step treated BC and two-step
gle-step treated BC has also disappeared. This bleaching agent is treated BC sheet.
 S. Gea et al. / Bioresource Technology 102 (2011) 9105–9110 9109

Table 1
Summary of mechanical properties of untreated BC, single-step treated BC, and two-
step treated BC sheet (E = Young’s modulus, r = tensile strength, e = elongation at
break).

Samples E (GPa) r (MPa) e ()

Untreated BC 7.6 ± 1.2 88.9 ± 9.3 0.0124 ± 0.009
Single-step treated BC 14.1 ± 1.6 139.1 ± 12.6 0.0134 ± 0.006
Two-step treated BC 18.8 ± 2.0 207.0 ± 13.8 0.0159 ± 0.002

Table 2
Summary of the main degradation (peak temperature and % value) and the final
residue in percentage of untreated BC, single-step treated BC, and two-step treated BC
sheet.

Samples Peak temp (°C) % Residue at 1000 °C

Untreated BC 289 34.0
Single-step treated BC 322 28.1
Two-step treated BC 359 14.5

Table 3
FTIR peak assignments for bacterial cellulose.

Wave Assignment References

number
(cm1)
3495 t(OAH) cellulose II Moharram and Mahmoud
(2008)
3443 t(OAH) cellulose II Moharram and Mahmoud
(2008)
3420 t(NAH) free Oh et al. (2005), Movasaghi
et al. (2008)
3345 t(OAH) cellulose I Moharram and Mahmoud
(2008)
3278 t(OAH) cellulose I Moharram and Mahmoud
(2008)
3245 t(H bonded OAH) Focher et al. (2001)
3150–3220 ts(NAH) Movasaghi et al. (2008)
2930 ta(CH2) Oh et al. (2005), Movasaghi
et al. (2008) Fig. 4. (a) TGA thermograms of untreated BC, single-step treated BC, and two-step
2860 ta(CH2) Oh et al. (2005), Movasaghi treated BC sheet at a heating rate of 20 °C min1 and (b) the their derivative in
et al. (2008) temperature.
1730–1735 t(C@O) form proteins and Movasaghi et al. (2008)
lipids
1710–1702 t(C@O) Kacurakova et al. (2002),
served for the final mass residue. The residue value increases from
Movasaghi et al. (2008)
1650 Water absorbed Moharram and Mahmoud about 15% for two-step treated BC to 34% for untreated BC. This can
(2008) be explained by the presence of phosphorous compounds, as initial
1635–1645 C(OAH of absorbed water) Oh et al. (2005) impurities, which can greatly enhance the char formation.
1538 Protein amide II absorption Movasaghi et al. (2008) The purification process can also be followed by FTIR spectros-
1425–1435 In plane d(HCH,OCH) Oh et al. (2005)
1358–1375 w(CH) Oh et al. (2005)
copy (refer to Table 3 and Fig. S1 in Supplementary material). The
1325 Benzene ring mixed with the Movasaghi et al. (2008) hydrogen-bonded OH stretching shifts to higher wave numbers
CH in-plane bending and the intensity of OAH stretch of cellulose type I increases due
1146–1160 ta(CAOAC) ,CH deformation Kacurakova et al. (2002), to the purification process. At the same time the NAH stretch peak
Movasaghi et al. (2008)
from proteins and amino acids disappears. No evidence of change
1111 t(CAC) ring (polysaccharides, Movasaghi et al. (2008)
cellulose in cellulose type (I to II) due to the purification treatment was ob-
1071–1067 t(CAO) Kacurakova et al. (2002) served in the FTIR spectrum at this region.
1046 d(CAO) of the CAOH of Movasaghi et al. (2008)
carbohydrates
870–900 CH out-of-plane bending Kacurakova et al. (2002) 3.4. Structural analysis
vibrations
665–670 Out of plane d(CAOH) Oh et al. (2005)
It can be shown (refer to Fig. S2 in Supplementary material) that
t = stretching; d = bending; ta = asymmetric stretching; ts = symmetric stretching. the X-ray diffractogram of untreated BC, single-step treated BC and
two-step treated BC sheet show three main peaks (located at
2h = 14.7°, 16.2° and 22.4°) which correspond to the primary dif-
stands a lower temperature before the main degradation happens fraction of the ð1 1 0Þ, (1 1 0) and (2 0 0) planes of polymorph cellu-
(peak at 320 °C with a shoulder at about 400 °C). The untreated lose I in agreement with previously reported data (Sugiyama et al.,
as-produced BC presents a more complex behaviour. The main cel- 1991; Klechkovskaya et al., 2003). No reflections, instead, are
lulose degradation can be found at temperatures as low as 290 °C, found in correspondence of 2h = 12.1° and 20.8°, which are charac-
while the series of degradations between 150 and 250 °C can be teristic of cellulose II (Laszkiewicz, 1997; Mansikkamäki et al.,
attributed to proteinous impurities. A singular behaviour is ob- 2005). This again demonstrates that the two-step purification pro-
9110 S. Gea et al. / Bioresource Technology 102 (2011) 9105–9110

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