Bioresource Technology 104 (2012) 503–508

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Bioresource Technology

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Bacterial cellulose production from cotton-based waste textiles: Enzymaticsaccharification enhanced by ionic liquid pretreatment

Feng Hong a,b,⇑, Xiang Guo a, Shuo Zhang c, Shi-fen Han a, Guang Yang a, Leif J. Jönsson d

a Group of Microbiological Engineering and Industrial Biotechnology, College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, Chinab State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, Chinac School of Environmental Science and Engineering, Donghua University, Shanghai 201620, Chinad Department of Chemistry, Umeå University, SE-90187 Umeå, Sweden

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

Article history:Received 10 July 2011Received in revised form 5 October 2011Accepted 7 November 2011Available online 25 November 2011

Keywords:Bacterial celluloseIonic liquidCotton-based waste textilesEnzymatic saccharificationBiorefinery

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.11.028

Abbreviations: [AMIM]Cl, l-allyl-3-methylimidazocellulose; [BMIM]Cl, 1-butyl-3-methylimidazolium chcellulose; DNS, 3,5-dinitrosalicylic acid; DP, degreeliquid; RTIL, room temperature ionic liquid.⇑ Corresponding author at: College of Chemistry

Biotechnology, Donghua University, North Ren MinShanghai 201620, China. Tel./fax: +86 21 67792649.

E-mail address: fhong@dhu.edu.cn (F. Hong).

Cotton-based waste textiles were explored as alternative feedstock for production of bacterial cellulose(BC) by Gluconacetobacter xylinus. The cellulosic fabrics were treated with the ionic liquid (IL) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl). [AMIM]Cl caused 25% inactivation of cellulase activity at aconcentration as low as of 0.02 g/mL and decreased BC production during fermentation when presentin concentrations higher than 0.0005 g/mL. Therefore, removal of residual IL by washing with hot waterwas highly beneficial to enzymatic saccharification as well as BC production. IL-treated fabrics exhibiteda 5–7-fold higher enzymatic hydrolysis rate and gave a seven times larger yield of fermentable sugarsthan untreated fabrics. BC from cotton cloth hydrolysate was obtained at an yield of 10.8 g/L whichwas 83% higher than that from the culture grown on glucose-based medium. The BC from G. xylinusgrown on IL-treated fabric hydrolysate had a 79% higher tensile strength than BC from glucose-based cul-ture medium which suggests that waste cotton pretreated with [AMIM]Cl has potential to serve as a high-quality carbon source for BC production.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Bacterial cellulose (BC) is an extracellular biomaterial producedby some bacteria such as acetic acid bacteria (Bielecki et al., 2002).BC has many desirable properties, such as a high purity (free of lig-nin and hemicellulose), a high crystallinity, a high degree of poly-merization, a nano-structured network, a high wet tensile strength,a high water-holding capacity, and good biocompatibility (Bieleckiet al., 2002), and is therefore considered for applications in bio-medicine, cosmetics, advanced acoustic diaphragms, paper-mak-ing, and the food and textile industries (Bielecki et al., 2002).

Carbon sources utilized in fermentation processes for BC pro-duction include monosaccharides (such as glucose and fructose),disaccharides (such as sucrose and maltose), and alcohols (suchas ethanol, glycerol, and mannitol). However, these feedstocksare usually expensive, and sometimes give low yields of BC, which

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lium chloride; BC, bacterialloride; CMC, carboxymethylof polymerization; IL, ionic

, Chemical Engineering andRoad, No. 2999, Songjiang,

leads to high BC production costs (Bielecki et al., 2002). The prob-lem with high production cost limits the scale of industrial manu-facture of BC and becomes a bottleneck for extending theapplications for BC. In recent years, many studies have focusedon attempts to produce BC by using alternative feedstocks, suchas food processing effluents, hemicelluloses in waste liquor fromatmospheric acetic acid pulping, molasses, konjak glucomannan,fruit juice, rice bark, and wheat straw (Hong et al., 2011). Theseraw materials are abundant and either industrial/agriculturalwaste or relatively inexpensive agricultural products. Old cottongarments or clothes and used regenerated cellulose (such as vis-cose) fabrics mainly consist of cellulose polymers, which couldpotentially be another kind of inexpensive polysaccharide resourcefor BC production. Other types of textile wastes, which arise duringmanufacturing of yarn and fabric or apparel-making processes,could also be used with possible benefits including decreasingthe production costs of BC, saving natural resources and reducingenvironmental problems.

Some form of pretreatment of cotton-based waste textiles is re-quired before their use as feedstock for microbial cultivation or fer-mentation. Ethanol was produced by fermentation with baker’syeast of such waste treated with sodium hydroxide or concen-trated phosphoric acid and enzymatic hydrolysis (Jeihanipourand Taherzadeh, 2009). Kuo et al. (2010) used 85% phosphoric acid,

504 F. Hong et al. / Bioresource Technology 104 (2012) 503–508

N-methylmorpholine-N-oxide (NMMO) monohydrate, the ionic li-quid 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), andNaOH/urea to pretreat waste cotton textiles. The highest sugaryield (90%) was achieved with 85% phosphoric acid, and yields of80–90% were produced with [BMIM]Cl-treated materials, NMMO-treated materials, and NaOH/urea-treated materials.

Imidazolium-based room-temperature ionic liquids (RTILs) haveadvantages over other pretreatment reagents, since they are non-volatile, exhibit low viscosity, can be recovered and reused, andhave minimum environmental impact (Cao et al., 2009; Pinkertet al., 2009). Imidazolium-based chloride RTILs have been studiedwith regard to dissolution of cellulose (Swatloski et al., 2002; Lauset al., 2005; Zhang et al., 2005; Bentivoglio et al., 2006; Zhu, 2008;Cao et al., 2009; Pinkert et al., 2009) and pretreatment prior to enzy-matic hydrolysis (Dadi et al., 2006, 2007; Kuo and Lee, 2009; Zhaoet al., 2009) or acid hydrolysis (Li et al., 2008; Zhang et al., 2009;Kim et al., 2010). [BMIM]Cl has been utilized most often, but it isa corrosive, toxic and extremely hygroscopic solid (m.p. � 70 �C)(Liebert and Heinze, 2008; Pinkert et al., 2009). 1-Allyl-3-methyl-imidazolium chloride ([AMIM]Cl) is less toxic, less viscous andmore readily synthesized, and has a better ability than [BMIM]Clto solubilize cellulose (Zhang et al., 2005). [AMIM]Cl has been usedto pretreat microcrystalline cellulose before enzymatic hydrolysis(Dadi et al., 2007) as well as before acid hydrolysis (Zhang et al.,2009), but not to treat cellulosic materials with a high crystallinityindex and degree of polymerization (DP), such as cotton-basedtextiles.

In the present study, [AMIM]Cl was chosen to dissolve cotton-based waste textiles as a pretreatment prior to enzymatic hydroly-sis, and the optimum conditions of the pretreatment were evalu-ated. The effects of IL residues on cellulase activity and BCproduction were studied. Finally, the hydrolysate was utilized forthe cultivation of Gluconacetobacter xylinus for the production ofbacterial cellulose pellicle. Moreover, the properties of BC culturedin cloth hydrolysate, e.g. dry weight, water-holding capacity andtensile strength, were examined.

2. Methods

2.1. Materials and microorganism

The cotton-based materials employed in the study were used/old undyed 100% cotton T-shirts. N-methylimidazole was pur-chased from Luer Chemical Trading Co. Ltd. (Shanghai, China)and allyl chloride was obtained from Sinopharm Chemical ReagentCo. Ltd. (Shanghai, China). The cellulase preparation was from Bo LiBiological Products Co. Ltd. (Wuxi, China). The activity of the cellu-lase preparation was 11 U/mg as determined by using carboxy-methyl cellulose (CMC) as the substrate. All other reagents wereof analytical grade and used without further purification.

The microorganism, G. xylinus (formerly Acetobacter aceti subsp.xylinus or A. xylinus) ATCC 23770 (obtained from American TypeCulture Collection, Manassas, VA, USA), was maintained on agarplates containing a seed culture medium. The culture medium,which contained (w/v) 2.5% D-mannitol, 0.5% yeast extract, 0.3%peptone (Oxoid, UK), and 2.5% agar, had an initial pH of 5.0.

2.2. Synthesis of [AMIM]Cl

[AMIM]Cl (MW 158.63 g/mol) was synthesized according toZhang et al. (2005). First, 22.9 g N-methylimidazole and 25.2 gallyl chloride were added to a round-bottom flask fitted to a re-flux condenser, and allowed to react in an ice bath for 4 h. Thetemperature was raised to 55 �C and the reaction was allowedto proceed for 7–8 h with vigorous stirring. The unreacted

reagents were extracted with ether, and the ionic liquid waspurified by vacuum distillation to remove the residual etherand other impurities, such as water. The [AMIM]Cl, was slightlyamber.

2.3. Dissolution of cotton cloth in [AMIM]Cl and regeneration

Waste cotton cloth was cut into small pieces (approximately1 � 5 mm) and dried at 40 �C for 48 h. A 0.05 g piece of cotton clothwas added to10 g of IL in a 50 mL round-bottom flask under agita-tion (500 rpm). The flask was incubated in an oil-bath heater. In or-der to study the effects of the dissolution temperature on thepretreatment of waste cotton fibers in [AMIM]Cl, the maximal dis-solved amount of cotton cloth and the corresponding dissolutiontime were investigated at 90 �C, 110 �C, and 130 �C, respectively.When the cloth had dissolved, an additional cloth piece (0.05 g)was added and allowed to dissolve. This process was repeated untilno further dissolution was observed. The maximum amount thatcould be dissolved was determined and the time required to dis-solve 2% (w/w) of the cellulosic sample was recorded. Deionizedwater was used as an anti-solvent for regenerating cotton cellulosefrom the IL. Fifty milliliters of anti-solvent were added to the cot-ton/IL solution with vigorous stirring, and a precipitate immedi-ately formed. The precipitate was collected by centrifugation at2200�g for 10 min and washed with deionized water 5 times un-der vigorous mixing.

2.4. Enzymatic hydrolysis of the regenerated cotton cellulose

Wet cotton cellulose regenerated from 1 g cotton cloth wasplaced in 20 mL of 50 mM citrate buffer (pH 5.0) containing3.3 U/mL cellulase and incubated at 50 �C with shaking at80 rpm. The reaction was monitored by withdrawal of samplesfrom the supernatant and measuring the amount of soluble reduc-ing sugars by using an assay based on 3,5-dinitrosalicylic acid(DNS) with D-glucose as the standard. The yield of reducing sugarsfrom regenerated cotton cloth was calculated as follows:

Sugar yield ð%Þ ¼ Reducing sugar weightCotton cloth weight

� 100

2.5. Effect of [AMIM]Cl on enzymatic hydrolysis

Hydrolysis was performed at 60 �C and pH 5.0 for 10 min in areaction mixture of 1 mL containing 0.02, 0.03, 0.05, 0.1 or 0.2 gof IL. Then reducing sugar was determined by using the DNS meth-od. The hydrolytic reaction mixture consisted of 2.5 U cellulase, 1%(w/w) CMC, and 50 mM citrate buffer at pH 5.0. A sample withoutany IL was used as a control.

2.6. Effect of [AMIM]Cl on BC production

G. xylinus was cultured in medium consisting of (w/v) 2.5% glu-cose, 0.5% yeast extract, and 0.3% peptone. The initial pH of themedium was 5.0 (Hong and Qiu, 2008; Hong et al., 2011). Additionsof 0.005, 0.01, 0.05, 0.1 or 0.5 g of [AMIM]Cl to 100 mL of the glu-cose-based culture medium were made to investigate the effects ofthe presence of IL. The cultures were placed at 30 �C in a staticincubator for 7–14 days. After cultivation, the BC pellicle was col-lected by filtration in G3 crucibles (pore size 16–30 lm, HeqiChemical and Science Co. Ltd., Shanghai, China) and dried at105 �C for 24 h. Each culture was performed in duplicate and meanvalues of the yield of BC are given.

F. Hong et al. / Bioresource Technology 104 (2012) 503–508 505

2.7. Bacterial cellulose production in enzymatic hydrolysate ofregenerated cotton cellulose

The seed culture medium contained (w/v) 0.3% peptone, 0.5%yeast extract, and 2.5% glucose. In the production culture medium,cotton cloth hydrolysate with a reducing sugar concentration of17 g/L replaced glucose as the carbon source. Cultures with 17 g/Lglucose were used as controls. After static cultivation at 30 �C for7–14 days, the BC pellicle was collected by filtration. After thoroughwashing with deionized water, the weight of the cellulose pelliclewas determined after drying at 105 ± 0.5 �C for 24 h. The result isreported as the dry weight of the BC pellicle.

Before tensile strength measurement, the bacterial cellulosepellicle was soaked in 1% NaOH and heated at 80 �C for 120 minto remove impurities such as culture medium and trapped bacte-rial cells. The bacterial cellulose pellicle was cut into 40 mm longand 10 mm wide strips for analysis of tensile strength. The tensilestrength of wet BC was measured by using a universal testing ma-chine (H5K-S, Hounsfield Test Equipment Ltd., UK) operating at acrosshead speed of 50 mm/min. All data for determination of ten-sile strength were collected under the same conditions. The tensilestrength (in megapascal, MPa, or N/mm2) was calculated by divid-ing the tensile force by the area of the cross section of the BC strips.Each test was performed by using 10 samples and mean values ofthe yield of BC are given.

100

3. Results and discussion

3.1. Influence of dissolution temperature and time on IL pretreatment

As seen in Fig. 1, the dissolution of cotton in [AMIM]Cl increasedwith temperature and time; however, the yield of regenerated cot-ton cloth obtained after the 130 �C pretreatment was relativelylow. The weight of regenerated cotton obtained from 2.4 g of cot-ton cloth dissolved at 130 �C was almost equal to that obtainedfrom 2 g cotton cloth dissolved at 110 �C (about 1.86 g). This canbe attributed to the destruction of cotton fiber occurring at highertemperatures during the dissolution process. During the pretreat-ment at 130 �C, the color of the cotton/IL solution became darkeras the incubation time increased. It was concluded that underthe conditions used the pretreatment temperature should be re-stricted to 110 �C in order to reduce the damage to the cotton cloth.

The largest amount of dissolved cotton at 90 �C was 1.2 g, whichrequired 470 min. The time periods required dissolving 1.2 g at110 �C and 130 �C were 96 and 27 min, respectively. It is suggestedthat dissolution of cellulose at 90 �C takes much longer time. The

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Dissolution time (min)

Cot

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clot

h w

eigh

t (g)

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Fig. 1. Influence of the dissolution temperature on the solubility of cotton cloth in[AMIM]Cl. The data are based on duplicate tests.

maximal amounts dissolved at 110 �C and 130 �C was 2 and2.4 g, respectively. Thus, the amount dissolved at 130 �C was 2-foldlarger than the amount dissolved at 90 �C.

In order to investigate if the dissolved cellulose was damaged at110 �C due to carbonization at high temperature and prolongeddissolution time, the yield of reducing sugar obtained from enzy-matic hydrolysis of 1 g of regenerated cellulose pretreated for dif-ferent periods of time was determined. The results showed that thehighest sugar yield was obtained when 1 g of cotton cloth was dis-solved for 90 min (Fig. 2). At that time point, the cotton cloth hadjust been dissolved (Fig. 1) and as judged from the yield of reducingsugar there was still no great loss. The low sugar yields obtainedwhen the dissolution time was less than 90 min (Fig. 2) can beattributed to incomplete treatment in IL. The current results dem-onstrate that the dissolving temperature and the period of time arecrucial for the yield. Higher temperature and longer reaction timewould result in more sugar loss. Since dissolution of cotton at130 �C would result in loss of more cellulose and since dissolutionat 90 �C would take too long time, treatment at 110 �C for 90 minwas selected for further experiments.

3.2. Comparison of the weight of regenerated cellulose afterpretreatment

Fig. 3 shows that the weight of the regenerated cellulose ob-tained from initial amounts of cotton cloth ranging from 0.5 to1.0 g was larger than that of the added cotton cloth which is indic-ative of residual IL (Zhao et al., 2009). The weight of the regener-ated cellulose was lower (14% loss) than that of the originalcotton when 1.2 g cotton cloth was dissolved at 110 �C (Sample 4in Fig. 3) perhaps due to damage to the cotton fibers during thetime required to dissolve this amount of material. Hence, in orderto obtain a high yield after the treatment at 110 �C, 1.0 g was se-lected as the most suitable amount of cotton cloth to add.

Even though the cellulose precipitate was washed with deion-ized water 5 times, IL may still have remained entrapped in the cel-lulose. The result indicates that the washing process used could notremove all of the IL from the regenerated cellulose. Zhao et al.(2009) found that chloride ions (0.08 M) were detected in the glu-cose solution after complete hydrolysis of cellulose regeneratedfrom [BMIM]Cl treatment. This indicates that residual [BMIM]Clwas tightly bound to the cellulose despite thorough washing afterthe regeneration. The entrapment of IL could be a consequence of

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Dissolution time (min)

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ucin

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Fig. 2. Influence of the dissolution time on the sugar yield after 48 h of hydrolysis ofregenerated cotton cloth after pretreatment in [AMIM]Cl at 110 �C.

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Fig. 3. Effect of IL pretreatment at 110 �C on the weight of the regeneratedcellulose. The data are based on duplicate tests.

506 F. Hong et al. / Bioresource Technology 104 (2012) 503–508

the precipitation of cellulose during rapid regeneration, or could bedue to strong hydrogen bonding between cellulose and IL mole-cules (Zhao et al., 2009).

3.3. Enzymatic hydrolysis of regenerated cotton cellulose

Fig. 4 shows the yield of reducing sugar from the original cottoncloth and after pretreatment with [AMIM]Cl at 110 �C for 90 min.After 4 h, the yields of reducing sugar from pretreated and un-treated cotton cloth were 22.4% and 4.0%, respectively. Afterhydrolysis for 24 h, the yield of reducing sugar from untreated cot-ton was only 12.1%, whereas that of pretreated cotton was 81.6%.This indicates that the pretreatment with [AMIM]Cl is a very effi-cient approach to increase the hydrolytic rate of cotton cloth.

Enzymatic saccharification of cellulose to glucose suffers fromslow reaction rates mainly due to the highly crystalline structureand high DP of cellulose and the inaccessibility of enzyme adsorp-tion sites (Zhang and Lynd, 2004; Dadi et al., 2006; Chandra et al.,2007). It has been reported that the crystallinity of cellulose coulddecrease after IL pretreatment and the cellulosic fibers attain a lar-ger surface area to become more exposed to cellulase and water(Dadi et al., 2006). The data from this study confirm that the regen-erated cellulose is less crystalline (CrystallinityXRD = 37.4%) thanthe untreated cotton (CrystallinityXRD = 86.3%, X-ray diffraction

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Original cloth Treated cloth

Yie

ld o

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ugar

(%

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Fig. 4. Enzymatic hydrolysis of regenerated cotton at 50 �C with 3.3 U/mL cellulase.

spectra not shown). The results from this study and those of otherresearchers (Kuo and Lee, 2009) show that the enzymatic sacchar-ification of cotton materials can be improved by pretreatment with[AMIM]Cl or [BMIM]Cl.

3.4. Effect of [AMIM]Cl concentrations on enzymatic hydrolysis

It is known that ILs can affect cellulase activity (Turner et al.,2003; Zhao et al., 2009), and the [AMIM]Cl is no exception sincethe hydrolytic efficiency of cellulase decreased at concentrationshigher than 0.01 g/mL (around 60 mM) (Fig. 5). When the concen-tration of IL was 0.05 g/mL (320 mM), around half of the activitywas lost. The IL concentration in pretreated waste cotton fabricsin a reaction volume of 20 mL is likely about 0.005 g/mL (32 mM)provided that 0.1 g of IL was entrapped in the regenerated cellulose(cf. Sample 2 in Fig. 3), and thus should not cause inhibition.

The cellulase preparation is a complex mixture of several en-zymes (including endo- and exo-cellulases, b-glucosidase, oxida-tive cellulases, etc.). Residual [AMIM]Cl might have differenteffect on different enzymes, resulting in varied corresponding su-gar concentrations in the hydrolysate. Further studies are neededto investigate the effect of residual IL on the inactivation of sepa-rate components in the cellulase preparation.

3.5. Effect of [AMIM]Cl on BC production

Fig. 6 demonstrates that BC production was affected if the con-centration of IL was 0.001 g/mL (6.3 mM) or higher and is thereforemore sensitive to [AMIM]Cl than enzymatic hydrolysis (Fig. 5).Washing the regenerated cellulose thoroughly in 60 �C water un-der vigorous agitation produced a substrate suitable for productionof bacterial cellulose. A concentration of 0.005 g/mL did not influ-ence the enzymatic hydrolysis (Fig. 5) but did affect BC production.The results strongly suggest that the regeneration and washingprocess must be performed thoroughly to remove residual[AMIM]Cl from the regenerated cellulose.

Zhao et al. (2009) tried to suspend the cellulose regeneratedfrom [BMIM]Cl in boiling water for 2 h to get rid of residual IL.However, no improvement was observed. Kuo et al. (2010) didnot give any information about whether the enzymatic hydrolysateof the regenerated cellulose from [BMIM]Cl contained any residualIL or if it was useful to produce BC, which was instead producedfrom a hydrolysate of cellulose pretreated with 85% phosphoric

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Fig. 5. Effect of the concentration of [AMIM]Cl on cellulase hydrolysis.

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Fig. 6. Effect of the concentration of [AMIM]Cl on the BC production.

F. Hong et al. / Bioresource Technology 104 (2012) 503–508 507

acid. The results from this study demonstrate that hot water wash-ing is an effective method and that agitation is a key factor for re-moval of residual [AMIM]Cl to a level that is too low to affect BCproduction.

3.6. Production of bacterial cellulose in enzymatic hydrolysate

The properties of BC from cultures of cotton cloth hydrolysatewere investigated and are shown in Table 1. The water-holdingcapacity of BC produced in a glucose-based medium was almostthe same as that of BC produced in hydrolysate; however, thedry weight of BC produced in the hydrolysate was much higherthan that of BC produced in glucose-based medium. The tensilestrength of BC produced in glucose-based medium was0.039 MPa, while that of BC produced in hydrolysate was0.070 MPa, these properties are perhaps due to the high contentof nano-cellulose fibers (10.8 g/L) in the BC pellicle from the hydro-lysate medium. The BC would be able to form a more compact net-work than in the glucose medium, where the BC yield was only5.9 g/L. Higher yield but thinner thickness of the BC pellicle(1.48 mm) would lead to higher tensile strength.

An enhancing effect on the BC yield has been observed previ-ously. Higher BC yields (40–65%) were obtained in cotton enzymatichydrolysate cultures compared to that achieved with G. xylinus inglucose-based cultures (Kuo et al., 2010). It was proposed that theculture with cotton hydrolysate should have a higher carbon sourceconcentration than the glucose culture, since not only glucose butalso soluble cello-oligosaccharides would be present in the cottonhydrolysate. The amount of sugars in the cultures was based onreducing sugars determined by the DNS method. It is well-knownthat one soluble cello-oligosaccharide molecule has only one reduc-ing, as has a glucose molecule that is analyzed with the DNS method.Oligosaccharides may be utilized by G. xylinus. G. xylinus is reportedto have a CMC-hydrolyzing enzyme (CMCase; endo-1,4-b-glucan-ase) (Husemann and Werner, 1963) and the activity is reported tobe found during growth (Oikawa et al., 1997; Koo et al., 1998; Kaw-ano et al., 2002). Two different genes encoding endo-1,4-b-glucanas-es have been reported by two groups and the amino-acid sequencesencoded by these genes are different in the two strains (Standal et al.,1994; Okamoto et al., 1994). The endo-1,4-glucanase could hydro-

Table 1Characterization of BC prepared in a hydrolysate of waste cotton fabric.

Culture medium Glucose Hydrolysate

Dry weight of BC (g/L) 5.9 ± 0.5 10.8 ± 0.4Water-holding capacity of BC (%) 99.4 ± 0.1 98.8 ± 0.3Thickness of BC strip (mm) 1.63 ± 0.01 1.48 ± 0.09Tensile strength (MPa) 0.039 ± 0.015 0.070 ± 0.011

lyze water-soluble cellulose such as CMC, hydroxyethyl celluloseand cellodextrin. It hydrolyzes cellohexaose to cellobiose, cellotrioseand cellotetraose, and also cellopentaose to cellobiose and cellotri-ose, but does not act on cellobiose, cellotriose, or cellotetraose(Husemann and Werner, 1963). But when bacterial cellulose wasadded into a reaction mixture containing cellohexaose, the hydroly-sis products disappeared in the reaction mixture. It is suggested thatendo-1,4-glucanase might have transglycosyl activity and that thisactivity might be closely related to cellulose synthesis (Amanoet al., 2005). Therefore, soluble cello-oligosaccharides would behydrolyzed into oligosaccharides with low DP during the growthofG. xylinus, and then they might enhance bacterial cellulose biosyn-thesis by transglycosylation. It is interesting that bacterial cellulosecould not be degraded by its own cellulase after ribbon formation,which is crystalline cellulose. These properties are different fromthose of endo-glucanases from fungi (Amano et al., 2005).

The results that BC from cotton cloth hydrolysate was ob-tained in higher yields and had higher tensile strength than BCfrom conventional culture medium suggest that waste cotton pre-treated with [AMIM]Cl has potential to serve as a high-qualitycarbon source for BC production. The key factor influencing theapplication of the IL-pretreatment technology is that the cost ofILs is still very high. Therefore, it is necessary to make large ef-forts to decrease the production cost of ILs and to recycle ILs inindustrial processes. With respect to chemical synthesis of ILs,many chemists are investigating new approaches for low-costsynthesis of ILs. Further studies aiming at the recovery and reuseof ILs are underway.

4. Conclusions

Treatment of cotton cloth with [AMIM]Cl produced a substratefor the production of cellulose by G. xylinus provided that traces ofthe IL were removed after cellulose regeneration. The bacterial cel-lulose had desirable physical properties. For practical applicationof the IL-pretreatment technology, the cost of the ILs has to becomelower and recovery and reuse have to be improved.

Acknowledgements

The project was funded by the Science and Technology Com-mission of Shanghai Municipality (11230700600 and08520750200), the Shanghai Municipal Education Commission(09ZZ68), the 111 Project (B07024), the State Key Laboratory forModification of Chemical Fibers and Polymer Materials (DonghuaUniversity), Swedish Research Links Programme (Swedish Re-search Council, Project 348-2006-6705) and the Fundamental Re-search Funds for the Central Universities. All the financialsupports are gratefully acknowledged.

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