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

Bacterial cellulose (BC) has attracted a great deal of academic
and commercial interests, with numerous applications in biotech-
nology, pharmacy, biobased packaging and other fields of biomed-
ical science (Iguchi et al., 2000). The reason of such interests
resides in the ease in producing a fully biobased cellulosic material
which has good mechanical properties such as high tensile
strength and modulus, high purity, high water-binding capacity,
and an ultra-fine network structure. The relative high mechanical
properties of BC microfibrils have also led to their use as reinforc-
ing agents in composite materials (Soykeabkaew et al., 2009; Gea
et al., 2010). The Young’s modulus of a single filament or microfi-
bril of BC as estimated using a Raman spectroscopy technique is
130–140 GPa (Hsieh et al., 2008; Eichhorn et al., 2010).
Many strains of bacteria possess the ability to produce cellulose
at the surface of a medium containing carbon and nitrogen as food
source. These include Sarcina, Agrobacterium, Rhizobium, and Aceto-
bacter (Deinema and Zevenhuizen, 1971). However, the rod-shaped
⇑ Corresponding author at: School of Engineering and Material Science and
Centre for Materials Research, Queen Mary University of London, London E1 4NS,
UK.
E-mail address: t.peijs@qmul.ac.uk (T. Peijs).
to be capable of producing cellulose in commercial quantities.
Therefore, methods of producing BC have been developed with
the aims of improving yield, structure, and other desired physical
properties (Yamanaka et al., 2000). Besides using different produc-
tion methods, the medium used for culture, the pH level, and the
source of nitrogen and phosphate as the main food source for A.
xylinum, as well as several additives, have also all been studied
(Huang et al., 2010).
A crucial step in the production of any cellulose product is its
purification, which is traditionally, known as a chemical pulping
process in the paper-making industry. This process is intended to
remove essentially all of the undesired residual insoluble lignin
and other chemicals bound to the cellulose fibre as well as impuri-
ties occurring during processing and converting them into com-
pounds which are soluble in alkali water. If lignin is not
removed, photo-oxidation, which causes discolouration of paper
with age, is likely to occur, and also the final paper product is brit-
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).
Alkaline is one of the most commonly used chemicals in the
mercerization process since it is able to hydrolyse and remove
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
the crystal structure from cellulose I to cellulose II. The concentra-
tion of NaOH used for this transformation varies, depending on the
type and the origin of cellulose being treated. Previous researchers
have reported that the transformation of cellulose I to cellulose II
occurs at concentrations of NaOH above 6% (Borysiak and Gar-
barczyk, 2003; Moigne and Navard, 2010). Many researchers have
investigated the effect of immersion time on the purification of cel-
lulose, from few minutes to 48 h (Moharram and Mahmoud, 2008;
Mansikkamäki et al., 2005; Nakagaito and Yano, 2008; Oh et al.,
2005; Shibazaki et al., 1997; Aziz and Ansell, 2004).
Research into the change of cellulose I to cellulose II as a result
of alkalisation is still the subject of debate (Zhou et al., 2002; Oh
et al., 2005). Dinand et al. (2002) has hypothesised and tested that
samples immersed in NaOH solutions for 2–24 h, with a given con-
centration, yielded the same results.
There are three steps in the transformation process, which be-
gins with fibre swelling due to water absorption, leading to a sig-
nificant increase in mobility of the cellulose chains. Then the
alkaline solution penetrates the amorphous areas of the cellulose,
before saturating the cellulose and disrupting the crystalline re-
gions, finally forming new crystalline lattices after the mercerising
solution is washed off (Lee et al., 2004). Usually, the amorphous
and crystalline regions which cover the upper surface of the cellu-
lose structure react with the alkali solution and are removed when
the solution is finally washed off (Gassan and Bledzki, 1999; Liu
and Hu, 2008).
During the polymorphic transformation from cellulose I to cel-
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
transformation is a secondary phenomenon, which could not occur
without first breaking the primary hydrogen bonds in cellulose I
(Laszkiewicz, 1997). The result of this transformation is a signifi-
cant decrease in mechanical properties. Ishikawa and Okano
(1997) have reported a drop in Young’s modulus of a single ramie
fibre from 27 GPa (cellulose I) to 21 GPa (cellulose II). The appear-
ance of cellulose polymorphs is easily detected by conventional
wide angle X-ray diffraction (Mansikkamäki et al., 2005). The mea-
surement of single cellulose fibres/fibrils has been performed by
Matsuo et al. (1990) using an X-ray diffraction method with cellu-
lose I having a modulus of 120–135 GPa and cellulose II 106–
112 GPa. Kroon-Batenburg and Kroon (1997) have also reported
the moduli of both polymorphs and found them to be 130–
137 GPa and 71–90 GPa for cellulose I and II, respectively.
Bleaching is another very common process in the paper-making
industry. This stage is aimed at enhancing the brightness of the
pulp to provide a bright white paper for applications such as print-
ing and tissue paper. It also eliminates the problem of yellowing of
the paper exposed to light due mainly to residual lignin in the pulp
(3–6 wt.%) and removes resin and other extractives present in
unbleached pulp (Bajpai, 2005). Bleaching agents are often used
based on their reduction and oxidation behaviour in nature. Chem-
icals which are usually employed include bisulphite, dithionite and
borohydride, while also peroxide, hypochlorite, paracetic acid and
ozone have been used (Robert, 1996).
Within this research, both treatments (pulping/mercerization
and bleaching) have been performed on BC, in analogy with tradi-
tional paper-making practice, in order to remove cellulose by-prod-
ucts and other organic compounds (like nucleic acids and proteins
remaining from the culture medium) but, at the same time, prevent-
ing the polymorphic transformation from cellulose I to cellulose II.
Although the effect of purification on the improvement of mechan-
ical properties of processed BC sheet has already being reported ear-
lier, a further enhancement in mechanical and thermal properties of
these sheets is envisaged by retaining the more high-performance
cellulose I crystal phase in the BC microfibrils.
2. Methods
2.1. Materials
In this study, strains of A. xylinum for the production of BC were
supplied by the Microbiology Laboratory of the Institute of Agricul-
ture, Bogor, Indonesia. The chemicals used for the culture medium
were glucose, ammonium sulphate, potassium hydrogen ortho-
phosphate, magnesium sulphate and yeast extract. In the purifica-
tion process, sodium hydroxide, sodium hypochlorite and
additional vitamins such as B1, B3 and B5 used to assist the growth
of the bacteria were used as received from VWR International (UK).
2.2. Sample preparation
2.2.1. Preparation of BC pellicle
The culture medium for the production of BC was prepared by
the procedure reported by Gea et al. (2007). For every litre of dis-
tilled water, 50 g glucose, 5 g (NH3)3PO4, 4 g KH2PO4, 5 g yeast ex-
tract, and 0.1 g MgSO47H2O were added. This culture medium was
autoclaved at 121 °C for 2 h, and, once cooled to ambient temper-
ature, vitamins B1, B2 and B5 were added. The acidity of the med-
ium was adjusted to pH = 4 using glacial acetic acid. A strain of
A. xylinum was inoculated into the static culture medium for
21 days at 28 °C.
2.2.2. Treatment of BC pellicle
After harvesting, some of the pellicles of BC were left on the
sieve to allow the liquid from the pellicles to escape through the
sieve without any treatment. These samples are called untreated
BC. The remaining pellicles were washed under running tap water,
and then these pellicles were immersed overnight in 2.5 wt.%
NaOH. These are hereafter referred to as NaOH-treated BC (sin-
gle-step treated BC). A third sample was prepared in the same
way as the second sample, i.e. NaOH-treated BC, and successively
treated also with 2.5 wt.% NaOCl (two-step treated BC). The pelli-
cles were then rinsed under running tap water to remove any sol-
vent until neutral pH conditions.
2.2.3. Processing of BC sheet
For the preparation of specimens for morphological analysis,
samples were left in the form of a gel, while, for other tests, such
as mechanical, thermal, and structural analysis, the samples were
processed into sheet form. For the preparation of BC sheet, the pel-
licles (90–95% water) was sandwiched between two stainless steel
plates of 20 mm thickness along with a wire mesh, and compres-
sion moulded for 5 min at 115 °C at 70 MPa pressure to remove
any remaining water.
2.3. Characterisation
2.3.1. Morphological analysis of BC gel
The morphology of untreated BC, single-step treated BC and
two-step treated BC was investigated using a cryo-field emission
scanning electron microscope (FE-SEM) Quanta 3D ESEM from
FEI (Eindhoven, The Netherlands). Each gel was cut with a sharp
knife, securely mounted on a sample holder under ambient condi-
tions, and then quickly frozen in liquid nitrogen. The sample was
subsequently transferred under vacuum into the cryo-SEM sample
preparation chamber, with the sample temperature constantly
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

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
40 kV with a filament of 40 mA. The sheet was directly mounted
on the sample holder. The aperture slits were set at 0.1° and the
scanning step was 0.02° with a scan time of 2.5 s per step.
2.3.5. Infrared analysis
Fourier transfer infrared (FTIR) spectra of the BC sheets were
obtained using a Nicolet 8700 FTIR spectrometer (Thermo Electron
Corporation, UK) in conjunction with an MTEC Photoacoustic Spec-
trum (PAS) cell. Spectra were obtained in the mid infrared region
(4000–500 cm1) at 8 cm1 resolution and averaging of 128 scans.
3. Results and discussion
3.1. Morphology of the BC gels
Fig. 1 shows the contrast between BC gel before (a) and after (b)
two-stage purification. Unlike plant cellulose, which is bound with
lignin, hemicellulose and other chemicals which occur naturally
with cellulose, BC is not bound to other chemicals and is of relative
high purity (Klemm et al., 2006). Therefore the purification process
applied in this work differs from plant cellulose purification as it is
aimed at removing only the remaining organic material, which is a
source of food for the microbes in the medium. Proteins and nu-
cleic acids, either from yeast extract or produced by microbes dur-
ing incubation, cause impurities as well as give the gel a yellowish
colour as shown in Fig. 1a. Vitamin B also contributed to the col-
ouration. The remaining rod-shape bacteria of A. xylinum, as shown
on the surface of untreated BC gel, are the other main target of
alkalisation (Fig. 2a).
There are two stages in the purification process. First, BC gel
was washed in 2.5 wt.% NaOH solution and then with 2.5 wt.%
Fig. 1. Pellicles of (a) untreated BC (b) and two-step treated B.
NaOCl overnight consecutively. The two samples, single-step trea-
ted BC and two-step treated BC, were compared with native un-
treated BC. A fixed concentration of 2.5 wt.% NaOH solution was
used in this study based on the intent to maintain the cellulose I
polymorph of BC and prevent the formation of cellulose II which
is characterised by lower mechanical properties. It has been widely
reported that concentrations of over 6% NaOH can potentially
change the crystal structure of BC from cellulose I to cellulose II
(Shibazaki et al., 1997; Dinand et al., 2002; Mansikkamäki et al.,
2005; Oh et al., 2005; Gomes et al., 2007). From the work of Las-
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
accompanied by the breaking of many inter- and intra-molecular
hydrogen bonds which are naturally present in cellulose.
By using a two-step purification process, i.e. an alkaline solution
followed by a NaOCl solution, non-cellulose materials such as pro-
tein and nucleic acids derived from bacterial cells and the culture
broth are removed from the pellicle, allowing the formation of
strong inter- and intra-fibrillar hydrogen bonds. Besides being free
from impurities, this two-step treated BC gel shown in Fig. 1b was
odourless with a neutral pH. Within a few weeks, mould starts to
grow on untreated BC sheet, resulting in a colour change with
the untreated BC becoming darker. Two-step treated BC sheet
can instead be stored for a long time without experiencing a
change in colour and/or quality.
The reason for choosing this method is to maintain the three-
dimensional structure of BC gel, allowing the inside of the gel to
be observed easily. The surface of untreated BC is still opaque
meaning that it is impossible to study the internal structure of
the untreated BC network directly, as shown in Fig. 2a. The organic
impurities coming from the medium which are used in the culture
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
cess does not change the structure of BC from cellulose I to cellu-
lose II. A slight increase in the relative peak intensity of the diffrac-
tion peaks relative to the treated BC samples suggested a change in
the orientation of the BC microfibril crystals, relative to the X-ray
beam.
4. Conclusions
A two-step purification process is described for the production
of impurity-free bacterial cellulose (BC) samples, as demonstrated
by SEM images, TGA and FTIR, which greatly improves the mechan-
ical and thermo-gravimetric performance of processed BC sheets.
The treatment with 2.5 wt.% NaOH followed by 2.5 wt.% NaOCl pre-
vented any structural changes of the native fibre from cellulose I
type to cellulose II, as demonstrated by XRD and FTIR analysis,
which are detrimental for mechanical properties. The Young’s
modulus of NaOH-treated BC sheet increased by a factor of two
compared to untreated BC, and even further after bleaching.
Acknowledgements
S.G. would like to acknowledge the Indonesian Government and
the University of Sumatra Utara, Medan, for financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2011.04.077.
References
Aziz, S.H., Ansell, M.P., 2004. The effect of alkalization and fibre alignment on the
mechanical and thermal properties of kenaf and hemp bast fibre composites:
part 2- cashew nut shell liquid matrix. Compos. Sci. Technol. 64, 1231–1238.
Bajpai, P., 2005. Environmentally Benign Approaches for Pulp Bleaching, vol. 1.
Elsevier, Amsterdam.
Kroon-Batenburg, L.M.J., Kroon, J., 1997. The crystal and molecular structures of
cellulose I and II. Glycoconjugate J. 14, 677–690.
Borysiak, S., Garbarczyk, J., 2003. Applying the WAXS method to estimate the
supermolecular structure of cellulose fibres after mercerization. Fibres Text East
Eur. 11 (5), 104–106.
Deinema, M.H., Zevenhuizen, L.P.T.M., 1971. Formation of cellulose fibrils by gram-
negative bacteria and their role in bacterial flocculation. Arch. Microbiol. 78,
42–51.
Dinand, E., Vignon, M., Chanzy, H., Heux, L., 2002. Mercerization of primary wall
cellulose and its implication for the conversion of cellulose I to cellulose II.
Cellulose 9, 7–18.
Eichhorn, S.J., Dufresne, A., Aranguren, M., Marcovich, N.E., Capadona, J.R., Rowan,
S.J., Weder, C., Thielemans, W., Roman, M., Renneckar, S., Gindle, W., Veigel, S.,
Keckes, J., Yano, H., Abe, K., Nogi, M., Nakagaito, A.N., Mangalam, A., Simonsen, J.,
Benight, A.S., Bismarck, A., Berglund, L.A., Peijs, T., 2010. Review: current
international research into cellulose nanofibres and nanocomposites. J. Mater.
Sci. 45, 1–33.
Focher, B., Palma, M.T., Canetti, M., Torri, G., Cosentino, C., Gastaldi, G., 2001.
Structural differences between non-wood plant celluloses: evidence from solid
state NMR, vibrational spectroscopy and X-ray diffractometry. Ind. Crop. Prod.
13, 193–208.
Gassan, J., Bledzki, A.K., 1999. Influence of fibre surface treatment on the creep
behaviour of jute fibre-reinforced polypropylene. J. Thermoplast. Compos.
Mater. 12, 388–398.
Gea, S., Bilotti, E., Reynolds, C.T., Soykeabkeaw, N., Peijs, T., 2010. Poly(vinyl)
alcohol-bacterial cellulose composites prepared using an in-situ process. Mater.
Lett. 64, 901–904.
Gea, S., Torres, F.G., Tronscoso, O.P., Reynolds, C.T., Vilasecca, F., Iguchi, M., Peijs, T.,
2007. Biocomposites based on bacterial cellulose and apple and radish pulp. Int.
Polym. Proc. 22, 497–501.
Gomes, A., Matsuo, T., Goda, K., Ohgi, J., 2007. Development and effect of alkali
treatment on tensile properties of curaua fiber green composites. Compos. Part
A-App. S. 38, 1811–1820.
Huang, H.-C., Chen, L.-C., Lin, S.-B., Hsu, C.-P., Chen, H.-H., 2010. In situ modification
of bacterial cellulose network structure by adding interfering substances during
fermentation. Bioresour. Technol. 101, 6084–6091.
Hsieh, Y.C., Yano, H., Nogi, M., Eichhorn, S.J., 2008. An estimation of the Young’s
modulus of bacterial cellulose filaments. Cellulose 15, 507–513.
Iguchi, M., Yamanaka, S., Budhiono, A., 2000. Review bacterial cellulose–a
masterpiece of nature’s arts. J. Mater. Sci. 35, 261–270.
Ishikawa, A., Okano, T., 1997. Fine structure and tensile properties of ramie fibres in
the crystalline form of cellulose I, II, III1 and IV1. Polymer 38, 463–468.
Kacurakova, M., Smith, A.C., Gidley, M.J., Wilson, R.H., 2002. Molecular interactions
in bacterial cellulose composites studied by 1D FT-IR and dynamic 2D FT-IR
spectroscopy. Carbohyd. Res. 337, 1145–1153.
Klechkovskaya, V.V., Baklagina, Y.B., Stepina, N.D., Khripunov, A.K., Buffat, P.A.,
Suvorova, E.I., Zanaveskina, I.S., Tkchenko, A.A., Gladchenko, S.V., 2003.
Structure of cellulose Acetobacter xylinum. Crystallogr. Rep. 48, 755–762.
Klemm, D., Schumann, D., Kramer, F., Hebler, N., Hornung, M., Schmauder, H.P.,
Marrsch, S., 2006. Nanocellulose innovative polymers in research and
application. Adv. Polym. Sci. 205, 49–96.
Laszkiewicz, B., 1997. Solubility of bacterial cellulose and its structural properties. J.
Appl. Polym. Sci. 67, 1871–1876.
Lee, M.H., Park, H.S., Yoo, K.J., Hauser, P.J., 2004. Enhancing the durability of linen-
like temperature mercerized cotton. Text Res. J. 74, 146–154.
Liu, Y., Hu, H., 2008. X-ray diffraction study of bamboo fibres treated with NaOH.
Fibers Polym. 19, 735–739.
Mansikkamäki, P., Lahtinen, M., Rissanen, K., 2005. Structure changes of cellulose
crystallites induced by mercerization in different solvent system; determined
by powder X-ray diffraction method. Cellulose 12, 233–242.
Matsuo, M., Sawatarie, C., Iwai, Y., Ozaki, F., 1990. Effect of orientation and
crystallinity of the measurement by X-ray diffraction of the crystal lattice
moduli of cellulose I and II. Macromolecules 23, 3266–3275.
Moharram, M.A., Mahmoud, O.M., 2008. FTIR spectroscopic study of the effect of
microwave heating on the transformation of cellulose I into cellulose II during
mercerization. J. Appl. Polym. Sci. 107, 30–36.
Moigne, N.Le., Navard, P., 2010. Dissolution mechanism of wood cellulose fibres in
NaOH–Water. Cellulose 17, 31–45.
Movasaghi, Z., Rehman, S., Rehman, I., 2008. Fourier transform infrared (FTIR)
spectroscopy of biological tissues. Appl. Spectrosc. Rev. 43, 134–179.
Nakagaito, A.N., Yano, H., 2008. Toughness enhancement of cellulose
nanocomposites by alkali treatment of the reinforcing cellulose nanofibers.
Cellulose 15, 323–331.
Oh, S.Y., Yoo, D.Il., Shin, Y., Kim, H.C., Kim, H.Y., Chung, Y.S., Park, W.H., Youk, J.H.,
2005. Crystalline structure analysis of cellulose treated with sodium hydroxide
and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy.
Carbohyd. Res. 340, 2376–2391.
Robert, J.C., 1996. The Chemistry of Paper. The Royal society of Chemistry,
Cambridge.
Roma, M., Winter, M.T., 2004. Effect of sulfate groups from sulfonic acid hydrolysis
on the thermal degradation behaviour of bacterial cellulose.
Biomacromolecules 5, 1671–1677.
Shibazaki, H., Kuga, S., Okano, T., 1997. Mercerization and acid hydrolysis of
bacterial cellulose. Cellulose 4, 75–87.
Soykeabkaew, N., Sian, C., Gea, S., Peijs, T., 2009. All-cellulose nanocomposites by
surface selective dissolution of bacterial cellulose. Cellulose 16, 435–444.
Sugiyama, J., Vuong, R., Chanzy, H., 1991. Electron diffraction study on the two
crystalline phases occurring in native cellulose from an algae cell wall.
Macromolecules 24, 4168–4175.
Yamanaka, S., Ishihara, M., Sugiyama, J., 2000. Structural modification of bacterial
cellulose. Cellulose 7, 213–225.
Yamanaka, S., Watanabe, K., Kitamura, N., Iguchi, M., Mitsuhashi, Y., Nishi, Y., 1989.
The structure and mechanical properties of sheets prepared from bacterial
cellulose. J. Mater. Sci. 24, 3141–3145.
Zhou, L.M., Yeung, K.W.P., Yuen, C.W.M., 2002. Effect of NaOH mercerization on the
crosslinking of ramie yarn using 1,2,3,4-butanetetracarboxylic acid. Text Res. J
72, 531–538.