Bacterial Cellulose from Simple and Low Cost Production Media by Gluconacetobacter xylinus

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ArticleTitle Bacterial Cellulose from Simple and Low Cost Production Media by Gluconacetobacter xylinusArticle Sub-Title

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Journal Name Journal of Polymers and the Environment

Corresponding Author Family Name VazquezParticle

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Division Polymer and Composite Material Group, Laboratorio de Materiales yEstructuras, Facultad de Ingeniería, Instituto de Tecnologías y Ciencias de laIngeniería (INTECIN) CONICET

Organization Universidad de Buenos Aires

Address Las Heras 2214, Buenos Aires, CP 1127, Argentina

Email [email protected]

Author Family Name ForestiParticle

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Division Polymer and Composite Material Group, Laboratorio de Materiales yEstructuras, Facultad de Ingeniería, Instituto de Tecnologías y Ciencias de laIngeniería (INTECIN) CONICET


Address Las Heras 2214, Buenos Aires, CP 1127, Argentina

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Division Departamento de Ingeniería Química, Facultad de Ingeniería


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Abstract Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus using non conventional low-costcarbon sources, such as glycerol remaining from biodiesel production and grape bagasse, a residue of wineproduction. The carbon sources assayed showed their suitability for microbial cellulose production, withrelatively high production values such as 10.0 g/l for the culture medium with glycerol from biodiesel ascarbon source and corn steep liquor as nitrogen source; and 8.0 g/l for the culture medium containing grapebagasse and corn steep liquor. Glucose, commercial glycerol and cane molasses were also assayed as carbonsources for comparison. The bacterial celluloses produced were characterized by means of scanning electronmicroscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric analysis.Morphological analysis showed that bacterial cellulose microfibrils produced from the non-conventionalmedia used were several micrometers long and had rectangular cross-sections with widths and thicknesses inthe range of 35–70 and 13–24 nm, respectively. X-ray patterns showed crystallinity levels in the range of 74–79 % (area method), whereas both X-ray patterns and infrared spectroscopy evidenced the presence of bandscharacteristic of Cellulose I polymorph. Thermal properties were similar to those found for the pellicleobtained from glucose. The study performed showed the suitability of using wine residues or glycerolremaining from increasing biodiesel production as cheap carbon sources for production of bacterial cellulosemicrofibrils, with similar characteristics as those obtained by use of more expensive carbon sources such asglucose or commercial glycerol. On the other hand, the low cost nitrogen sources used (corn steep liquor ordiammonium phosphate) also contributed to the economy of the bioprocess.

Keywords (separated by '-') Bacterial cellulose - Low cost carbon sources - Grape bagasse - Glycerol from biodieselFootnote Information

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Journal: 10924

Article: 541

UNCORRECTEDPROOF

ORIGINAL PAPER1

2 Bacterial Cellulose from Simple and Low Cost Production Media

3 by Gluconacetobacter xylinus

4 Analıa Vazquez • Marıa Laura Foresti •

5 Patricia Cerrutti • Miguel Galvagno

6

7 � Springer Science+Business Media New York 2012

8 Abstract Bacterial cellulose pellicles were produced by

9 Gluconacetobacter xylinus using non conventional low-cost

10 carbon sources, such as glycerol remaining from biodiesel

11 production and grape bagasse, a residue of wine production.

12 The carbon sources assayed showed their suitability for

13 microbial cellulose production, with relatively high pro-

14 duction values such as 10.0 g/l for the culture medium with

15 glycerol from biodiesel as carbon source and corn steep

16 liquor as nitrogen source; and 8.0 g/l for the culture medium

17 containing grape bagasse and corn steep liquor. Glucose,

18 commercial glycerol and cane molasses were also assayed

19 as carbon sources for comparison. The bacterial celluloses

20 produced were characterized by means of scanning electron

21 microscopy, X-ray diffraction, Fourier transform infrared

22 spectroscopy and thermogravimetric analysis. Morpholog-

23 ical analysis showed that bacterial cellulose microfibrils

24 produced from the non-conventional media used were sev-

25 eral micrometers long and had rectangular cross-sections

26 with widths and thicknesses in the range of 35–70 and

27 13–24 nm, respectively. X-ray patterns showed crystallinity

28 levels in the range of 74–79 % (area method), whereas both

29X-ray patterns and infrared spectroscopy evidenced the

30presence of bands characteristic of Cellulose I polymorph.

31Thermal properties were similar to those found for the

32pellicle obtained from glucose. The study performed

33showed the suitability of using wine residues or glycerol

34remaining from increasing biodiesel production as cheap

35carbon sources for production of bacterial cellulose micro-

36fibrils, with similar characteristics as those obtained by use

37of more expensive carbon sources such as glucose or com-

38mercial glycerol. On the other hand, the low cost nitrogen

39sources used (corn steep liquor or diammonium phosphate)

40also contributed to the economy of the bioprocess.

41

42Keywords Bacterial cellulose � Low cost carbon

43sources � Grape bagasse � Glycerol from biodiesel

44Introduction

45Cellulose is a linear polysaccharide consisting of a chain of

46b(1 ? 4) linked D-glucose units. Although generally

47obtained from wood and natural fibers, it is now well

48established that cellulose is also produced by a family of

49sea animals called tunicates, several species of algae, and

50by some species of bacteria. In the last decade, nanosized

51constituents of cellulose have triggered a revived interest

52on the well-known natural polymer. During biosynthesis of

53cellulose chains, van der Waals forces and hydrogen

54bonding between hydroxyl groups and oxygens of adjacent

55molecules promote parallel stacking of multiple cellulose

56chains forming elementary fibrils that further aggregate

57into larger microfibrils [1], with diameters within 2–20 nm

58and several microns length.

59In the last decade much research has been devoted to the

60extraction of cellulose microfibrils and nanocrystals from

A1 A. Vazquez (&) � M. L. Foresti

A2 Polymer and Composite Material Group, Laboratorio de

A3 Materiales y Estructuras, Facultad de Ingenierıa, Instituto de

A4 Tecnologıas y Ciencias de la Ingenierıa (INTECIN) CONICET,

A5 Universidad de Buenos Aires, Las Heras 2214,

A6 CP 1127 Buenos Aires, Argentina

A7 e-mail: [email protected]

A8 P. Cerrutti � M. Galvagno

A9 Departamento de Ingenierıa Quımica, Facultad de Ingenierıa,

A10 Universidad de Buenos Aires, Buenos Aires, Argentina

A11 M. Galvagno

A12 Instituto de Investigaciones Biotecnologicas CONICET,

A13 UNSAM, Buenos Aires, Argentina

123Journal : Large 10924 Dispatch : 15-9-2012 Pages : 10

Article No. : 541h LE h TYPESET

MS Code : JOOE-1080 h CP h DISK4 4

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61 wood and plant fibers by use of mechanical treatment or acid

62 hydrolysis, respectively. A number of reviews on the

63 extraction, properties and applications of microfibrillated

64 cellulose and cellulose nanocrystals have been published in

65 the last years [1–6]. On the other hand,microbial synthesis of

66 cellulose in which specific bacteria synthesize cellulose

67 microfibrils as a primary metabolite, appears as a promising

68 route for the obtention of cellulose microfibrils. During

69 bacterial cellulose (BC) synthesis, cellulose molecules are

70 synthesized in the interior of the bacterial cell and spun out to

71 form nanofibrils of ca. 2–4 nm in diameter which then

72 aggregate in the form of ribbon-shaped microfibrils of ca.

73 80 9 4 nm [7]. The overlapping and intertwisted cellulose

74 ribbons form a non-wovenmat with very high water content.

75 Bacteria-derived cellulose is of particular interest due to its

76 light weight, high mechanical properties, non-toxicity,

77 renewability, and biodegradability. Besides, the high

78 chemical purity of the cellulose mat produced by bacteria

79 avoids chemical treatments frequently used in plant-derived

80 celluloses for the removal of hemicellulose and lignin.

81 BC is produced as an extracellular primary metabolite

82 by bacteria belonging to the genera Acetobacter, Agro-

83 bacterium, Alcaligenes, Pseudomonas, Rhizobium, Aero-

84 bacter, Achromobacter, Azotobacter, Salmonella or

85 Sarcina [8, 9]. However, its most efficient producers are

86 gram-negative acetic acid bacteria Acetobacter xylinum

87 which has been reclassified and included within the novel

88 genus Gluconacetobacter, as G. xylinus [10, 11]. Culture is

89 carried out in static or agitated conditions at temperatures

90 around 28–30 �C. Since bacteria used are aerobic, in static

91 fermentations cellulose pellicle is formed only in the

92 vicinity of the oxygen-rich air–liquid surface. The reason

93 why the bacteria generate cellulose is unclear, but it has

94 been suggested that it is a mean that bacteria use to

95 maintain their position close to the surface of culture

96 solution [12], as well as a protective coating that guard

97 bacteria from ultraviolet radiation [13], or to prevent the

98 entrance of enemies and heavy-metal ions whereas nutri-

99 ents diffuse easility along the pellicle [7].

100 Although the first report on an extracellular gelatinous mat

101 whose chemical composition and reactivity was the same as

102 cell-wall cellulose dates from 1886 [14, 15], BC did not

103 receivedmuch attention until themid-1980swhen remarkable

104 mechanical properties of its pellicle were discovered [16, 17].

105 In the nineties the use of BC for composite materials was

106 reported by several authors [18, 19], and in the following years

107 the possibility of obtaining nanosized cellulosemicrofibrils of

108 high purity with low energy input brought a resurgence in the

109 area. Nowadays, proposed applications for bacterial cellulose

110 include membranes for filtration, paper reinforcement,

111 acoustic membranes, medicinal pads, hydraulic fracturing

112 fluids for recovery of hydrocarbons, absorption composites

113 such as nappies and sanitary products, reinforcement of

114polymer matrices, preparation of optically transparent films,

115tissue scaffolds, artificial skin bone cement, flexible displays

116screens, etc. [20–28].

117In microbial fermentations, the cost of substrates nor-

118mally accounts for up to 50–65 % of the total cost of

119production. BC production is not the exception, and thus in

120recent years much work has been devoted to find new low

121cost carbon sources. Recently, Castro et al. [29] produced

122BC microfibrils (2.8 g/l of medium) from non-conventional

123sources such as pineapple peel juice and sugarcane juice by

124use of a strain from Gluconacetobacter swingsii sp [29].

125Moosavi-Nasab and Yousefi [30] studied the feasibility of

126using low quality date syrup—a fruit largely produced in

127the hot arid regions of Southwest Asia and North Africa—,

128for the production of BC using G. xylinus, obtaining a yield

129of 43.5 g/l of fermentation medium. To economize the BC

130production, Rani et al. [31] used coffee cherry husk extract

131(a byproduct from the coffee processing) and corn steep

132liquor (a byproduct from the starch processing industry)

133as less expensive sources of carbon and nitrogen, respec-

134tively, obtaining 6.24 g/l of BC in optimized conditions.

135Carreira et al. (2011) studied the effect of several industrial

136residues (grape skins aqueous extract, cheese whey, crude

137glycerol and sulfite pulping liquor) on BC production with

138G. Sacchari. They reported that productions can be sub-

139stantially improved by the addition of N and/or P sources.

140They also reported that the responses in BC production

141were dependent on the raw material used, and also on the

142different N and P sources tested [32].

143In the current work, production of bacterial cellulose by

144G. xylinus using as carbon sources glycerol remaining from

145biodiesel production and grape bagasse (a residue of white

146wine production in Mendoza (Argentina)) was studied,

147with the aim of formulating a general, simple and inex-

148pensive medium to produce BC. More common carbon

149sources such as glucose, commercial glycerol and cane

150molasses were also assayed for comparison. Carbon and

151nitrogen sources used were compared not only in terms of

152cellulose yield and production, but also the morphology

153and structure of the BC obtained were studied. In this

154context, BC microfibrils produced in the selected media

155were characterized by means of scanning electron

156microscopy (SEM), X-ray diffraction (XRD), Fourier

157transform infrared spectroscopy (FTIR), and thermogravi-

158metric analysis (TGA).

159Materials and Methods

160Microorganism

161The bacterial strain of G. xylinus (syn. Acetobacter aceti

162subsp. xylinus, A. xylinum) NRRL B-42 used in this work

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163 was gently provided by Dr. Luis Ielpi (Fundacion Instituto

164 Leloir, Buenos Aires, Argentina).

165 Cultivation Media and Conditions

166 Inocula were cultured for 48 h in Erlenmeyer’s flasks con-

167 tainingHestrin andSchramm(HS)medium (%,w/v): glucose,

168 2.0; peptone, 0.5; yeast extract, 0.5; disodiumphosphate, 0.27;

169 citric acid, 0.115. The pH was adjusted to 6.0 with dil. HCl or

170 NaOH. Agitation (200 rpm) was provided by an orbital sha-

171 ker. Culture media used for BC production were HSmodified

172 by replacing D-glucose by other carbon sources at the same

173 concentration such as glycerol (commercial and that coming

174 from biodiesel production) or cane molasses (50.75 % w/w

175 fermentable sugars). Glycerol from biodiesel and grape

176 bagasse alone or in the presenceof 0.5 %w/v corn steep liquor

177 (CSL) or 0.7 % w/v diammonium phosphate (DAP) as

178 nitrogen source, were also assayed.

179 For BC production, static incubations were performed in

180 Erlenmeyer’s flasks for 14 days. The initial pH of the

181 production media was 5.0. All cultures were incubated at

182 28 ± 1 �C and a ratio ‘‘volume flask: volume medium’’ of

183 5:1 was maintained. All media were sterilized by auto-

184 claving (121 �C, 15 min).

185 To calculate cellulose production, pellicles were rinsed

186 with water to remove the culture medium, and then boiled

187 in 2 % w/v NaOH solution for 1 h in order to eliminate the

188 bacterial cells from the cellulose matrix. Then, pellicles

189 were washed with distilled water till neutralization. Dry

190 weight was measured after drying the films at 37 �C till

191 constant weight. Results were reported as ‘‘production’’

192 and expressed as gram dry weight of cellulose per litre of

193 the original medium (g/l) [30]. Yield was calculated as

194 gram dry weight of cellulose per gram of consumed sub-

195 strate (g/g).

196 Pretreatment of glycerol remaining from biodiesel pro-

197 duction was done as described by Chi et al. [33]. The

198 product obtained (pretreated glycerol) contained 70 % (w/

199 v) authentic glycerol. Glycerol concentration was deter-

200 mined enzymatically (Boehringer-Mannheim/R-Biopharm,

201 AG, D-64293 Darmstadt, Ger.). Grape bagasse was

202 homogenized in a blender with an appropriate volume of

203 distilled water. The extract was then filtered through paper

204 Whatman No3 and pH was adjusted to pH 5.0 with NaOH.

205 Glucose and fructose contents were determined enzymati-

206 cally (Boehringer-Mannheim/R-Biopharm, Cat. No. 10 139

207 106 035).

208 Conditioning of Bacterial Cellulose

209 for Characterization Studies

210 For characterization studies the cellulose pellicles obtained

211 were washed with water and homogenized in a blender for

2125 min. The microfibrils thus obtained were treated for 14 h

213in a 5 % w/v KOH solution, rinsed with water till neu-

214tralization (pH = 7.0), and homogenized in water for

2155 min [29]. For XRD, FTIR and TGA analysis, BC sus-

216pensions were dried on plastic Petri dishes at 45 �C during

2173 h. For scanning electron microscopy the homogenate was

218conveniently diluted with distilled water.

219Scanning Electron Microscopy (SEM)

220Drops of BC/water suspensions were deposited on micro-

221scope glasses and dried at 45 �C for 3 h. Samples were

222coated with gold using an ion sputter coater, and observed

223by use of an scanning electron microscope Zeiss Supra 40

224with field emission gun operated at 3 kV. Average diam-

225eter distributions of bacterial cellulose microfibrils were

226obtained from microscopic images by use of the ImageJ

227software, which measures the number of pixels in the

228picture and the scales lengths according to the calibration

229provided by the user.

230X-ray Diffraction (XRD)

231The structure of bacterial cellulose was analysed with a

232Bruker/Siemens automated wide-angle powder X-ray dif-

233fractometer. The X-ray diffraction pattern was recorded in

234a 2h angle range of 0–40�. The wavelength of the Cu/Ka

235radiation source used was 0.154 nm, generated at acceler-

236ating voltage of 40 kV and a filament emission of 30 mA.

237X-ray diffraction data were analysed using MDI Jade 5.0

238software. Curve-fitting was performed to find individual

239peak regions.

240The crystallinity index (CI) of produced BC was deter-

241mined by the following equation [34].

CIð%Þ ¼Ic � Iam

Ic� 100 ð1Þ

243243where Ic or I200 corresponds to the maximum intensity of

244the lattice diffraction, and Iam corresponds to the intensity

245of the peak at 2h = 18�, which accounts for the amorphous

246part of cellulose. The intensity of the peaks was measured

247as the maximum value obtained for the peak taking into

248account a baseline.

249Additionally, crystallinity of bacterial celluloses was

250determined by integration of each XRD peaks taking into

251account a baseline for each peak (area assigned to the

252crystalline part), and the total area under the diffractogram

253considering a straight line from 0 to 40� 2h as baseline.

254Fourier Transform Infrared Spectroscopy (FTIR)

255Attenuated total reflectance Fourier transform infrared

256spectroscopy (ATR-FTIR) was performed using a Nicolet

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257 6700 Thermo Scientific Spectrometer equipped with a Smart

258 Orbit ATR accessory. The reflectance element was a dia-

259 mond crystal. Spectra were collected in the 4,000–400 cm-1

260 range with 32 scans and at 4 cm-1 resolution.

261 Thermogravimetric Analysis (TGA)

262 Dynamic thermogravimetric analysis of dried samples was

263 conducted in a TGA-50 Shimadzu instrument. Temperature

264 programs were run from 25 to 550 �C at a heating rate of

265 10 �C/min, under nitrogen atmosphere (30 ml/min) in

266 order to prevent thermoxidative degradation.

267 Results and Discussion

268 Bacterial Cellulose Production

269 As previously introduced, the cost of the carbon source

270 plays a key role on bacterial cellulose production costs. In

271 the current manuscript, low cost carbon sources such as

272 glycerol obtained as byproduct of biodiesel production, and

273 grape bagasse from regional wine production, were

274 assayed. The volumetric productions (g/l) and the yield

275 (g/g) of BC in HS culturing medium modified by replacing

276 glucose by other carbon sources such as cane molasses

277 (10.0 g/l of fermentable sugars) or glycerol (10.0 g/l of

278 commercial grade or obtained from biodiesel production)

279 are shown in Fig. 1. As it is indicated in the Figure, the

280 highest production and yield were obtained for commercial

281 glycerol. Results obtained agree with those of Keshk and

282 Sameshima [35] who found a 155 % higher cellulose yield

283 for BC production by G. xylinus in HS containing glycerol

284 instead of glucose. Carreira et al. (2011) reported higher

285 productions of BC in the presence of analytical glycerol

286with respect to crude glycerol (2.07 and 0.10 g/l respec-

287tively) [32]. Kornmann et al. [36] also reported that glucose

288concentration had to be maintained at low levels during

289exopolysaccharides production by G. xylinus, in order to

290reduce the production of by-products as (keto)-gluconates

291that significantly diminished yields. The costs of carbon

292sources employed are also shown in Fig. 1, with the min-

293imum found for glycerol obtained from biodiesel produc-

294tion. Although BC production using analytical grade

295glycerol was three times higher than production achieved

296using glycerol from biodiesel; production costs using the

297biodiesel byproduct could lead to values up to 18-folds

298lower.

299BC production from a waste of white wine production

300such as grape bagasse (see Materials and Methods for

301details) containing 14.4–15.4 and 22.2 g/l fructose, was

302also assayed. The effect of the addition of compounds

303usually employed as nitrogen sources such as DAP and

304CSL was evaluated (Fig. 2). Results show that both pro-

305ductions and yields increased in the presence of DAP or

306CSL. Production values obtained were only slightly lower

307than in the presence of commercial glycerol as carbon

308source. Moreover, productions obtained with grape bagasse

309without supplementation were higher than those achieved

310with glucose or glycerol from biodiesel (see Fig. 1).

311When experiments with glycerol obtained from biodie-

312sel (20.0 g/l) alone or in the presence of DAP were carried

313out, pellicle production was not observed. In this sense,

314Carreira et al. (2011) have also reported that (NH4)2SO4

315inhibited completely the cellulose production from crude

316glycerol by the bacterial strain G. sacchari isolated from a

317commercial food source [32]. However, when glycerol

318supplemented with CSL was used, the highest production

319was obtained, leading to values similar to commercial

320glycerol.

0

2

4

6

8

10

12

glucose (0.22-0.24

U$D/kg)

cane molasses (0.12

U$D/kg)

commercial glycerol

(0.46-0.75 U$D/kg)*

glycerol from

biodiesel (0.04-0.15

U$D/kg)

Pro

du

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on

(g

/l)

0

0.2

0.4

0.6

0.8

1

1.2

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g/g

)

Fig. 1 Volumetric productions

and yields of BC in the presence

of different carbon sources:

filled square Production (g/l);

open square Yield (g/g).

Asterisk Current international

prices

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321 Productivities achieved only with N supplementation

322 (0.021–0.030 g/l h) largely encouraged the use of the non-

323 conventional production media proposed, when comparing

324 with the range of values reported in bibliography (0.009–

325 0.018 g/(l h) [29, 31, 32]. In the case of grapes skins, Carreira

326 et al. (2011) found that the highest increment on BC produc-

327 tion (nearly 85 %) was obtained when 4 g/l yeast extract and

328 2 g/l KH2PO4 were added as supplements. This condition

329 corresponded to the maximum productivity of BC achieved

330 by these authors (0.0104 g/l h) [32]. It is worth to notice

331 that grape bagasse allows the production of an added-value

332 product such as BC decreasing on the way environmental

333 problems associated with its disposal, since only a very small

334 portion of winery by-products are used world-wide [37]. On

335 the other hand, as large quantities of glycerol are generated

336 worldwide by the biodiesel industry and several other indus-

337 tries, its conversion to high-value added products is of great

338 interest [38, 39]. The results obtained herein for both grape

339 bagasse and glycerol from biodiesel are encouraging for the

340 cost-effective industrial production of BC.

341 Scanning Electron Microscopy (SEM)

342 Figure 3a–f shows scanning electron microscopy images of

343 BC obtained from the different carbon sources assayed.

344 Ribbon like microfibrils forming a highly fibrous network-

345 like structure can be seen (Fig. 3a). The bacterial strain of

346 G. xylinus is also shown in Fig. 3a (see magnification

347 square). Determination of microfibrils width distribution is

348 not straightforward due to the frequent twisting of bacterial

349 cellulose ribbons. Image analysis was then made manually

350 in order to distinguish between widths, apparent thickness

351 of edged on ribbons, and ‘‘intermediate’’ views. The

352 analysis performed did not show the existence of signifi-

353 cant differences among the dimensions of nanofibers

354 obtained from the different carbon sources assayed, with

355 nanofibers widths in the range of 35–70 nm, and thickness

356 values in the 13–24 nm interval. Nanofibers widths are

357similar to those produced by other authors, whereas mea-

358sured thicknesses are slightly higher [1, 29]. In terms of

359length, all microfibrils were several micrometers long since

360the microfibrils-ends were not seen even at low magnifi-

361cation (i.e. 5 K9).

362X-ray Diffraction (XRD)

363Cellulose has several crystalline polymorphisms (I, II, III,

364IV). Cellulose I is the crystalline cellulose that is naturally

365produced by a variety of organisms, i.e. trees, plants,

366tunicates, algae and bacteria. The structure of cellulose I is

367thermodynamically metastable and can be converted to

368cellulose II or III. All the cellulose strands are ordered in a

369highly ordered parallel arrangement [40]. The conversion

370from cellulose I to cellulose II can be produced by regen-

371eration (solubilization and recrystallization) and merceri-

372zation (alkaline solution). Cellulose II has a monoclinic

373structure. Cellulose III is obtained from Cellulose I and II

374by means of liquid ammonia and thermal treatments.

375Cellulose IV is formed from Cellulose III [1].

376One interesting characteristic of bacterial derived cel-

377lulose microfibrils is that it is possible to adjust culturing

378conditions in order to alter microfibril formation and

379crystallization [41, 42]. Diffraction patterns obtained for

380BC obtained from glucose, commercial glycerol, glycerol

381remaining from biodiesel production, grape bagasse and

382cane molasses are shown in Fig. 4. Three peaks shown in

383Fig. 4 at 2h = 14.62� (1–10), 16.29� (110) and 22.48�

384(200) (direction ‘‘c’’ of the unit axis is along the axis of the

385polymer for Miller indices) confirmed that only cellulose I

386was present in all BC samples [43–46]. No peaks, instead,

387are found at 2h = 12.1� and 20.8�, which are characteristic

388of cellulose II [47, 48]. Cellulose I is the crystal structure

389with the highest axial elastic modulus [1].

390Crystallinity of cellulose can be determined by different

391methods, and the results are known to be dependant on the

392method applied [49]. However, results obtained by each

0

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8

10

12

grape bagasse grape bagasse +DAP grape bagasse+CSL glycerol biodiesel

+CSL

Pro

du

cti

on

(g

/l)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Yie

ld (

g/g

)

Fig. 2 Volumetric productions

and yields of BC using as

carbon source two

agroindustrial wastes with and

without supplementation: filled

square Production (g/l); open

square Yield (g/g). CSL corn

steep liquor, DAP diammonium

phosphate

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393 particular method are useful for comparing samples iden-

394 tically analyzed. In the current manuscript, crystallinity

395 was calculated by dividing the area of crystalline peaks by

396 total diffractogram area (e.g. amorphous and crystalline

397 zones). The areas were calculated using Origin software by

398 fitting of multiple peaks, and by considering a connected

399 baseline for each peak, and a straight line from 2h = 0� to

400 2h = 40� for the total area (see example in Fig. 5).

401 It is worth noting that the method described is

402 undoubtedly influenced by the baselines that the operator

403takes into account, both at the base of each peak and also

404for the whole diffractogram. Calculated crystallinity values

405are shown in the first results column of Table 1. Results

406show that bacterial celluloses obtained from wine produc-

407tion residues or from the glycerol remaining from biodiesel

408production, have similar crystallinity values than those

409obtained from commercial glycerol or glucose (i.e.

41074–79 %). The use of cane molasses as carbon source

411apparently leads to cellulose microfibrils with lower crys-

412tallinity (67 %).

Fig. 3 a Bacterial strain of G. xylinus trapped in bacterial cellulose network (carbon source: grape bagasse). Bacterial cellulose from b glucose,

c commercial glycerol, d glycerol remaining from biodiesel production, e grape bagasse, f cane molasses

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413 Besides, the crystallinity index (CI %) of BC samples

414 was calculated as described in Materials an Methods after

415 subtraction of the background signal measured without

416 cellulose using the XRD patterns [34]. The I200 intensity was

417 measured from the top of the 22.48� peak to the straight

418 baseline of the diffractogram, and Iam was measured at 18�

419 as a representative of the amorphous cellulose fraction.

420 Results have been included in Table 1. Despite the method

421 used provides a simple tool to measure crystallinity, there

422 are some issues that should be taken into account: (a) the

423 measure of the height of the peak at 18� as the amorphous

424part is underestimated because the maximum of the amor-

425phous cellulose part was found to be shifted to higher angle

426[49]; (b) the measurement of the height of the peak without

427taking into account its area is an oversimplification of the

428determination; (c) the method considered only the highest

429peak (I200) which accounts for the aligned cellulose crystal

430lattice, without taking into account the others peaks. Table 1

431shows that determination of CI with the method proposed

432led to higher crystallinity values than the area method. On

433the other hand, both set of crystallinity determinations evi-

434dence that results obtained for different carbon sources

435assayed are very similar. However, it seems that cane

436molasses cultivating medium produces a bacterial cellulose

437with a slightly lower crystallinity than the others carbon

438sources. The behaviour could be ascribed to the glucan chain

439hydroxyl group are not allowing to self-assembly and there

440is a reduction in the crystallinity.

441It has been reported that cellulose I structure is made of

442parallel chains characterized by an intermolecular hydro-

443gen bond network extending from the O2–H hydroxyl to

444the O6 ring oxygen of the next unit [50]. Cellulose I has

445two polymorphs: a triclinic structure (Ia) and a monoclinic

446structure (Ib), which coexist in various proportions

447depending on the cellulose source [2, 51, 52]. The Ia unit

448cell contains one chain, whereas cellulose the Ib unit cell

449contains two parallel chains [53]. The ratio between Ia and

450Ib polymorphs depends on the source of the nanocellulose

451[54]. However it is controversial due to the difficulties in

452structural characterization of individual nanocellulose [35].

453Moon et al. [1] and Keshk and Sameshima [35], showed

454that Ia/Ib ratio can be modified by culturing conditions

455(stirring, temperature, and additives). The presence of

456additives interferes with the aggregation of the elementary

457fibrils into the normal ribbon assembly, and chemically

458addition media may result in increased contents of Ib

459crystal structure [42]. Ib polymorph is preferentially

10 12 14 16 18 20 22 24 26 28 30

2θ

abc

d

e

(110)Iα

Iβ (200)

(110)

Iam

Fig. 4 XRD patterns of BC obtained from different cultivation

medium. The spectra were vertically moved for a clearer view of each

diffractogram. (a) glucose, (b) commercial glycerol, (c) glycerol

remaining from biodiesel production, (d) grape bagasse, (e) cane

molasses. Diffraction patterns have been normalized with respect to

the peak at 2h = 22.48�

10 15 20 25 30 35 40

2θ

crystalline zones

amorphous zone

Fig. 5 Method used for determining the crystallinity based on

crystalline peaks and total pattern area comparison. The area of the

peak at 2h = 16.29� was included in the area below the peak at

2h = 14.62�

Table 1 Crystallinity, crystallinity index (CI) and Ib content of BC

obtained by use of different carbon sources

Carbon source Crystallinity

(%)aCI

(%)bIb/Ia Ib (%)c

Glucose 77 92 1.45 31

Commercial glycerol 79 95 1.79 44

Glycerol from biodiesel 76 94 1.73 42

Grape bagasse 74 89 1.39 28

Cane molasses 67 89 1.39 29

a Crystallinity (%) was calculated as the ratio of the crystalline peaks

and total diffractogram areas (Fig. 5)b Crystallinity index, CI (%) was calculated from the maximum

intensity of the lattice diffraction and the intensity at 2h = 18�c Ib % was calculated as (Ib - Ia)/Ib 9 100 where I is the height of

the peak taking into account a baseline at the peak

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460 formed in the isolated elementary fibrils that are free from

461 constraint present when aggregated in the normal microfi-

462 bril ribbon assembly. It has been suggested that this added

463 constraint is necessary for the formation of the metastable

464 Ia phase [55]. The ratio between both polymorphisms

465 affects the inter-hydrogen bonding and may affect

466 mechanical properties of BC. Moreover, the Ia/Ib could

467 also alter the width of the microfibrils leading to square

468 instead of rectangular cross-sections of the BC ribbons [1].

469 Table 1 shows Ia/Ib and the percent fraction of Ib poly-

470 morph in BC calculated as (Ib - Ia)/Ib. Results show

471 similar contents of the Ib polymorph for glucose, cane

472 molasses and grape bagasse carbon sources (28–31 %). On

473 the other hand, commercial glycerol and glycerol from

474 biodiesel production showed increased contents of the Ib

475 polymorph with values in the 42–44 % range. Changes in

476 Ia/Ib in the range found did not produce changes in ribbons

477 widths sections that could be distinguished from the SEM

478 images obtained.

479 Fourier Transform Infrared Spectroscopy (FTIR)

480 Figure 6 shows the FTIR spectra collected for the bacterial

481 celluloses produced by use of the different carbon sources

482 assayed. Spectra showed bands typical of cellulose I. The

483 band centered at 896 cm-1 is typical of b-linked glucose

484 polymers [29]. The band at 1,050 cm-1 could be associated

485 with ether C–O–C functionalities [30]. The band at

486 1,109 cm-1 is assigned to C–O bond stretching [29]. The

487 band at 1,159 cm-1 is assigned to cellulose C–O–C bridges

488 [31]. The weak band found at 1,211, 1,275, 1,314 and

489 1,334 cm-1, can be ascribed to O–H in plane vibration,

490 C–H bending, CH2 wagging, and O–H in-plane bending,

491respectively [29]. The band centered at around 1,428 cm-1

492could be associated with either CH2 symmetrical bending or

493surface carboxylate groups. The band at 1,644 cm-1 is due to

494the H–O–H bending vibration of absorbed water molecules

495[31, 56]. The band at 2,898 cm-1 is attributed to CH2

496stretching. The bands at 3,220–3,342 cm-1 indicate inter-

497molecular and intramolecular hydrogen bonds [43]. Hydro-

498xyl groups (–OH) appear at around 3,350 cm-1. However,

499the analysis of the zone from 3,000 to 3,600 cm-1 is not very

500clear. The bands centered at around 3,220 cm-1 and at

501750 cm-1 have been reported to account for the triclinic Ia

502allomorph, whereas bands centered at around 3,283 cm-1

503and at 710 cm-1 are said to belong to the monoclinic Ib

504allomorph [57]. Sugiyama et al. [58] worked with different

505modifiedBCand they said that the shrink or disappearance of

5063,240 cm-1 peak in the spectrum implies a lower Ia content,

507and indicates that the inter-molecular hydrogen bonds are

508weaker and crystallinity content is low. However, the

509increasing of the amorphous part influences the hydration

510ability of bacterial cellulose, and as consequence it could

511also produce a higher OH content and deformation of this big

512peak. As it is shown in Table 1, from our results the BC from

513cane molasses has the lower intensity peak with a greater

514number of exposed free OH groups as compared with the

515other BC. Ib peaks at 710 cm-1 in BC from cane molasses

516agree with its low crystallinity. The grape bagasse derived

517BC did not show the same behavior, which may be due to the

518higher water absorption of the sample in the air during the

519experiment. The amorphous part of the specimen also can

520absorb more water from the atmosphere. Park et al. [49] said

521that this method is influenced by the amorphous and crys-

522talline regions.

523Thermogravimetric Analysis (TGA)

524Figure 7 shows the TG curves of the pellicles obtained for

525the different carbon sources assayed, in terms of percent

526weight of the original sample versus temperature. In all the

527samples two significant mass losses are observed. The first

528one takes place from room temperature to 150 �C and it is

529assigned to membrane dehydration. Physically adsorbed

530and hydrogen bond linked water molecules can be lost at

531that first stage [59]. The second mass loss is observed from

532200 to 400 �C, and is assigned to thermal decomposition of

533cellulose [59].

534Table 2 summarizes data on the onset degradation tem-

535perature, the temperature of maximum weight loss rate, and

536the pyrolysis residue remaining after heating the samples up

537to 550 �C. The extrapolated onset temperature (Tonset),—

538which denotes the temperature at which the weight loss

539begins—was taken as the temperature in which the mass loss

540started, as it is shown in Fig. 7. The temperature ofmaximum

541weight loss rate (Tmax) was calculated from the first

4000 3500 3000 2500 2000 1500 1000 500

3342

3220

2898

1644

1428

1109

896

Ab

so

rba

nce

Wavenumber (cm-1)

1050

a

b

c

d

e

Fig. 6 FTIR-ATR of BC from different culture medium: (a) glucose,

(b) commercial glycerol, (c) glycerol remaining from biodiesel

production, (d) grape bagasse, (e) cane molasses

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542 derivative of the TGdata, and it indicates the point of greatest

543 rate of change on the weight loss curve.

544 Results obtained for the non-conventional carbon sour-

545 ces assayed are similar to those found for glucose derived

546 pellicles (Table 2). On the other hand, cane molasses BC

547 showed Tonset and Tmax values lower than the other BC

548 pellicles. Thermal degradation behaviour is known to be

549 affected by some structure parameters such as molecular

550 weight, crystallinity and orientation of the fibres [34, 59].

551 In the case of cane molasses BC, XRD results showed that

552 it had the lowest crystallinity (Table 1), which could be the

553 reason for thermal behaviour observed. In the case of the

554 mass loss profile of BC obtained from analytical glycerol,

555 the sharp decrease in weight evidenced for this sample

556 could be adscribed to its high crystallinity (Table 1). The

557 maximum decomposition temperature is a criterion of

558 thermal stability. BC from glucose has one of the highest

559 values of Tmax which indicates its higher thermal stability

560 when compared with cane molasses and analytical glycerol

561 BC. Glucose pellicle has the highest carbon residue content

562 as well. Glycerol from biodiesel and grape bagasse carbon

563 sources led to BC pellicles with Tmax values similar to

564 glucose BC.

565Conclusions

566Carbon source price plays a key role on the costs of

567industrial BC production. Aiming to reduce BC costs, low-

568priced carbon sources such as glycerol obtained as

569byproduct of biodiesel production, and grape bagasse from

570regional wine production, were assayed. Both, biodiesel

571glycerol and wine residues showed their suitability for high

572BC production (10.0 and 8.0 g/l, respectively) when sup-

573plemented with corn steep liquor for nitrogen requirements.

574Production values obtained were much higher than those

575achieved with HS culture medium, and similar to the ones

576found with HS culture medium modified by replacing

577D-glucose by commercial glycerol. With respect to nitrogen

578sources added (DAP or CSL), they also contributed to the

579economy of the bioprocess, as high values of productivity

580were obtained without the use of more expensive raw

581materials such as yeast extract.

582BC ribbons obtained in non-conventional culture media

583showed widths values in the range of 35–70 nm and

584thicknesses values in the 13–24 nm interval, which were

585similar to cross-sections dimensions of ribbons found for

586BC obtained from glucose or commercial glycerol media.

587X-rays patterns and FTIR spectra confirmed the presence of

588cellulose I crystalline polymorphism. Crystallinity and

589crystallinity index values determined for bacterial cellu-

590loses obtained from wine production residues and from the

591glycerol remaining from biodiesel, were similar to those

592obtained for commercial glycerol or glucose BC. Mor-

593phological, structural and thermal similarities between

594microbial cellulose microfibrils derived from the low-

595priced media assayed and those obtained from traditional

596culture media (i.e. glucose), as well as the high production

597values achieved, suggest that grape bagasse and abundant

598glycerol remaining from increasing biodiesel production

599are attractive carbon sources for reducing the costs of

600industrial bacterial cellulose production.

601Acknowledgments Authors acknowledge Dr. Luis Ielpi (Fundacion602Instituto Leloir, Buenos Aires, Argentina) for the bacterial strain,603Dr. Mariana Combina (Estacion Experimental Agropecuaria, Instituto604Nacional de Tecnologıa Agropecuaria, Mendoza, Argentina) and Dr.605CeciliaRojo (EstacionExperimentalAgropecuaria, InstitutoNacional de606Tecnologıa Agropecuaria, Mendoza and CONICET, Argentina) for607grape bagasse, and Agencia Nacional de Promocion Cientıfica y Tec-608nologica for financial support (PICT 00223 2008—PRESTAMO BID).

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0 50 100 150 200 250 300 350 400 450 500 5500

10

20

30

40

50

60

70

80

90

100R

esid

ual m

ass (

%)

Temperature (°C)

a

e

d

b

c

Fig. 7 TG curves of BC from different culture medium: (a) glucose,

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Bacterial Cellulose from Simple and Low Cost Production Media by Gluconacetobacter xylinus