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Research ArticleReceived: 26 August 2010 Revised: 30 November 2010 Accepted: 30 November 2010 Published online in Wiley Online Library: 6 January 2011

(wileyonlinelibrary.com) DOI 10.1002/jctb.2567

Wheat straw acid hydrolysate as a potentialcost-effective feedstock for productionof bacterial celluloseFeng Hong,a,b∗ Ying Xue Zhu,a Guang Yanga and Xue Xia Yanga

Abstract

BACKGROUND: Bacterial cellulose (BC) is an extracellular biopolymer product of vinegar bacteria, which is widely used in manyareas. However, problems of high production cost have prevented widescale extension of BC applications. In this work, BC wasproduced using wheat straw hydrolysates prepared by dilute acid hydrolysis instead of the usual carbon sources, with the aimof decreasing the production costs of BC.

RESULTS: In order to remove microbial growth inhibitors, wheat straw hydrolysates were detoxified by treatment with variousalkalis including calcium hydroxide, sodium hydroxide and ammonia, and their combination with activated charcoal or laccase.Results showed that the detoxification effect using calcium hydroxide was much better than that with the other alkalis. The BCyield using hydrolysate treated with Ca(OH)2 and activated charcoal was at least 50% higher than that using routine carbonsources. Additionally, the ions of Ca2+ and Na+ in the hydrolysates had important and positive effects on BC production whileCl− exhibited negative effects.

CONCLUSION: Wheat straw was shown to be a suitable feedstock for BC production, and a process was established for BCproduction from lignocellulosic feedstocks using a detoxification treatment.c© 2011 Society of Chemical Industry

Keywords: bacterial cellulose; Acetobacter aceti subsp. xylinus; wheat straw; hydrolysate; detoxification

INTRODUCTIONBacterial cellulose (BC) synthesized by the acetic acid bacteriumAcetobacter aceti subsp. xylinus (formerly A. xylinum) is a valuableproduct having excellent properties such as transparency, tensilestrength, water-binding capability, adaptability to the livingbody, and biodegradability. This kind of material has prospectiveapplications in many fields incuding biomedical materials, foods,fuel cells, high-quality audio membranes and functional papersincluding electronic paper.1 – 3 However, problems with the highcosts of culture media and relatively low-yield strain have, todate, prevented widescale takeup of BCs for industrial productionand extended applications.4 Therefore it is important to developmethods to produce BC at the lowest cost possible.5 In orderto resolve these problems, especially the costs of feedstock,other kinds of low-cost carbon sources need to be developed.In recent years, some relatively cheap agricultural products orwaste, including food process effluents,6 hemicelluloses in wasteliquor from atmospheric acetic acid pulping,7 molasses,8, konjakglucomannan,9 fruit juices10 and rice bark11 as well as wastecellulosic fabrics12 have been developed as alternative low-cost feedstocks for BC production and would simultaneouslybe capable of reducing environmental problems. The use ofagricultural residues such as wheat straw is another interestingalternative.

Owing to the requirement for environmentally sustainableenergy sources, wheat straw has been hydrolyzed to sugarsby dilute acid at high temperature and used for bioethanol

production as the fermenting carbon sources, which might also beconsidered a good carbon source to biosynthesize BC. However,a problem associated with efficient conversion of cellulose andhemicellulose to sugars is that a broad range of compoundsthat inhibit the fermenting microorganism are liberated orformed during dilute acid hydrolysis. Inhibitory compounds inlignocellulose hydrolysates include phenolic compounds, furanderivatives such as furfural and 5-hydroxymethyl-furfural (5-HMF),as well as aliphatic acids such as acetic acid, formic acid, andlevulinic acid.13 – 15 Detoxification treatments to remove microbialgrowth inhibitors with Ca(OH)2, NaOH and NH4OH are wellestablished methods to improve the fermentability of hydrolysatesfor ethanol production. Pretreatment with Ca(OH)2 is one of themost efficient detoxification methods and has been the focus ofseveral studies.15 – 18 Adsorption by activated charcoal has alsobeen widely used to remove toxic compounds from lignocellulose

∗ Correspondenceto:Feng Hong,CollegeofChemistry,ChemicalEngineeringandBiotechnology, Donghua University, North Ren Min Road, No.2999, Songjiang,Shanghai 201620, China. E-mail: fhong@dhu.edu.cn

a Group of Microbiological Engineering and Industrial Biotechnology, Collegeof Chemistry, Chemical Engineering and Biotechnology, Donghua University,Shanghai 201620, China

b State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,Donghua University, Shanghai 201620, China

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hydrolysates.15 A method to use hemicellulose acid hydrolysatesfrom wheat straw as a feedstock for the production of bacterialcellulose has been successfully demonstrated.19

In this research, in order to develop a low-cost feedstockfor BC production from whole wheat straw, hydrolysates wereprepared using a two-stage dilute acid hydrolytic technology andsubsequently detoxified by different alkaline treatment methodsto remove microbial growth inhibitors. In this way, wheat strawhydrolysates could be developed as a suitable carbon source,which would be helpful in decreasing the cost of BC production,especially industrial scale.

MATERIALS AND METHODSChemicalsUnless otherwise stated, all chemicals used in the preparationof culture media were obtained from Sinopharm ChemicalReagent Co. Ltd. (Shanghai, China). Distilled water was used inall experiments. Activated charcoal (analytical grade, productNo.10006619), sodium hydroxide and calcium hydroxide werepurchased from Sinopharm Chemical Reagent Co. Ltd. Ammoniasolution (30%) was obtained from Shanghai Boer ChemicalReagent Co. Ltd. Laccase from Trametes versicolor (product no.53 739) was obtained from Sigma–Aldrich (Steinheim, Germany).One unit of laccase activity corresponds to the amount ofenzyme which forms 1 µmole radical cation of 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid per minute at pH 5.2 and30 ◦C.

Microorganism, culture media and growth conditionsThe microorganism, Acetobacter aceti subsp. xylinus (formerlyGluconacetobacter xylinus or A. xylinum) ATCC 23 770 (obtainedfrom American Type Culture Collection, Manassas, VA.) was usedas a model strain and maintained on agar plates containing aseed culture medium. The culture medium contained (w/v) 2.5%D-mannitol, 0.5% yeast extract, 0.3% peptone (Oxoid, UK) and2.5% agar at initial pH 5.0.20

Two loops of the strain were inoculated into a conical flaskcontaining the seed culture medium (without agar) detailed above.The seed culture was incubated at 30 ◦C and 160 rpm on a rotaryshaker for 1 day and the pH value of the medium was not regulatedduring cultivation.9 The seed culture was then inoculated into a50 mL liquid production medium of 6 v/v% capacity, which wascultivated at 30 ◦C in a static incubator for 11 days. The liquidproduction medium used for BC production consisted of 0.5 w/v%yeast extract, 0.3 w/v% peptone (Oxoid, UK) and wheat strawhydrolysate as sole carbon source, in which the concentration oftotal reducing sugars was diluted to 2.5 w/v%. For comparison,the wheat straw hydrolysate was simply replaced by glucose,sucrose or mannitol of the same concentration (2.5 w/v%) in othertests. Each culture with different carbon sources was performed induplicate and a mean value of BC yield is given.

Hydrolysis of wheat strawThe wheat straw was obtained from farmland in Wuxi, China. A two-step dilute acid hydrolytic technology was applied to hydrolyze thewheat straw following an earlier method18 with some modification.First, 10 g of the chipped and ground wheat straw (40–60 mesh)was impregnated with 60 mL of 1.5% (w/v) sulfuric acid in a batchreactor overnight. Then the mixture was treated at 190 ◦C and12 × 105 Pa for 10 min and the hydrolysate collected by filtration.

Second, the residual solid fraction was re-impregnated with 30 mLof 3% (w/v) sulfuric acid and was hydrolyzed for 10–20 min at210 ◦C and 19 × 105 Pa. After filtration, the liquid fractions fromthe two-step treatment were mixed as the hydrolysates. To collectenough hydrolysates for later experiments the hydrolytic processwas carried out several times and the hydrolysates obtained fromeach hydrolytic process were pooled together to be referred to asthe final hydrolysates. The concentration of total reducing sugar inthe wheat straw hydrolysate was assayed by a 3,5-dinitrosalicylicacid method21 and found to be about 40 mg mL−1.

Detoxification of wheat straw hydrolysatesDifferent types of alkaline treatments were demonstrated toremove efficiently some inhibitors in konjak glucomannan acidhydrolysates.9 In the study, alkaline treatments supplemented witha new ammonia treatment17 were investigated to get rid of specificinhibitors in the hydrolysates and details of the detoxificationprocedures were as follows:

1. A1: First, adjusting the pH value of the hydrolysate to 10.0by NaOH and incubating it at 30 ◦C for 12 h followed byadjusting the pH value to 5.0 by addition of 5 mol L−1 H2SO4

again. Second, adding 2% (w/v) activated charcoal into thehydrolysate and mixing them for 5 min. Finally, removing theactivated charcoal from the hydrolysate and adjusting the pHvalue to 5.0 by addition of 0.1 mol L−1 H2SO4 or 0.1 mol L−1

NaOH again.2. B1: First, adjusting the pH value of the hydrolysate to 10.0

by Ca(OH)2 and incubating at 30 ◦C for 12 h followed byadjusting pH value to 5.0 by addition of 5 mol L−1 H2SO4

again. Second, adding 2% (w/v) activated charcoal into thehydrolysate and mixing them for 5 min. Finally, removing theactivated charcoal from the hydrolysate and adjusting the pHvalue to 5.0 by addition of 0.1 mol L−1 H2SO4 or 0.1 mol L−1

NaOH again.3. C1: First, adjusting the pH value of the hydrolysate to 10.0

by ammonia and incubating at 30 ◦C for 12 h followed byadjusting the pH value to 5.0 by addition of 5 mol L−1 H2SO4

again. Second, adding 2% (w/v) activated charcoal into thehydrolysate and mixing them for 5 min. Finally, removing theactivated charcoal from the hydrolysate and adjusting the pHvalue to 5.0 by addition of 0.1 mol L−1 H2SO4 or 0.1 mol L−1

NaOH again.4. A2: First, adjusting the pH value of the hydrolysate to 5.0 by

NaOH and then adding 10% (v/v) laccase (2.75 U mL−1) tothe hydrolysate. Second, incubating the hydrolysate at 30 ◦Cfor 12 h and adjusting the pH value to 5.0 by addition of0.1 mol L−1 H2SO4 or 0.1 mol L−1 NaOH again.

5. B2: First, adjusting the pH value of the hydrolysate to 5.0 byCa(OH)2 and then adding 10% (v/v) laccase (2.75 U mL−1) tothe hydrolysate. Second, incubating the hydrolysate at 30 ◦Cfor 12 h and adjusting the pH value to 5.0 by addition of0.1 Mol L−1 H2SO4 or 0.1 mol L−1 NaOH again.

6. C2: First, adjusting the pH value of the hydrolysate to 5.0 byammonia and then adding 10% (v/v) laccase (2.75 U mL−1) tothe hydrolysate. Second, incubating the hydrolysate at 30 ◦Cfor 12 h and adjusting the pH value to 5.0 by addition of0.1 mol L−1 H2SO4 or 0.1 mol L−1 NaOH again.

Element analysis of wheat straw hydrolysatesThe concentrations of Na+, Ca2+, Fe3+, Ni2+, Cu2+ and Cr6+in wheat straw hydrolysates treated by different detoxification

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methods were assayed using an element analysis instrument (VarioELO, Elmentar, Germany). Samples were prepared by addition of5% nitric acid prior to determination. Two tests were carried outand mean values are given.

Harvest and weighing of BCAfter cultivation, bacterial cellulose membrane was collected byfiltration in G3 crucibles (pore size 16–30 µm, Heqi Chemical andScience Co. Ltd, Shanghai, China) and dried to constant weight at105 ◦C using the standard process often used in analytic chemistry.Subsequently, the mass of BC was weighed and the BC yieldcalculated.

RESULTS AND DISCUSSIONDetoxification of wheat straw hydrolysates and productionof BCIn preliminary studies it was found that lignocellulose hy-drolysates contained many compounds toxic to microbialgrowth, such as 2-furaldehyde, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzaldehyde, phenol, ferulic acid, cinnamic acid and5-hydroxymethyl-2-furaldehyde (HMF) (data not shown), as previ-ously reported by Persson et al.16 The growth of the bacteria usedin the study was inhibited and no BC was formed in non-detoxifiedhydrolysate. Therefore, it is necessary to detoxify the hydrolysatebefore utilization. In this work, different detoxification methodswere investigated to remove microbial growth inhibitors, andtheir effects on the production of BC were compared with thoseof a regular carbon source such as glucose, sucrose and mannitol.Figure 1 clearly shows that the Ca(OH)2 treated hydrolysates, i.e.B1 treated hydrolysate, enabled the highest production of BC,with a value of 15.4 mg mL−1, followed by A1, B2, A2, C1 andC2. Based on the same concentration of total reducing sugars ascarbon source in static cultures, the yield of BC produced usinghydrolysate B1 was 70%, 65% and 50% higher than that usingglucose, sucrose and mannitol, respectively, as carbon source.

Dahman et al.22 investigated the production of bacterialcellulose by using pure single sugars and sugar mixtures withthe components simulating the sugar compositions in theacid hydrolysates of some agriculture residues, and gave someinteresting results on the sugar metabolism of G. xylinus. Theyfound that the highest BC yield of 17.7 g g−1 sugars was achievedin sugar mixtures with composition similar to that in wheat strawacid hydrolysates, while the yield obtained in single fructose sugar

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Figure 1. Effects on BC yield of different types of hydrolysates detoxifiedby various methods. The growth of bacteria used in the study was inhibitedand no BC was produced in a non-detoxified hydrolysate. Two trials werecarried out and mean values are given. The error bars show the absolutedeviations.

medium was only 14.8 g g−1 fructose. The single fructose mediumgave the highest volumetric BC yield of 5.7 g L−1 among all sugarmedia examined. Large amounts of the metabolized sugars wereused mainly for bacterial cell growth and maintenance, ratherthan BC production. This was clearly observed with single sugarsgiving low BC production, while sugar consumption was usedfor BC production with sugar mixtures. This can be attributedto the possibility that the higher sugar concentration in singlesugar media may have caused catabolite repression, not presentwith sugar mixtures.22 Because the authentic acid hydrolysates ofwheat straws also contain multiple sugar components, the highestyield of BC in B1-treated hydrolysate may have benefited from thesugar composition.

Besides saccharides, however, agricultural lignocellulosicresidues contain other complex components including lignin,ash, organic extractives and protein, whose hydrolytic productswould have a very complicated effect on bacterial growth and cel-lulose synthesis in cells. No possible approach has been proposedthus far to see how to use real agricultural residues as feedstocksfor BC production. It is also noteworthy that the study on sugarmetabolism in sugar mixtures was made in ‘shaking’ cultivationwhile this current study was in static cultivations. This differencewould affect results and should be investigated further in future.

The detoxification with activated charcoal (techniques A1,B1 and C1) had more marked effects on BC production thanthat with laccase (techniques A2, B2 and C2, as seen in Fig. 1).Jonsson et al.23 showed that wood hydrolysates detoxified withlaccase from the white-rot fungus Trametes versicolor promotedan increase in glucose consumption and ethanol productivity byyeast, probably because laccase involves oxidative polymerizationof low-molecular-weight phenolic compounds present in thehydrolysate. Activated charcoal exhibits a stronger detoxificationcapability since more toxic compounds may be adsorbed onactivated charcoal. The adsorption effectiveness of activatedcharcoal depends mainly on the variables used for the adsorptionprocess: pH, temperature, contact time, and activated charcoalconcentration.15 Gong et al.24 showed that most toxic compoundswere removed by activated charcoal. The combination of pHadjustment and activated charcoal adsorption was especiallygood for removing lignin derivatives, but charcoal alone removed95.4% of these compounds.25 Weak organic acids are most readilyadsorbed in the non-ionized state and consequently a low pH(acids) favors adsorption. For example, phenols are weak acids,and at low pH, the neutral or non-ionized phenolic molecules arehighly adsorbed.15 Since activated charcoal is a low-cost materialwith a high capacity to adsorb compounds, its application wouldbe suitable at industrial scale.

Element analysis of wheat straw hydrolysatesSome studies have shown that the concentration of metal ionwas the key factor for the yield of bacterial cellulose.26,27 Herein,the ion concentrations of detoxified hydrolysates by methods A1and B1 and that without detoxification (termed the ‘control’) weredetermined, respectively, and the results are shown in Table 1.It was found that the concentrations of Fe3+, Ni2+ and Cu2+ inthe hydrolysates after detoxification were lower than those in thecontrol sample. The concentrations of Ca2+ in sample B1 and Na+in sample A1 were dramatically increased due to the extra additionof Ca(OH)2 and NaOH during the detoxification. The appearance ofion of Cr6+ is probably from an impurity in the added alkalis sinceit did not appear in the control. As the concentration of Fe3+, Ni2+,Cu2+ and Cr6+ in the detoxified hydrolysates was much lower than

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Figure 2. Effects of Na2SO4 concentration on BC yield. Two trials werecarried out and mean values are given. The error bars show the absolutedeviations.

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Figure 3. Effects of NaCl concentration on BC yield. Two trials were carriedout and mean values are given. The error bars show the absolute deviations.

the concentration of Ca2+ and Na+ , the effects of Ca2+ and Na+from the added alkalis on BC production were studied in furtherexperiments.

Effects of the concentrations of Ca2+ and Na+ on BC productionThe effects of the concentrations of Ca2+ and Na+ on BC productionwere investigated by adding Na2SO4, NaCl and CaCl2, respectively,into the culture medium using glucose as sole carbon source,and the results are shown in Figs 2–4. Measurements in eachculture supplemented with salts of different concentration wereperformed in duplicate, and the mean values of BC yield are given.

As shown in Fig. 2, much higher BC yield was obtainedby the extra addition of Na2SO4, compared with that for noaddition. When the concentration of Na2SO4 was 10 mmol L−1, i.e.20 mmol L−1 of Na+ ion concentration, the highest BC yield wasobtained, at 11.9 mg mL−1. However, adding extra NaCl into themedium brought about different influences on BC yield. In Fig. 3,compared with BC yield without NaCl addition, it was improved forNaCl concentrations in the range 1–20 mmol L−1, but decreasedclearly at higher concentrations. The highest yield of BC was9.0 mg mL−1 when the concentration of NaCl was 5 mmol L−1,which was the same as for the Na+ ion. The data in Figs 2 and 3indicate that the Na+ ion indeed had a strong influence on theyield of BC, and so did the anions. Additionally, excessively highconcentrations of chloride anion, e.g. 50 and 100 mmol L−1 of

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Figure 4. Effects of CaCl2 concentration on BC yield. Two trials were carriedout and mean values are given. The error bars show the absolute deviations.

NaCl addition, had an obvious negative effect on BC production;nevertheless, sulphate anion did not affect BC yield negativelyeven at a concentration of 100 mmol L−1. Figure 3 also shows ahigh error at 20 mmol L−1 NaCl. This might be due to a largeexperimental error when the experiment was performed only induplicate since the BC harvest was not so large.

Figure 4 shows that Ca2+ cation considerably improved BC yieldunder certain concentrations (≤7 mmol L−1) and gave the highestyield (8.5 mg mL−1) at 7 mmol L−1. BC production was restrainedwhen the concentration of CaCl2 was more than 10 mmol L−1

(10–100 mmol L−1).The BC yield of pellicles formed in the static cultures using

the Ca(OH)2-detoxified hydrolysates after 11 days was highest.The finding described here implies that the reason why BCproduction using the Ca(OH)2-treated hydrolysates was improvedshould be ascribed to the detoxification efficiency, multiple sugarcomplex metabolism (as discussed above) and the effects ofcalcium cation. The effect of calcium ion may be attributedto stimulation of the activity of cellulose synthase because thecalcium ion inhibits phosphodiesterase-A (PDE-A), a key regulatorof bacterial cellulose synthesis. PDE-A hydrolyzes the allostericactivator of the cellulose synthase cyclic bis(3′,5′)diguanylic acid(c-di-GMP) in the membrane of A. xylinum to the linear ineffectual5′-phosphoguanylyl-(3′,5′)-guanosine (pGpG).28,29

The concentration of Ca2+ in the B1 hydrolysate detoxified byaddition of Ca(OH)2 was 8.3 × 102 µg mL−1 (namely 21 mmol L−1,Table 1), which exceeded 7 mmol L−1, but did not inhibit thegeneration of BC. The BC yield of hydrolysate B1 was the highest(15.4 mg mL−1) among all the detoxified hydrolysates (Fig. 1). Thisis because the anion in the Ca(OH)2-treated hydrolysates is SO4

2−but not Cl− since the hydrolysis of wheat straw was performedusing sulfuric acid. Figures 2 and 3 show that Cl− had greaternegative effects on BC production than SO4

2−. Therefore thenegative effect of Cl− should be considered in the analysis ofFig. 4. It was found that the addition of CaSO4 up to 50 mmol L−1

in single sugar medium was able to enhance the generation ofBC (data not shown). Keeping in mind that the effect of Ca2+ andNa+ (<7 mmol L−1) on BC yield was roughly equal (Figs 3 and 4),detoxification technique B1 (had Ca2+) gave higher BC yield thanA1 (had Na+). This is because technique B1 brought more Ca2+into the hydrolysate. The concentration of Ca2+ (21 mmol L−1)in the B1-detoxified hydrolysate was around three times higherthan the total concentration of Ca2+ (1.5 mmol L−1) and Na+(4.8 mmol L−1, Table 1) in the A1-detoxified hydrolysate.

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Table 1. Element analysis of wheat straw hydrolysates

Element concentration (µg mL−1)

Wheat straw hydrolysates Ca2+ Na+ Fe3+ Cr6+ Ni2+ Cu2+

Control 1.5 × 102 2.4 × 101 1.9 × 101 0 8.9 × 10−2 2.7 × 10−1

A1 6.0 × 101 1.1 × 102 5.8 1.1 × 10−2 4.3 × 10−2 5.2 × 10−2

B1 8.3 × 102 2.9 × 101 1.5 × 101 1.5 × 10−1 8.0 × 10−2 2.4 × 10−2

CONCLUSIONSIn this research, six detoxification methods were used to treatthe acid hydrolysates of wheat straws for BC production. Theresults indicated that detoxification with Ca(OH)2 treatmentworked better than treatment with NaOH and NH4OH. The BCyield with the Ca(OH)2-detoxified hydrolysates was much betterthan the yields with several conventional carbon sources. The BCyield obtained (15.4 mg mL−1) was much higher than that usingglucose (70% higher), sucrose (65% higher) and mannitol (50%higher). Therefore, treatment of wheat straw hydrolysates withCa(OH)2 gave a satisfactory detoxification effect. The additionof extra calcium and sodium in a glucose medium in the formof CaCl2 (1–7 mmol L−1), Na2SO4 (1–100 mmol L−1) and NaCl(1–20 mmol L−1) at initial pH 5.0 resulted in an improvement ofBC production. The mechanisms behind the improvement of BCyield is not only related to efficient detoxification but also toother factors such as concentration of metal ions, multiple sugarcomplex metabolism, different alkaline treatments, and formationof possible stimulatory factors in the alkaline-treated hydrolysates.Study of the complicated mechanisms behind these findingsto determine, if possible, stimulatory factors for the fermentingbacterium formed during alkaline processing as well as analysisof the sugar composition in acid hydrolysates before and afterfermentation are of great interest.

Whole wheat straw was successfully developed as a newfeedstock for BC production in the study. The cost of the feedstockis relatively low since wheat straw is a cheap, abundant resourcethroughout the world. Thus, this new carbon source is promisingfor mass industrial production of nano-structured BC materials. Inaddition, wheat straw normally causes heavy air pollution in Chinabecause most are burned after harvest. The proposed approachwould solve this environmental problem. This novel approachestablishes a strong and key foundation for utilization of otherrenewable agricultural residues including rice straw, corn stover,etc., as cost-effective feedstocks to produce BC materials at largescale.

ACKNOWLEDGMENTSThe project was funded by the Science and Technology Commis-sion of Shanghai Municipality (0852 nm03500 and 08520750200),the Shanghai Municipal Education Commission (09ZZ68), the 111Project (B07024), the Key Laboratory of Science & Technologyof Eco-Textile (Donghua University), Ministry of Education, China,and the Fundamental Research Funds for the Central Universities.All the financial support is gratefully acknowledged.

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