ORIGINAL PAPER

In situ production of nanocomposites of poly(vinyl alcohol)and cellulose nanofibrils from Gluconacetobacter bacteria:effect of chemical crosslinking

Cristina Castro • Arja Vesterinen • Robin Zuluaga •

Gloria Caro • Ilari Filpponen • Orlando J. Rojas •

Galder Kortaberria • Piedad Ganan

Received: 25 October 2013 / Accepted: 20 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Nanocomposites of poly(vinyl alcohol)

(PVA) reinforced with bacterial cellulose (BC) were

bioproduced by Gluconacetobacter genus bacteria. BC

was grown from a culture medium modified with

water-soluble PVA to allow in situ assembly and

production of a novel nanocomposite that displayed

synergistic property contributions from the individual

components. Chemical crosslinking with glyoxal was

performed to avoid the loss of PVA matrix during

purification steps and to improve the functional

properties of composite films. Reinforcement with

BC at 0.6, 6 and 14 wt% content yielded nanocom-

posites with excellent mechanical, thermal and dimen-

sional properties as well as moisture stability. Young’s

modulus and strength at break increased markedly with

the reinforcing BC: relative to the control sample (in

absence of BC), increases of 15, 165 and 680 % were

determined for nanocomposites with 0.6, 6 and 14 %

BC loading, respectively. The corresponding increase

in tensile strengths at yield were 1, 12 and 40 %,

respectively. The results indicate an exceptional rein-

forcing effect by the three-dimensional network struc-

ture formed by the BC upon biosynthesis embedded in

the PVA matrix and also suggest a large percolation

within the matrix. Bonding (mainly hydrogen bonding)

and chemical crosslinking between the reinforcing

phase and matrix were the main contributions to the

properties of the nanocomposite.

Keywords Nano composites � Poly(vinyl

alcohol) � Bacterial cellulose �Gluconacetobacter medellinensis � Thermal

stability � Mechanical properties

Introduction

Various composite materials reinforced with cellulose

have emerged in light of recent advances in the areas

of nanotechnology and bioengineering (Samir et al.

2004; Habibi et al. 2010; Iwamoto et al. 2007;

C. Castro � R. Zuluaga (&) � G. Caro � P. Ganan

School of Engineering, Universidad Pontificia

Bolivariana, Circular 1 # 70-01, Medellın, Colombia

e-mail: robin.zuluaga@upb.edu.co

A. Vesterinen

Department of Biotechnology and Chemical Technology,

School of Chemical Technology, Aalto University,

P.O. Box 16100, 00076 Espoo, Finland

I. Filpponen � O. J. Rojas

Department of Forest Products Technology, School of

Chemical Technology, Aalto University, P.O. Box 16100,

00076 Espoo, Finland

O. J. Rojas

Departments of Forest Biomaterials and Chemical and

Biomolecular Engineering, North Carolina University,

Campus Box 8005, Raleigh, NC 27695, USA

G. Kortaberria

‘‘Materials?Technologies’’ Group, Chemical and

Environmental Engineering Department, Universidad del

Paıs Vasco, 20018 San Sebastian, Spain

123

Cellulose

DOI 10.1007/s10570-014-0170-1

Iwamoto et al. 2005). The most abundant source of

cellulose is vascular plants wherein it forms an

intermixed and tight system with hemicelluloses and

lignin. Chemical, enzymatic or mechanical treatments

(or their combinations) are required to deconstruct the

basic components of the cell walls (Janardhnan and

Sain 2006; Zuluaga et al. 2009). Many of these

processes deteriorate the fiber and produce microfi-

brils with a wide size dispersion and high tendency to

agglomerate, which may limit the performance of

composites obtained after their incorporation and

homogenization into a matrix by compression, injec-

tion, extrusion or casting (Bondeson et al. 2006;

Berglund 2005).

One possible solution to this problem is the use of

cellulose of bacterial origin (bacterial cellulose, BC).

BC is excreted extracellularly by bacteria of the genus

Gluconacetobacter (formerly Acetobacter) by enzy-

matic mechanisms that lead to the polymerization of

glucose into chains and to their assembly into bundles

of microfibrils and ribbons (Iguchi et al. 2000; Klemm

et al. 2001). In general, BC has the same chemical

composition as cellulose from vascular plants, but BC

is produced free of other polymers (such as hetero-

polysaccharides or lignin); thus, BC is chemically pure

and forms fibrils of uniform lateral dimensions (Brown

et al. 1976; Nakagaito et al. 2005). In addition, BC has

excellent mechanical strength, high water-holding

capacity and crystallinity (Bielecki et al. 2005).

Bacterial cellulose is produced as a film infinitely

interconnected as a percolating cluster. Such a

network has been the subject of investigations on the

effect of water-soluble substances that are added to the

culture medium, mainly in relation to the rate of

cellulose synthesis and fibril dimensions (Uhlin et al.

1995; Tokoh et al. 2002). Brown and Laborie (2007)

adopted a biomimetic approach for the production of

nanocomposites of BC and poly(vinyl alcohol) (PVA)

as well as polyethylene oxide (PEO). It was found that

the reinforcing BC phase was evenly distributed over

the entire matrix. However, loss or solubilization of

the polymer matrix (PVA or PEO) occurred upon post

processing (washing and purification). This observa-

tion was ascribed to the fact that the PVA or PEO

matrix and the reinforcing BC interacted via hydrogen

bonding, which was insufficient to prevent polymer

solubilization (Laborie 2009). Consequently, the

purity and functional properties of the nanocomposites

were affected by the presence of substances from the

culture medium, such as sugars, organic acids and

proteins. Gea et al. (2010) prepared in situ PVA/BC

composites through the addition of PVA into the

culture media and compared them with materials

obtained after impregnation of BC gels with PVA

solutions. Compared to composites prepared in situ,

those prepared by impregnation have a higher PVA

content (3.7 compared to 1.4 PVA wt%) for the same

initial component composition. Obviously, the differ-

ence was due to the effect of purification. Interest-

ingly, the in situ process resulted in composites with

better mechanical and optical properties due to the

more effective component intermixing and homoge-

nization. Other composites with BC as the primary

component have been obtained after immersion of BC

in solutions of host compounds such as acrylic acid,

gelatin, silanol, phosphate and fibrin (Choi et al. 2004;

Yasuda et al. 2005; Lin et al. 2009; Barud et al. 2007;

Brown et al. 2011; Wan et al. 2006). In situ manufac-

ture of composites of BC and PVA and other

polymers, such as acemannan (from aloe vera),

chitosan and starch, during BC synthesis was recently

reported (Saibuatong and Phisalaphong 2010; Phisal-

aphong and Jatupaiboon 2008; Grande et al. 2008).

However, a distinctive feature in these systems was

again the fact that the host polymer was partially or

fully removed during the purification process; there-

fore, as was the case in impregnation processes, BC

was the main constituent of the final material. Overall,

it is not surprising that the properties that have been

assessed in BC-based composites are mainly attributed

to the BC component and affected only slightly by the

residual polymer.

In the present work, we prepared PVA/BC nano-

composites in situ followed by chemical crosslinking.

The culture medium of a new strain of Gluconacetob-

acter bacteria (Castro et al. 2012, 2013) was modified

with PVA that was added together with a crosslinking

agent to act as the matrix (and main component) of the

composite. Therefore, cellulose ribbons were synthe-

sized by the bacteria in contact with the PVA resulting

in highly crosslinked nanocomposites that resisted

matrix material (PVA) losses during washing and

purification processes. The main morphological and

physical properties of the obtained composites were

determined and discussed as a function of BC content.

To the best of our knowledge, no reports are

available describing the BC synthesis in the presence

of a polymer matrix with simultaneous crosslinking to

Cellulose

123

yield highly reinforced matrices that would otherwise

be solubilized (for example, during purification steps).

Materials and methods

Pre-conditioning of the microbial strain

The recently discovered Gluconacetobacter strain

used in this study was isolated from a pellicle of

homemade vinegar (Castro et al. 2012, 2013). The

purified bacterium was incubated in a static Hestrin-

Schramm culture medium (HS) containing 2 w/v %

glucose, 0.5 w/v % peptone, 0.5 w/v % yeast extract

and 0.27 w/v % Na2HPO4, adjusted to pH 3.5 by

phosphoric acid and autoclaved at 121 �C. The

toxicity of the crosslinking agent (glyoxal) to bacteria

was minimized by a methodology that is currently in

the patent process.

In situ production of PVA/BC nanocomposites

Poly(vinyl alcohol) (Sigma-Aldrich, Saint Louis, MO)

with a reported molecular mass of 146-186 kDa and

98–99 % hydrolysis degree was added to the HS

medium under stirring at 90 �C to obtain aqueous

solutions of 0, 3, 4.5 and 6 wt% PVA concentrations.

The respective solution was allowed to reach room

temperature, and respective volumes of glyoxal aque-

ous solution (40 wt%) were added under stirring until

a final glyoxal content of 10 wt% with respect to PVA

was reached. The modified HS media was inoculated

with 10 v/v % of the preconditioned inoculum and

statically incubated for 8 days at 28 �C. The collected

pellicles were dried at 40 �C for 48 h and then cured

(crosslinked) at 120 �C for 5 min. The crosslinked

pellicles were then washed with distilled water,

immersed for 14 h in 5 wt% aqueous KOH solution

and finally rinsed until reaching pH 7 to remove any

bacteria and residual components from the culture

media.

Six sample batches were prepared for each PVA

concentration; three of the samples were crosslinked,

and the remaining ones were used as reference to

quantify (gravimetrically) the amount of cellulose in

the nanocomposites. Control samples of crosslinked

PVA (in the absence of BC) were also prepared

following the same procedure used for the manufac-

ture of the PVA/BC nanocomposites. A schematic

diagram summarizing the manufacture procedures and

obtained samples is provided in Fig. 1. The different

specimens are referred to as ‘‘PVA X’’ or ‘‘PVA/BC X’’

reference (matrix) or composite samples, respectively;

here ‘‘X’’ refers to the actual reinforcing BC content

on a dry basis of the final solid material, which was

measured gravimetrically, as noted in Table 1 and

elsewhere in the discussion.

Morphology on PVA/BC nanocomposites

Scanning electron microscopy (SEM, Jeol JSM 5910

LV operated at 10 kV) was used to image the fracture

surfaces of dry nanocomposites deformed in tension.

Before SEM analyses, all specimens were precondi-

tioned at 75 % RH (relative humidity) using an

atmosphere saturated with NaCl solution for 8 days

and then coated with gold/palladium using an ion

sputter coater for 5 min.

Chemical characterization

Attenuated total reflection Fourier transform infrared

spectroscopy (ATR-FT-IR) was used to identify the

main chemical features of the PVA/BC nanocompos-

ites. Before the measurement, the nanocomposites were

dried for 2 h at 40 �C to precondition the test samples.

ATR-FT-IR spectra were recorded on a Nicolet 6700

spectrophotometer in the 4,000–400 cm-1 range ATR

with a diamond crystal. The spectra were recorded with

a resolution of 4 cm-1 and an accumulation of 64 scans.

Mechanical properties

Tensile strength was measured on specimens cut into

dimensions according to the ASTM D-1708 standard

and preconditioned at 24 �C and 75 % RH. The

strength at break and elastic modulus were determined

with an Instron Instrument according to ASTM D-882

using a load cell of 200 N at 5 mm/min and 22 mm

grip distance.

Dynamic mechanical analysis (DMA) with humid-

ity control was carried out with a Q 800 DMA (TA

Instruments) equipped with a humidity chamber.

Oscillatory measurements (1 Hz frequency) were

performed at 23 �C using 5.3 mm 9 12-mm strips

cut from the crosslinked nanocomposites and refer-

ence films. Samples were loaded to the chamber and

conditioned in 0 %RH for 240 min. Thereafter, two

Cellulose

123

humidity cycles of varying % RH (between 0 and

90 %) were applied using 480 min as equilibrium

time. The data were collected every 2 s until reaching

equilibrium.

Water absorbency

Water absorption was evaluated after immersion of the

samples in pure water at room temperature for 48 h.

All samples were cut into the same dimensions (circles

with 20 mm in diameter), dried (40 �C for 48 h) and

weighed before immersion. The weight gain after

immersion and removal of excess water with an

absorbent paper were determined. The water uptake

per gram of dry sample (swelling fraction) wc was

calculated using the equation:

wc ¼ ðws � wdÞ=wd ð1Þ

where ws and wd are the weights of the samples after

swelling and drying, respectively.

Thermal properties

Thermogravimetrical analysis (TGA, Mettler Toledo)

was performed to study the thermal degradation

behavior of the composite samples. The TGA appa-

ratus was flushed with nitrogen atmosphere, and

10 mg of sample was used. Each specimen was heated

from room temperature to 800 �C at a rate of 10 �C/min.

Differential scanning calorimetry (DSC, Metter Toledo)

was used to acquire thermograms under N2 flow.

Samples (5 mg) were placed in hermetically closed

Fig. 1 Scheme for the

production of

nanocomposites PVA/BC

during cellulose synthesis

by Gluconacetobacter

bacteria

Table 1 Nanocomposite composition and reference names

Nanocomposite Reference PVA in

culture

medium

(wt%)

BC

content

(wt%)

Glyoxal

in culture

medium

(wt%)

PVA/BC 0.6 PVA 0.6 6 0.6 0.60

PVA/BC 6 PVA 6 4.5 6.0 0.60

PVA/BC 14 PVA 14 3 14.0 0.30

Cellulose

123

DSC crucibles and heated from -40 to 250 �C at

10 �C/min to erase the thermal history, after which

they were cooled to -40 �C at 10 �C/min. The

second heat scan was conducted from -40 to 250 �C

at 10 �C/min, and the glass transition temperature (Tg)

was taken as the inflection point of the specific heat

increment at the glass-rubber transition, while the

melting temperature (Tm) was taken as the peak

temperature of the melting endotherm.

Two crystallinity parameters were determined from

Eqs. 2 and 3; the first one was calculated from the

sample weight (Xc), and the other one (Xp) took into

account the amount of matrix material in the compos-

ite. In these equations DHm� = 161.6 Jg-1 is the heat

of fusion for 100 % crystalline PVA (Roohani et al.

2008), and w is the weight fraction of polymeric

matrix material in the composite.

Xc ¼ DHm=DH0m ð2Þ

Xp ¼ Xc=w ð3Þ

Dielectric spectroscopy

Dielectric spectroscopy measurements were carried

out with a Novocontrol Alpha high-resolution dielec-

tric analyzer performing temperature sweeps from

-50 to 150 �C at a constant frequency of 1 kHz with a

heating rate of 3 �C/min. The instrument was inter-

faced to a computer and equipped with a Novocontrol

Novocool cryogenic system for temperature control.

Results and discussion

Poly(vinyl alcohol)/bacterial cellulose nanocompos-

ites were produced during the biosynthesis of cellulose

in static conditions by bacteria of the genus Glu-

conacetobacter. PVA added to the culture medium

was crosslinked with the reinforcing BC fibrils by

glyoxilation to improve bonding and prevent losses of

the PVA matrix during material washing and purifi-

cation. Table 1 includes the composition of the

nanocomposites obtained from different PVA and

BC ratios (glyoxal was always added at 10 % based on

PVA mass). The precursor PVA concentration in the

culture media was initially 3, 4.5 or 6 wt% resulting in

nanocomposites with BC weight percent based on

total dry mass of 14, 6 and 0.6 wt%, respectively.

Therefore, the composites were manufactured with

PVA as the main component and BC as the minority,

reinforcing phase. As noted, a decrease in BC

production was observed in the presence of larger

amounts of PVA in the culture medium, mainly owing

to the increased viscosity of the medium, which

hindered the transfer of microorganisms to the surface

where they consume oxygen for metabolism. Addi-

tional factors that may prevent BC production include

the possibility of PVA acting as a barrier to oxygen

diffusion through the medium and the relatively large

concentration of glyoxal used, which can limit the cell

growth. More importantly, there is an indication that

nanocomposites with different components can be

manufactured from a wide range of reinforcing/matrix

compositions, for example, through the addition of

water-soluble polymers to the culture medium at

different concentrations.

It is well known that light scattering in composite

materials increases with the amount of reinforcing

material caused by differences in the refractive indexes.

Furthermore, composites reinforced with BC have been

shown to display optical properties that depend heavily

on the refractive indexes of the components (Yano

et al. 2005; Iwamoto et al. 2005). In the present case,

the transparency of films produced with the PVA

matrix after crosslinking was not affected by the

presence of BC (Fig. 2a, b). The composite retains the

transparency of the PVA matrix even at cellulose

concentrations of 14 wt%, largely because of the

intimate contact and strong interfacial adhesion

between the ultrafine cellulose nanofibrils and PVA

matrix. Compared to films of pure BC (Fig. 2c), the

crosslinked composites exhibit yellowing. This is due

to the presence of glyoxal. Thus, during the crosslink-

ing reaction, the samples take on a yellowish appear-

ance. Figure 3 includes ATR-FT-IR spectra of PVA,

BC and PVA/BC nanocomposites after the respective

washing and purification steps. Nanocomposite spectra

(Fig. 3b–d) include the characteristic bands of PVA

observed in Fig. 3a. The characteristic cellulose signals

of C–O–C pyranose ring skeletal vibration at 1,060 and

1,030 cm-1 (indicated by the dotted line) is observed in

the spectrum of BC (Fig. 3e), but only observed in the

composite highly loaded with BC. This is mainly

because of the amount of reinforcing BC relative to

PVA in the nanocomposite; when the cellulose content

decreases the intensity of the band also decreases. In

addition, PVA presents characteristic bands at the same

wavelength, which creates overlap bands (see spectra

Cellulose

123

for PVA/BC 6 and PVA/BC 0.6). Unfortunately, the

esterification band from carbonyl groups at 1,730 cm-1

generated by the reaction of PVA and glyoxal was not

evident, mainly because this reaction yields a five-

member ring whose bands overlap with those of PVA

(Choi et al. 1999). Interestingly, the relative intensity of

the band at 1,140 cm-1 corresponding to symmetric

C–C stretching increases with BC content from the

PVA/BC 0.6 to the PVA/BC 6 composite but decreases

in PVA/BC 14. This band is strongly related with the

PVA crystallinity (Choi et al. 1999; Ngui and Malla-

pragada 1998; Peppas and Hansen 1982; Kenney and

Willcockson 1966). Therefore, it is possible that the

presence of BC in the matrix improves the PVA

crystallinity up to a given concentration, after which it

decreases. This decrease in crystallinity with cellulose

addition has been observed in the case of PVA

reinforced with CNCs (Peresin et al. 2010).

Results from tensile tests of PVA and PVA/BC

composite films are included in Fig. 4. Under the same

testing conditions, the reference matrices display a

highly elastic behavior. The nanocomposite remains

ductile after incorporation of BC; for example,

samples PVA/BC 0.6 have a yield point at an

elongation close to that of the reference film. The

nanocomposite eventually becomes very stiff when

the BC content increases to 14 % (PVA/BC 14). It is

observed that both Young’s modulus (YM) and

strength at break increase markedly with the reinforc-

ing BC: relative to the control sample (in absence of

BC), YM increases of 15, 165 and 680 % are

determined for nanocomposites with 0.6, 6 and 14 %

BC concentration, respectively. The corresponding

increase in tensile strengths at yield are 1, 12 and

40 %. The results indicate an exceptional reinforcing

effect by the three-dimensional network structure

formed by the BC upon biosynthesis embedded in the

PVA matrix and also suggest a large percolation

within the matrix (Samir et al. 2005). In addition, it is

likely that after crosslinking covalent bonds were

formed between the reinforcing BC and the PVA

matrix.

The observed increases in mechanical strength

have not been reported for PVA reinforced with

different cellulosic elements (Zimmermann et al.

2004; Cheng et al. 2009; Lu et al. 2008; Roohani

et al. 2008; Lee et al. 2009; Zhang et al. 2011; Qiu and

Fig. 2 Visual appearance of PVA 14 without BC (a), PVA/BC 14 nanocomposite (b) and bacterial cellulose (c) crosslinked films

Fig. 3 ATR-FT-IR spectra of crosslinked nanocomposites with

different amounts of bacterial cellulose produced from cellulose

synthesis: a PVA, b PVA/BC 0.6, c PVA/BC 6, d PVA/BC 14

and e bacterial cellulose

Cellulose

123

Netravali 2012). This behavior is closely tied to the

manufacturing method of the nanocomposite, wherein

microorganism bioengineering allows developing

materials in which there are intimate contacts between

the reinforcing phase and matrix with a maximum

percolation.

Scanning electron microscopy images of the frac-

tured surfaces of crosslinked PVA reference films

(prepared from 3 wt% aqueous solution), PVA 14 (see

also Table 1), the corresponding BC-reinforced nano-

composite (PVA/BC 14) and films of BC are shown in

Fig. 5. Figure 5a shows a uniform fracture of PVA 14

film with flow lines in the matrix indicating that the

rupture was caused by a crack propagation on the

smooth brittle surface. The respective nanocomposite

fracture shows no evidence of agglomerates indicating

that the BC ribbons were homogeneously distributed

throughout the matrix (Fig. 5b). Moreover, good

compatibility between the reinforcing PVA and the

BC matrix (good fiber-matrix bonding) is suggested by

the absence of pull-out BC ribbons. Likewise, the

typical delamination behavior of the BC films

observed in Fig. 5c is absent in the composite

material, suggesting that there is improved contact

and adhesion between the layers comprising the BC

network and not only between the reinforcing and matrix

components. The enhancement over nanocomposite

delamination promotes better stress transfer within the

material (Quero et al. 2010, 2011). All in all, the

results indicate strong interactions between the two

components, which has a direct influence on the final

mechanical characteristics of the composite after

crosslinking.

In previous work, it was verified that crosslinking

reactions of BC-glyoxal-BC occur even at low con-

centrations of glyoxal in the culture medium (docu-

ment in the patent process). Furthermore, the

insolubility of PVA films after crosslinking with

glyoxal confirms effective PVA-glyoxal-PVA reac-

tions. Therefore, it is also likely that PVA-glyoxal-BC

crosslinking takes place in the nanocomposite.

Thermograms of PVA films and their nanocom-

posites with BC are shown in Fig. 6. TGA profiles of

PVA exhibit two degradation peaks, while the nano-

composites display only a single peak. This suggests

that a high chemical compatibility and entanglement

exist between PVA and BC in the nanocomposite. The

first weight loss at 40–200 �C is due to water

evaporation absorbed during the preconditioning at

75 % RH. The PVA matrices have a higher percentage

of water loss compared with their respective nano-

composites; therefore, they have a greater tendency to

absorb water, and this trend is decreased with increasing

BC content. More important to this discussion is the fact

Fig. 4 Stress-strain

behavior of tensile test result

on crosslinked

nanocomposites and their

matrices

Cellulose

123

that compared to the PVA reference film, the thermal

stability of the nanocomposite is higher and increases

with the cellulose content. The maximum temperature

of thermal degradation for PVA/BC 6 and PVA/BC

14 nanocomposites increases from around 5–10 and

7–16 �C, respectively. This is explained by the effect of

BC in the polymer matrix, the good dispersion of BC, and

strong chemical and mechanical interactions with PVA.

Fig. 5 SEM images of the tensile fracture surface of crosslinked materials: a PVA 14, b PVA/BC 14 nanocomposite and c bacterial

cellulose. The SEM images are shown with two magnifications, as indicated

Fig. 6 Thermal

degradation profiles of

crosslinked materials. The

black lines correspond to the

nanocomposite and the gray

lines to their matrices

Cellulose

123

The thermal behavior of the samples was also

studied by DSC (Table 2). The glass transition tem-

peratures of PVA/BC 6 and 14 composites are shifted

to higher values compared to the respective matrices.

This effect suggests that good miscibility exists

between the PVA matrix and the reinforcing BC,

and the presence of the BC network restricts the

movement of the polymer chains. Moreover, the

melting point (Tm) and degree of crystallinity (Xc) of

the composites are slightly increased when compared

to the corresponding matrices (Table 2). These results

are in agreement with the FTIR data: the cellulose

surface acts as a nucleating agent for PVA crystalli-

zation, and the molecular mobility of PVA chains

decreases in the interfacial zone. Similar results were

reported for PCL and PVA with cellulose nanocrystals

(Roohani et al. 2008; Zoppe et al. 2009).

Dielectric spectroscopy was performed in order to

investigate further the effect of temperature on the

molecular interaction of PVA and BC. PVA/BC 14

samples as well as the respective matrix were

analyzed, and the results are shown in Fig. 7 for the

evolution of dielectric modulus M with temperature at

1 kHz. Due to the fact that some dielectric relaxations

may be obscured by the conductivity contribution, it is

more convenient to use the electric modulus formal-

ism, which shifts the loss peaks to the region of

frequencies where most of the measuring equipment

operates and at the same time diminishes the values of

the abscissa because of the definition of the electric

modulus (M* = 1/e*, where e* is complex permittiv-

ity) (Kortaberria et al. 2011). Two peaks are observed

for PVA/BC 14 and PVA 14: the main relaxation a

associated with the glass transition and b relaxation

assigned to local motions around main-chain bonds

and relaxation in crystalline domains (De la Rosa et al.

2001). The shifting of a relaxation to higher temper-

atures when BC is added to the composite indicates an

increase in the glass transition temperature of the

matrix. In fact, the Tg increases by 20 �C with 14 wt%

BC reinforcement. This was also observed in the DSC

experiments discussed previously (Tg increase 8 �C

with 14 wt% BC reinforcement), which highlights the

PVA-BC interactions and related reduction of polymer

chain mobility. The temperature at which b relaxation

appears does not seem to be markedly affected by

the reinforcement. This indicates that local motions

are not affected by the presence of reinforcement

Table 2 Thermal transitions and crystallinity (*) of matrix

and in situ nanocomposite reinforced with different amounts of

bacterial cellulose

Sample Tg

(�C)

Tm

(�C)

DHm

(Jg-1)

Xc

(%)

Xp

(%)

PVA 6 63.78 212.01 40.27 0.25 0.25

PVA/BC 6 87.05 215.76 43.78 0.27 0.29

PVA 14 68.33 209.47 39.62 0.25 0.25

PVA/BC

14

76.30 210.81 36.70 0.23 0.26

(*) Xc = DHm/DHm� and Xp = Xc/w; where

DHm� = 161.6 Jg-1 is the heat of fusion for 100 %

crystalline PVA (Roohani et al. 2008), and w is the weight

fraction of polymeric matrix material in the compositeFig. 7 Dielectric spectroscopy results of crosslinked materials.

The black lines correspond to nanocomposite PVA/BC 14 and

the gray lines to its matrix PVA 14

Fig. 8 Bulk water uptake capacity of matrices and nanocom-

posites after 48 h of immersion

Cellulose

123

presenting the same mobility. The reinforcement

affects only the main chain motions, reducing their

mobility.

Water absorption measurements were carried out in

order to study the swelling capacity of the composites,

and the results at equilibrium are shown in Fig. 8.

Compared to PVA reference samples, it is observed

that the water absorption (measured as the amount of

water retained by the material) of the nancomposites

decreases by 24, 26 and 36 wt% in the case of PVA/

BC 0.6, PVA/BC 6 and PVA/BC 14, respectively. This

is explained by the decrease in the free volume and

chain flexibility upon addition of BC, which restricts

swelling of the matrix. PVA and BC interactions

through hydrogen-bonding and crosslinking are

expected to decrease the availability of hydroxyl

groups to bind with water molecules, as suggested

elsewhere (Peresin et al. 2010).

In order to investigate the effect of absorbed water

on the mechanical behavior of the nanocomposites,

composite films were exposed to humidity cycles

between 0 and 90 % RH in a DMA unit (see Fig. 9).

Changes observed in storage modulus were accompa-

nied by shifts in material strain as the relative humidity

of the surrounding environment changed. As expected,

the modulus decreases with increasing humidity

(Fig. 9). However, compared with the BC-free films,

the changes were smaller for the nanocomposites.

Thus, as the amount of reinforcing BC increases,

better mechanical stability is achieved. Likewise,

changes in the dimensions of the materials were

observed with the gain of moisture: Fig. 9b indicates

an increase in the strain of the system as the % RH

increases due to the plasticizing effect of water.

Moreover, a higher dimensional stability was

observed for the composites with increased content

of reinforcing BC. This is in agreement with the

strength and swelling measurements of composites

(Figs. 4, 8). The highest increase in dimensional

stability was observed with PVA/BC 6 and PVA/BC

14. These findings suggest that cellulose restrains the

flow of PVA polymer chains. However, the high

mobility remains with PVA/BC 0.6, which can also be

observed in tensile tests (Fig. 4), where the mobility of

PVA/BC 0.6 resists a breakage resulting in high strain.

The storage modulus and strain of the PVA

matrices and the respective nanocomposites fully

and reversibly recovered after humidity cycles. This

elastic behavior is not affected by dehydration and

hydration of the specimens. Consequently, the net

effect of cellulose network coupled with strong

reinforcing-matrix interactions reduces moisture

uptake and restricts the movement of PVA chains,

providing mechanical and dimensional stability to the

composite.

Conclusions

Poly(vinyl alcohol) nanocomposites were produced

in situ during cellulose synthesis by bacteria of the

Gluconacetobacter genus. The loss of the hydrosoluble

Fig. 9 Variation of the a storage modulus and b strain of matrices and nanocomposites after two humidity cycles from 0 to 90 % RH at

room temperature

Cellulose

123

PVA polymer matrix during post-processing was

prevented by chemical crosslinking and the benefits

in mechanical and thermal properties assessed. Com-

posites with different BC reinforcing levels were

produced. Both Young’s modulus and tensile strength

markedly increased with the BC loading: improve-

ments of 680 and 40 % were obtained when compared

with the respective BC-free systems. SEM of fractured

films, DSC, TGA and dielectric spectroscopy results

suggest that effective dispersion occurred between the

PVA matrix and reinforcement BC phase. The

molecular mobility of PVA chains decreased in the

interfacial zone because of the presence of the network

of cellulose fibrils. This effect led to an increase in the

crystallinity of the matrix in the nanocomposite. The

TGA analysis also indicated that the nanocomposites

had a higher thermal stability compared to the

respective matrix. This further supports the evidence

of a high entanglement between the two components

produced as a three-dimensional network of nanoscale

BC after biosynthesis by the microorganisms. Overall,

the BC network is postulated to afford an improved

percolation within the matrix accompanied by an

improved bonding between the reinforcing BC and

PVA matrix (hydrogen bonds and crosslinking). This

can explain the exceptional mechanical performance

of the nanocomposites. Finally, the BC reinforcement

was shown to reduce water uptake and to improve

moisture stability of the composites under cyclic

humidity conditions.

Acknowledgments The authors would like to acknowledge

Colombia’s COLCIENCIAS and SENA for financial support as

well as Prof. Janne Laine of the Department of Forest Products

Technology of Aalto University (Finland).

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