Biosynthesis of Bacterial Cellulose/Carboxylic ... - Semantic Scholar

materials Article

Biosynthesis of Bacterial Cellulose/Carboxylic Multi-Walled Carbon Nanotubes for Enzymatic Biofuel Cell Application Pengfei Lv 1 , Quan Feng 2 , Qingqing Wang 1 , Guohui Li 1 , Dawei Li 1 and Qufu Wei 1, * 1

2

*

Key Laboratory of Eco-textiles, Jiangnan University, Wuxi 214122, Jiangsu, China; [email protected] (P.L.); [email protected] (Q.W.); [email protected] (G.L.); [email protected] (D.L.) Key Laboratory of Textile Fabric, Anhui Polytechnic University, Wuhu 241000, Anhui, China; [email protected] Correspondence: [email protected]; Tel.: +86-137-7110-6262; Fax: +86-510-8591-3100

Academic Editor: Juergen Stampfl Received: 26 January 2016; Accepted: 7 March 2016; Published: 9 March 2016

Abstract: Novel nanocomposites comprised of bacterial cellulose (BC) with carboxylic multi-walled carbon nanotubes (c-MWCNTs) incorporated into the BC matrix were prepared through a simple method of biosynthesis. The biocathode and bioanode for the enzyme biological fuel cell (EBFC) were prepared using BC/c-MWCNTs composite injected by laccase (Lac) and glucose oxidase (GOD) with the aid of glutaraldehyde (GA) crosslinking. Biosynthesis of BC/c-MWCNTs composite was characterized by digital photos, scanning electron microscope (SEM), and Fourier Transform Infrared (FTIR). The experimental results indicated the successful incorporation of c-MWCNTs into the BC. The electrochemical and biofuel performance were evaluated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The power density and current density of EBFCs were recorded at 32.98 µW/cm3 and 0.29 mA/cm3 , respectively. Additionally, the EBFCs also showed acceptable stability. Preliminary tests on double cells indicated that renewable BC have great potential in the application field of EBFCs. Keywords: bacterial cellulose; carbon nanotubes; laccase; glucose oxidase; enzyme biological fuel cell

1. Introduction More attention has been increasingly paid to eco-friendly materials because of an increased awareness of sustainable development and environmental protection [1]. Nowadays, enzymatic biofuel cells (EBFCs) as the new green energy devices have drawn much attention because they are capable of harvesting electricity from renewable and abundantly available sources by using enzymes as the catalysts for oxidation of biofuels (most commonly, glucose) and reduction of oxidizers (most commonly, oxygen) [2,3]. They are renewable energy without any harmful intermediates and side products. Most EBFCs cathodes involve the four-electron reduction of O2 to water [4]. O2 ` 4H+ ` 4e´ Ñ 2H2 O

(1)

Due to the fact that active centers of the enzymes are usually buried inside the protein matrix, electron transfer to the electrodes has been of crucial importance for EBFC performance [5]. Many types of carbon nanotubes (CNTs) have, therefore, been widely used as conductive nanowires in facilitating electron transfer from the catalytic centers of enzymes to the electrode surface because of their unique properties of being chemically inert with excellent conductivity, their electro-chemical stability, and molecular dimensions that enable intimate interaction with the enzymes [6–8]. These unique properties Materials 2016, 9, 183; doi:10.3390/ma9030183

www.mdpi.com/journal/materials

Materials 2016, 9, 183

2 of 10


of CNTs make them extremely attractive for electrochemical applications, protein electrochemistry, electrochemical sensors, and especially for biosensors or biofuel In the bioelectronics field,In CNTs electrochemistry, electrochemical sensors, and especially for[9,10]. biosensors or biofuel [9,10]. the have been usedfield, as supports for enzyme immobilization to enable direct electron transfer because of bioelectronics CNTs have been used as supports for enzyme immobilization to enable direct their large specific surface area and good conductivity [11]. electron transfer because of their large specific surface area and good conductivity [11]. Bacterial synthesized by Acetobacter xylinum, is a green, and low-cost Bacterial cellulose cellulose(BC), (BC), synthesized by Acetobacter xylinum, is a economical, green, economical, and biopolymer [12]. As [12]. a general BC has distinctive propertiesproperties includingincluding high ultrafine low-cost biopolymer As a material, general material, BC has distinctive high porosity, web-like structure, crystallinity, water absorbance, mechanical ultrafine three-dimentional porosity, three-dimentional web-like high structure, high crystallinity, water absorbance, properties, biocompatibility, which makeswhich it versatile of application many fields mechanical and properties, and biocompatibility, makesinit terms versatile in terms of in application in such as paper and paper-based products, audio components, tissue engineering, food, and electronic many fields such as paper and paper-based products, audio components, tissue engineering, food, industries [13,14], as well[13,14], as stretchable conductors lithium [15], ion battery [16],anodes conductive and electronic industries as well as stretchable[15], conductors lithiumanodes ion battery [16], and fire-resistant aerogels [17]. Furthermore, the hydroxyl groups on its backbone can provide can BC conductive and fire-resistant aerogels [17]. Furthermore, the hydroxyl groups on its backbone with a high hydrophilicity, which is crucial for the operation of polymer electrolyte membrane fuel provide BC with a high hydrophilicity, which is crucial for the operation of polymer electrolyte cells [18]. However, methods are notmethods convenient apply becausetothey require additional time membrane fuel cells these [18]. However, these are to not convenient apply because they require consuming, energy-intensive, or expensive steps, such as pyrolization very high temperatures additional time consuming, energy-intensive, or expensive steps, such asatpyrolization at very high with the application of curing agents or chemical modification. Post-treatments have been carried temperatures with the application of curing agents or chemical modification. Post-treatments have out cellulose a composite of cellulose andofreduced for applications in beenoncarried outtoonprepare cellulose to preparefilm a composite film cellulosegraphene and reduced graphene for supercapacitors [19], and flexible and conductive films [20]. applications in supercapacitors [19], and flexible and conductive films [20]. In multi-walled carbon carbon nanotubes nanotubes (c-MWCNTs) (c-MWCNTs) in In this this work, work, biosynthesis biosynthesis of of BC/carboxylic BC/carboxylic multi-walled in agitated agitated culture culture were were used used as as both both bioanode bioanode and and biocathode biocathode in in EBFCs EBFCs (Figure (Figure 1). 1). The The biocathode biocathode and and bioanode bioanode were were prepared prepared and and the the BC/c-MWCNTs BC/c-MWCNTs were were injected injected with with laccase laccase (Lac) (Lac) and and glucose glucose oxidase oxidase (GOD) (GOD) by by glutaraldehyde glutaraldehyde (GA) (GA) crosslinking, crosslinking, respectively. respectively. This This new new membrane membrane electrode electrode assemblies assemblies (MEAs) (MEAs) method method offers offers theoretical theoretical and and technological technological supports supports for for exploiting exploitinghigh-efficient high-efficientEBFCs. EBFCs.

Figure 1. Illustration of the EBFC equipped with 3D BC/c-MWCNTs hybrid electrodes (not to scale).

2. Experimental 2. ExperimentalMaterials Materialsand andProcedures Procedures 2.1. Chemicals The industrial laccase powder (3 U/mg) from Trametes Trametes was purchased from Wuhan Nuohui U/mg) from Pharmaceutical And Chemical Co., (Wuhan, Hubei, Hubei, China); the industrial glucose oxidase Co., Ltd. Ltd. (Wuhan, (800 U/mg) Chemical Co.,Co., Ltd.Ltd. (Zhenzhou, Henan, China); the U/mg) was wasobtained obtainedHenan HenanHuakang Huakang Chemical (Zhenzhou, Henan, China); the c-MWCNTs 95%) was supplied by XFNANO Nanjing XFNANO c-MWCNTs (OD,(OD, 95%) was supplied by Nanjing Materials Materials Tech(Nanjing, Co., Ltd. Jiangsu, (Nanjing,China), Jiangsu, China), and glutaraldehyde was purchased from Henan Tech Co., Ltd. and glutaraldehyde was purchased from Henan Huakang Huakang Chemical Co., Ltd. (Zhenzhou, Henan, China). Chemical Co., Ltd. (Zhenzhou, Henan, China). 2.2. Preparation of 2.2. Preparation of BC/c-MWCNTs BC/c-MWCNTs Composite Composite First, dispersed in culture media containing 2.5% (w/v) D-mannitol, 0.5% (w/v) First, c-MWCNTs c-MWCNTswere were dispersed in culture media containing 2.5% (w/v) D-mannitol, ˝ C in an autoclave yeast, 0.3% (w/v) bacto-peptone [21]. These culture media were sterilized at 120 0.5% (w/v) yeast, 0.3% (w/v) bacto-peptone [21]. These culture media were sterilized at 120 °C in an for 2 h andfor poured into Erlenmeyer flasks. Theflasks. bacterium was cultured Hestrinon and Schramm autoclave 2 h and poured into Erlenmeyer The bacterium wason cultured Hestrin and

Schramm (HS) (5% (w/v) glucose, 1.6% (w/v) bacto-peptone, 0.2% (w/v) citric acid, 0.2% (w/v) disodium hydrogen phosphate, 0.3% (w/v) potassium dihydrogen phosphate, 0.03% (w/v) magnesium sulfate) medium [22] by static incubation. The pre-cultured cells in a test tube containing 2


3 of 10

(HS) (5% (w/v) glucose, 1.6% (w/v) bacto-peptone, 0.2% (w/v) citric acid, 0.2% (w/v) disodium hydrogen phosphate, 0.3% (w/v) potassium dihydrogen phosphate, 0.03% (w/v) magnesium sulfate) Materials 2016, 9, 183 medium [22] by static incubation. The pre-cultured cells in a test tube containing small cellulose particles on the particles surface ofonthe into ainoculated 100 mL Erlenmeyer containing small cellulose themedium surface were of theinoculated medium were into a 100 flask mL Erlenmeyer 10 mL of the HS medium (in the presence of final 0.01 w/v% c-MWCNTs in culture media). The flask containing 10 mL of the HS medium (in the presence of final 0.01 w/v% c-MWCNTs in culture flasks were onincubated a rotary shaker operating at rotational of 100 rpm, forof6100 days at 30 media). Theincubated flasks were on a rotary shaker operatingspeed at rotational speed rpm, for˝ C. 6 The cellulose was separated the medium by the filtration andby were dipped into 1% dayssynthesized at 30 °C. The synthesized cellulose from was separated from medium filtration and were sodium solution for 2 h at solution 80 ˝ C in order eliminate theorder cells and medium embedded in dipped hydroxide into 1% sodium hydroxide for 2 to h at 80 °C in to eliminate the cells and the cellulose material, rinsed three timesthen to pH 7 in deionized water [23]. medium embedded inthen the cellulose material, rinsed three times to pH 7 in deionized water [23]. 2.3. Preparation of 2.3. Preparation of Enzyme Enzyme Electrode Electrode The biocathode was was prepared prepared by by the the following followingmethod: method:BC/c-MWCNTs BC/c-MWCNTs were injected The biocathode were injected by ´1 Lac with 1.5% (v/v) GA in 0.1 M acetic acid/sodium acetate buffer (pH = 5.5) solution by 40 mg mL −1 40 mg mL Lac with 1.5% (v/v) GA in 0.1 M acetic acid/sodium acetate buffer (pH = 5.5) solution for ´1 for h. The bioanode prepared similar method except that replaced −1 LacLac 1 h.1The bioanode waswas prepared by by similar method except that thethe 40 40 mgmg mLmL waswas replaced by ´ 1 by GOD. Then, electrode wasdipped dippedinto into0.1 0.1 M M acetic acetic acid/sodium acetate buffer −1 GOD. 20 20 mgmg mLmL Then, thethe electrode was acid/sodium acetate buffer (pH 5.5) solution solution containing containing 50 (pH = = 5.5) 50 mM mM glucose, glucose, as as shown shown in in Figure Figure 2. 2.

Figure 2. 2. Illustration Illustration of of the the Lac Lac and and GOD GOD immobilized immobilized on on BC/c-MWCNTs BC/c-MWCNTs by Figure by GA GA crosslinking. crosslinking.

2.4. Characterization and Electrochemical Electrochemical Measurements Measurements 2.4. Characterization and The morphology compositecomposite membranesmembranes surfaces were characterized The morphologyof the of BC the and BCBC/c-MWCNTs and BC/c-MWCNTs surfaces were by field emission scanning electron microscope (FESEM, S-4800, Hitachi, Tokyo, characterized by field emission scanning electron microscope (FESEM, S-4800, Hitachi, Tokyo, Japan). Japan). The morphology morphologyof of BC also wascharacterized also characterized by high-resolution transmission electron The the the BC was by high-resolution transmission electron microscope microscope (TEM, JEOL/JEM-2100, Tokyo, Japan). Dried BC and BC/c-MWCNTs composite (TEM, JEOL/JEM-2100, Tokyo, Japan). Dried BC and BC/c-MWCNTs composite were placed were over placed over aluminum support and sputtered with gold. The samples were coated with a thin layer aluminum support and sputtered with gold. The samples were coated with a thin layer of Au of Au nanoparticles to reduce the charging observation. nanoparticles to reduce the charging effects effects before before FESEMFESEM observation. The pure BC, c-MWCNTs, and BC/c-MWCNTs composite membranes prepared KBr The pure BC, c-MWCNTs, and BC/c-MWCNTs composite membranes werewere prepared in KBrinpellet pellet and scanned with Fourier transform infrared spectrophotometer (FTIR, Nicolet NEXUS, and scanned with Fourier transform infrared spectrophotometer (FTIR, Nicolet NEXUS, Hillsboro, Hillsboro, OR, USA). OR, USA). Electrochemical measurements measurements were were performed performed using using aa CHI CHI 660D 660D electrochemical electrochemical workstation workstation Electrochemical (CH Instruments, Instruments,Inc., Inc., Austin, TX, USA). The electrochemical was inmeasured in a (CH Austin, TX, USA). The electrochemical response response was measured a conventional conventional three-electrode system using BC/c-MWCNTs/Lac and BC/c-MWCNTs/GOD as working three-electrode system using BC/c-MWCNTs/Lac and BC/c-MWCNTs/GOD as working electrode, a electrode, a Pt wire auxiliary electrode and the Ag/AgCl as reference electrode. The electrocatalytic Pt wire auxiliary electrode and the Ag/AgCl as reference electrode. The electrocatalytic activity of activity of bioanode material was tested toward oxygen reduction reaction in 0.1 M sodium bioanode material was tested toward oxygen reduction reaction in 0.1 M sodium acetate/acetic acid acetate/acetic acid buffer solution (pH 5.5). The electrochemical measurements were carried out at buffer solution (pH 5.5). The electrochemical measurements were carried out at around 25 ˝ C. around 25 °C. 3. Results and Discussion 3.1. Culture Process Characterization Figure 3 illustrates a six-day track of bacterial cellulose growth in presence of c-MWCNTs under agitated culture. Firstly, G. xylinus was inoculated into the culture media contain c-MWCNTs. On the first day, culture solution fully presented black color as shown in Figure 3, but it could be


4 of 10

3. Results and Discussion 3.1. Culture Process Characterization Figure 3 illustrates a six-day track of bacterial cellulose growth in presence of c-MWCNTs under agitated culture. Firstly, G. xylinus was inoculated into the culture media contain c-MWCNTs. On the first day, culture solution fully presented black color as shown in Figure 3, but it could be seen that Materials 2016, 2016, 9, 1839, 183 Materials the color faded with the increasing number of days, revealing only a single black bacterial cellulose in theseen culture indicating the incorporation ofofc-MWCNTs into the bacterial cellulose. On the seen that the color faded with theincreasing increasing number revealing onlyonly a single blackblack bacterial that solution the color faded with the number ofdays, days, revealing a single bacterial cellulose in the culture solution indicating the incorporation of c-MWCNTs into the bacterial sixth cellulose day, the in culture solution became clearer (similar to the original culture color), the culture solution indicating the incorporation of c-MWCNTs into theindicating bacterial the cellulose. On the sixth day, the culturesolution solution became (similar to the original color),color), cellulose. On the sixth day, the culture becameclearer clearer (similar to the original culture incorporation of remaining c-MWCNTs into BC pallet. The sixth day growth ofculture bacterial cellulose in indicating the incorporation of remaining c-MWCNTs into BC pallet. The sixth day growth of indicating the incorporation of remaining c-MWCNTs structure. into BC pallet. The sixth day growth of the absence of c-WMCNTs presented a white cryptomere bacterial cellulose in the absence of c-WMCNTs presented a white cryptomere structure. bacterial cellulose in the absence of c-WMCNTs presented a white cryptomere structure.

Figure 3. Growth of bacterial cellulose in the presence of c-MWCNTs (50 mg/100 mL) sheets:

Figure 3. Photographic Growth ofimage bacterial cellulose presence ofgrowth c-MWCNTs (50ofmg/100 mL) sheets: of the six day trackinofthe bacterial cellulose in presence c-MWCNTs Photographic image of the six day track of bacterial cellulose growth in presence of c-MWCNTs Figure 3. Growth of bacterial cellulose in the presence of c-MWCNTs (50 mg/100 mL) under agitated conditions. The sixth day growth of bacterial cellulose in the absence of c-MWCNTssheets: (red font). Photographic image of the dayday track of bacterial cellulose growth in the presence of c-MWCNTs under agitated conditions. Thesix sixth growth of bacterial cellulose absence of c-MWCNTs under agitated conditions. The sixth day growth of bacterial cellulose in the absence of c-MWCNTs (red font).

Figure 4 displays schematic illustration of the formation of bacterial cellulose and the (red font). integration of c-MWCNTs into the 3D interconnected fibrous network of BC. Firstly, c-MWCNTs were dispersedschematic into culture illustration solution and then the formation free bacteriaof was attached cellulose to the surface bubbles Figure 4 displays of the bacterial and the integration Figure 4 displays schematic illustration of the formation of bacterial cellulose and the which into apparently underwent reproduction to synthesize bacterial cellulose fibers [24]. As thewere days dispersed of c-MWCNTs the 3D interconnected fibrous network of BC. Firstly, c-MWCNTs integration of c-MWCNTs the 3D interconnected fibrous network of BC.a Firstly, c-MWCNTs increased, the bacterial into cellulose attached itself to the c-MWCNTs to form more compact into culture solution and then the free bacteria was attached to the surface bubbles which apparently werestructure dispersed into culture the free bacteria was attached to the surface bubbles [25]. Due to the solution growth ofand thethen bacterial cellulose in the presence of c-MWCNTs under underwent reproduction tothe synthesize bacterial fibers [24]. with As the increased, thedays bacterial which apparently underwent reproduction tocellulose synthesize bacterial cellulose fibers [24]. As the agitated conditions, bacterial cellulose and c-MWCNTs entangled eachdays other and gradually gathered for the to dispersion of c-MWCNTs in culture solution. It was structure again observed the increased, the bacterial attached itself to the c-MWCNTs to form a due more compact cellulose attached itself thecellulose c-MWCNTs to form a more compact [25].that Due totothe growth of slow rate of BC-formation, c-MWCNTs therefore, absorbed on presence the BC whiles thebacterial bacteria under structure [25]. Due the growth of c-MWCNTs the was, bacterial cellulose in the of c-MWCNTs the bacterial cellulose intothe presence of under agitated conditions, the cellulose gathered around the the BC/c-MWCNTs composite and produced BC fibrils.with The each formation ofand newgradually BC agitated conditions, bacterial cellulose and c-MWCNTs entangled other and c-MWCNTs entangled with each other and gradually gathered for the dispersion of c-MWCNTs in fibrils on existing composite was continuous until a more compact, irregularly shaped, and gathered for the dispersion of c-MWCNTs in culture solution. It was again observed that due to the culture solution. It was again observed that due to the slow rate of BC-formation, c-MWCNTs was, randomly overlapped composite was obtained.

slow rate of BC-formation, c-MWCNTs was, therefore, absorbed on the BC whiles the bacteria

therefore, absorbed on the BC whiles the bacteria gathered around the BC/c-MWCNTs composite and gathered around the BC/c-MWCNTs composite and produced BC fibrils. The formation of new BC produced BC fibrils. The formation of new BC fibrils on existing composite was continuous until a fibrils on existing composite was continuous until a more compact, irregularly shaped, and more randomly compact,overlapped irregularlycomposite shaped, and was randomly obtained. overlapped composite was obtained.

Figure 4. Schematic illustration of formation of bacterial cellulose and the integration of carbon into the 3D interconnected fibrous network of BC.

3.2. Morphology Analysis The microstructure of BC was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 5a shows pure BC fibrils with ultrafine nanofiber Figure 4. Schematic illustration of formation of bacterial cellulose and the integration of carbon into high ultrafine porosity 3D web-like structure. As shown Figure 5a, BC of revealed Figurestructure, 4. Schematic illustration of formation of bacterial cellulose andinthe integration carbon into the 3D interconnected fibrous network of BC. the 3D interconnected fibrous network of BC. 4

3.2. Morphology Analysis The microstructure of BC was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 5a shows pure BC fibrils with ultrafine nanofiber structure, high ultrafine porosity 3D web-like structure. As shown in Figure 5a, BC revealed 4


5 of 10

3.2. Morphology Analysis The microstructure of BC was characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 5a shows pure BC fibrils with ultrafine nanofiber structure, porosity 3D web-like structure. As shown in Figure 5a, BC revealed Materialshigh 2016, 9,ultrafine 183 interconnecting pores which is in agreement with the report of refference [26]. The ultrafine network interconnecting pores whichsurface is in agreement with the favourable report of refference [26].for TheOultrafine network structure and larger specific area provided channels 2 transmission. The structure and larger specific surface area provided favourable channels for O2 transmission. high-resolution TEM image further revealed that these nanofibers were mainly consisted of randomly The high-resolution TEM image further revealed that these nanofibers were mainly consisted of orientated 3D web-like structure (Figure 5b) [27]. Figure 5c showed that c-MWCNTs was absorbed on randomly orientated 3D web-like structure (Figure 5b) [27]. Figure 5c showed that c-MWCNTs was 3D BC fibrils in which c-MWCNTs displayed the phenomenon of aggregation. As shown in Figure 5d, absorbed on 3D BC fibrils in which c-MWCNTs displayed the phenomenon of aggregation. the morphology of BC/c-MWCNTs samples revealed thatsamples c-MWCNTs were tightly wrapped As shown in Figure 5d, the morphology of BC/c-MWCNTs revealed that c-MWCNTs werein BC pellicle resulting in an spherical shaped structure. tightly wrapped in BC pellicle resulting in an spherical shaped structure.

Figure 5. (a) FESEM images of pure BC synthesized in shaking culture system; (b) TEM image of BC;

Figure 5. (a) FESEM images of pure BC synthesized in shaking culture system; (b) TEM image (c) FESEM images of BC/c-MWCNTs composite membranes; and (d) digital photos of of BC; (c) FESEM images of BC/c-MWCNTs composite membranes; and (d) digital photos of c-MWCNTs-dispersed HS medium a shaking culture system at 30 °C for 6 days (100 rpm). c-MWCNTs-dispersed HS medium a shaking culture system at 30 ˝ C for 6 days (100 rpm).

3.3. FTIR Analysis

3.3. FTIR Figure Analysis 6 shows the FT-IR spectra of the pure BC c-MWCNTs and BC/c-MWCNTs. The band at −1 for c-MWCNTs was attributed to the presence of hydroxyl groups(-OH) [28], as shown in 3465 cm Figure 6 shows the FT-IR spectra of the pure BC c-MWCNTs and BC/c-MWCNTs. The band −1 for c-MWCNTs was assigned to the presence of Figure The absorptionwas band at 1659 cm 1 for at 3465 cm´6b. c-MWCNTs attributed to the presence of hydroxyl groups(-OH) [28], as shown carboxyl functional groups (C=O) which was in´agreement with references [29,30]. The absorption 1 in Figure 6b. The absorption band at 1659 cm for c-MWCNTs was assigned to the presence of band at 2973 cm−1 for BC（Figure 6a）was also attributed to the presence of C-H stretching vibrations carboxyl functional groups (C=O) which was in agreement with references [29,30]. The absorption which was in´agreement with the characteristic bands of BC reported in the literature [31]. The main 1 banddifference at 2973 cm BC (Figure 6a) BC/c-MWCNTs was also attributed to theatpresence of C-Hpeak stretching in thefor spectra of BC and was found the absorption of 3465 vibrations cm−1, which in agreement with characteristic of BC reportedcomposite in the literature [31]. were The main −1 for BC/c-MWCNTs aswas shown in Figure 6c. Thethe peak at 3465 cmbands membranes difference in the spectra of BConly andphysical BC/c-MWCNTs foundbetween at the absorption peak of 3465 cm´1 , as enhanced, suggesting that interactionwas occurred BC and c-MWCNTs. shown in Figure 6c. The peak at 3465 cm´1 for BC/c-MWCNTs composite membranes were enhanced, suggesting that only physical interaction occurred between BC and c-MWCNTs.

5

Materials 2016, 9, 183 Materials 2016, 9, 183

6 of 10


Figure 6. FTIR of different (a) BC; (b) c-MWCNTs; and (c) BC/c-MWCNTs Figure 6. spectra FTIR spectra of composite. different composite. (a) BC; (b) c-MWCNTs; and composite. (c) BC/ c-MWCNTs composite.

3.4. Effect of Different Conditions on Power Output of the Figure 6. FTIR spectra of different composite. (a) BC; (b) EBFC c-MWCNTs; and (c) BC/c-MWCNTs composite. 3.4. Effect of Different Conditions on Power Output of the EBFC of Different ConditionsGlucose on PowerConcentrations Output of the EBFC 3.4.1.3.4. TheEffect Influence of Different 3.4.1. The Influence of Different Glucose Concentrations Figure displays the maximum power output of the EBFC with different glucose 3.4.1. The7a Influence of Different Glucose Concentrations concentration 15 mM topower 70 mM. Figure 6a EBFC showswith relatively higher power output in Figure 7a ranging displays from the maximum output of the different glucose concentration Figure 7a displays the maximum power output of the EBFC with different glucose lower region (15–506amM); when power the glucose concentration further rangingconcentrations from 15 mM to 70 mM. Figure showshowever, relatively higher output in lower concentrations concentration ranging from 15 mM to 70 mM. Figure 6a shows relatively higher power output in increased, the powerhowever, output greatly decreased. In general, power output showed afurther relatively region (15–50 mM); when the glucose concentration further increased, the power output lower concentrations region (15–50 mM); however, whenthe the glucose concentration higher enzyme activity among the range of 30–55 mM with an optimum concentration of about 50 greatly decreased. In general, the power output showed a relatively higher enzyme activity among the increased, the power output greatly decreased. In general, the power output showed a relativelymM. rangehigher of 30–55 mMactivity with an optimum concentration of about 50 mM.concentration of about 50 mM. enzyme among the range of 30–55 mM with an optimum

Figure 7. The maximum power output of the EBFC with different conditions: (a) glucose concentrations;

Figure 7. The maximum power output of the EBFC with different conditions: (a) glucose concentrations; (b) optimum pH; and (c) optimum temperature. (b) optimum and (c)power optimum temperature. Figure 7. The pH; maximum output of the EBFC with different conditions: (a) glucose concentrations;

(b) optimum pH; and (c) optimum temperature.

6

6

Materials 2016, 9, 183 Materials 2016, 9, 183

7 of 10

3.4.2. The Influence of Different pH 3.4.2. The Influence of Different pH The biofuel cells were incubated in buffer solution with pH ranging from 3 to 7 at room The biofuel cells were incubated in buffer solution with pH ranging from 3 to 7 at room temperature (Figure 7b). The immobilized laccase and glucose oxidase on BC/c-MWCNTs showed temperature (Figure 7b). The immobilized laccase and glucose oxidase on BC/c-MWCNTs showed relatively higher pH stability. Additionally, the immobilized enzyme on BC/c-MWCNTs composite relatively higher pH stability. Additionally, the immobilized enzyme on BC/c-MWCNTs composite showed a relatively higher activity among the range of pH 4–6, and the optimum pH was around 5.5, showed a relatively higher activity among the range of pH 4–6, and the optimum pH was around 5.5, as shown in Figure 7b. as shown in Figure 7b. 3.4.3. 3.4.3. The The Influence Influence of of Different Different Temperature Temperature The effectofoftemperature temperature power density of immobilized enzyme on BC/c-MWCNTs is The effect onon power density of immobilized enzyme on BC/c-MWCNTs is shown shown in 7c. Figure The immobilized was incubated buffer solution 5 min at in Figure The 7c. immobilized enzymeenzyme was incubated in bufferinsolution (pH 5.5) (pH for 55.5) minfor at different ˝ different temperatures varying from 15 to 75 °C. Figure 7c shows relatively higher activity retention temperatures varying from 15 to 75 C. Figure 7c shows relatively higher activity retention in lower ˝ C),(30–50 in lower temperature region °C),when however when the temperature further increased, power temperature region (30–50 however the temperature further increased, power output greatly output greatly decreased indicating the decrease of enzyme activity. The immobilized decreased indicating the decrease of enzyme activity. The immobilized enzymes showedenzymes a higher ˝ ˝ showed a higher enzyme activity among 45–60 °C with an optimum temperature at 55 °C. enzyme activity among 45–60 C with an optimum temperature at 55 C. In general, the immobilized In general, the immobilized enzyme possessed high activity in a broader temperature range, enzyme possessed high activity in a broader temperature range, enabling it a desirable material for enabling it a desirable material for enzymatic biofuel cells application and many other fields. enzymatic biofuel cells application and many other fields. 3.5. Electrochemical Electrode Electrochemical Behavior of the Lac/BC/c-MWCNTs Electrode As shown in Figure 8a, 8a, the the CVs CVs of of the theBC/c-MWCNTs/Lac BC/c-MWCNTs/Lac electrode electrode were were absent, absent, whereas whereas a pair of prominent redox peaks peaks (at (at 0.74 0.74 V V and and 0.11 0.11 V, respectively) can be observed [32], which was attributed attributed to to the the redox reaction of the Lac immobilized on the c-MWCNTs. This showed the good coupling between the enzymes and 3D BC/c-MWCNTs BC/c-MWCNTs substrate. substrate. The The redox redox of of Lac Lac on on the the electrode was a reversible and surface-confined process, which could be demonstrated demonstrated by the linear relationship relationship between redox currents and scan rate, as shown in Figure 8b. The bare 3D BC/c-MWCNTs electrode showed no catalytic action to O22 in in comparison comparison with with that that of of BC/c-MWCNTs/Lac BC/c-MWCNTs/Lacelectrode. electrode.

(a)

(b)

Figure 8. (a) Cyclic voltammograms of (I) the bare 3D BC/c-MWCNTs electrode and Figure 8. (a) Cyclic voltammograms of (I) the bare 3D BC/c-MWCNTs electrode and BC/c-MWCNTs/Lac electrode in a 0.1 M acetic acid/sodium acetate buffer solution (pH 5.5) at scan BC/c-MWCNTs/Lac electrode in a 0.1 M acetic acid/sodium acetate buffer solution (pH 5.5) at rates (mV s−1): (II) 50, (III) 150, (IV) 250, (V) 350; and (b) The inset is a plot of the oxidation and scan rates (mV s´1 ): (II) 50, (III) 150, (IV) 250, (V) 350; and (b) The inset is a plot of the oxidation and reduction peak currents reduction peak currents vs. vs. the the scan scan rates. rates.

3.6. Performance of the Biofuel Cell 3.6. Performance of the Biofuel Cell The EBFCs were fabricated with a 3D BC/c-MWCNTs/Lac cathode and 3D The EBFCs were fabricated with a 3D BC/c-MWCNTs/Lac cathode and 3D BC/c-MWCNTs/GOD ocv BC/c-MWCNTs/GOD anode by GA crosslinking. As demonstrated in Figure 9a, the Ecell of the EBFC anode by GA crosslinking. As demonstrated in Figure 9a, the Eocv of the EBFC was approximately cell was approximately 0.64 V. Through 30 days of open circuit voltage collected, a 51% decrease of open circuit voltage was observed indicating relatively satisfactory stability, as shown in Figure 9b. Linear 7


8 of 10

0.64 V. 2016, Through Materials 9, 183

30 days of open circuit voltage collected, a 51% decrease of open circuit voltage was observed indicating relatively satisfactory stability, as shown in Figure 9b. Linear sweep voltammetry sweep voltammetry (LSV) the waselectrochemical used to evaluate the electrochemical performance of enzymatic (LSV) was used to evaluate performance of enzymatic biofuel cell operating with biofuel operating with [33].the Aschange shownof in power Figure density 9c, the change offirst power density showed glucosecell [33]. As shown inglucose Figure 9c, showed increase and then a first increase a decrease thevoltage declinewhiles of open voltagevoltage whiles was the open-circuit decrease withand thethen decline of openwith circuit thecircuit open-circuit about 0.62 V voltage was about 0.62 V which was relatively consistent with the biggest open circuit in which was relatively consistent with the biggest open circuit voltage in Figure 9a. The voltage maximum 3 3 3 Figure The maximum current density was 0.29 mA/cm and the maximum density was current9a. density was 0.29 mA/cm and the maximum power density was 32.98power µW/cm implying 3 implying acceptable electric properties. 32.98 µ W/cm acceptable electric properties.

Figure Figure9.9.(a) (a)The Themaximum maximumopen opencircuit circuitvoltage voltageofofthe theEBFCs; EBFCs;(b) (b)The Theopen opencircuit circuitvoltage voltagefrom fromthe the EBFCs and (c) (c) The Thepower powerdensity densitycurve curveofofglucose/O glucose/O 2biofuel biofuel cell obtained by LSV in EBFCsover over 30 30 days; days; and cell obtained by LSV in 0.1 2 −1. ´1 0.1 m, pH 5.5, HAc/NaAc buffer containing 50 mM glucose, data were collected at 10 mV s m, pH 5.5, HAc/NaAc buffer containing 50 mM glucose, data were collected at 10 mV s .

4.4.Conclusions Conclusions Lac Lacand andGOD GODwere were respectively respectively immobilized immobilizedon on the the BC/c-MWCNTs BC/c-MWCNTscomposite compositetotoprepare preparethe the cathode and anode of EBFCs. The nanotopographic surface of c-MWCNTs networks ensured snug cathode and anode of EBFCs. The nanotopographic surface of c-MWCNTs networks ensured snug anchoring molecules by byGA GAcrosslinging. crosslinging.The Theinfluence influence different concentrations, anchoring of of enzyme enzyme molecules of of different concentrations, pH pH and and temperature on the performance of EBFCs was investigated, and the optimum concentration, temperature on the performance of EBFCs was investigated, and the optimum concentration, pH and pH and temperature 50 mM, 5.5, and 55 °C, respectively. The as-prepared EBFCs showed ˝ C, temperature were 50 were mM, 5.5, and 55 respectively. The as-prepared EBFCs showed satisfactory satisfactory electric properties. Our study demonstrated that the three-dimensional structure, electric properties. Our study demonstrated that the three-dimensional structure, controllable porosity controllable porosity as wellof asBC designable shapethe of BC could provide the application possibility as well as designable shape could provide application possibility in biofuel cell fields. in biofuel cell fields. Acknowledgments: This research was financially supported by the Priority Academic Program Development Acknowledgments: This research was financially supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions, the National Natural Science Foundation of China (51203064 and of Jiangsu Higher Education Institutions, National Natural Industry-Academia-Research Science Foundation of ChinaJoint (51203064 and 21201083), the Natural Science Fundation the of Jiangsu Province, Innovation 21201083), the Natural Science Fundation of Jiangsu Province, Industry-Academia-Research Joint Innovation Fund of Jiangsu Province (BY2014023-4, BY2014023-23 and BY2014023-29), Six talent peaks project in Jiangsu Province (2014-XCL001), DepartmentBY2014023-23 of Education in Anhui Province of Six China (2015LJRCTD001), China Fund of Jiangsu Provincethe (BY2014023-4, and BY2014023-29), talent peaks project inthe Jiangsu Postdoctoral Science Foundation (2014M560391), the China Postdoctoral Science Foundation (2015T80496). Province (2014-XCL001), the Department of Education in Anhui Province of China (2015LJRCTD001), the China Postdoctoral Science Foundation (2014M560391), the China Postdoctoral Science Foundation (2015T80496). Author Contributions: All authors contributed equally to this work.

Author Contributions: All authors contributed equally to this work. Conflicts of Interest: The authors declare no conflict of interest.

8


9 of 10

Conflicts of Interest: The authors declare no conflict of interest.

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11.

12. 13. 14. 15. 16. 17.

18. 19.

20. 21. 22.

Chen, Z.; Zhang, S.; Wu, F.; Yang, W.; Liu, Z.; Yang, M. Motion mode of poly (lactic acid) chains in film during strain-induced crystallization. J. Appl. Polym. Sci. 2015, 133, 42969–42978. [CrossRef] Cooney, M.J.; Svoboda, V.; Lau, C.; Martin, G.; Minteer, S.D. Enzyme catalysed biofuel cells. Energy Environ. Sci. 2008, 1, 320–337. [CrossRef] Yang, X.Y.; Tian, G.; Jiang, N.; Su, B.L. Immobilization technology: A sustainable solution for biofuel cell design. Energy Environ. Sci. 2012, 5, 5540–5563. [CrossRef] Tsujimura, S.; Kamitaka, Y.; Kano, K. Diffusion-Controlled Oxygen Reduction on Multi-Copper Oxidase-Adsorbed Carbon Aerogel Electrodes without Mediator. Fuel Cells 2007, 7, 463–469. [CrossRef] Prasad, K.P.; Chen, Y.; Chen, P. Three-dimensional graphene-carbon nanotube hybrid for high-performance enzymatic biofuel cells. ACS Appl. Mater. Inter. 2014, 6, 3387–3393. [CrossRef] [PubMed] Minteer, S.D.; Atanassov, P.; Luckarift, H.R.; Johnson, G.R. New materials for biological fuel cells. Mater. Today 2012, 15, 166–173. [CrossRef] Feng, W.; Ji, P.J. Enzymes immobilized on carbon nanotubes. Biotechnol. Adv. 2011, 29, 889–895. [CrossRef] [PubMed] Vashist, S.K.; Zheng, D.; Al-Rubeaan, K.; Luong, J.H.T.; Sheu, F.S. Advances in carbon nanotube based electrochemical sensors for bioanalytical applications. Biotechnol. Adv. 2011, 29, 169–188. [CrossRef] [PubMed] Gong, K.P.; Yan, Y.M.; Zhang, M.N.; Su, L.; Xiong, S.X.; Mao, L.Q. Electrochemistry and electroanalytical applications of carbon nanotubes: A review. Anal. Sci. 2005, 21, 1383–1393. [CrossRef] [PubMed] Baughman, R.H.; Zakhidov, A.A.; De Heer, W.A. Carbon nanotubes-the route toward applications. Science 2002, 297, 787–792. [CrossRef] [PubMed] Zhao, C.E.; Wang, Y.; Shi, F.J.; Zhang, J.R.; Zhu, J.J. High biocurrent generation in Shewanella-inoculated microbial fuel cells using ionic liquid functionalized graphene nanosheets as an anode. Chem. Commun. 2013, 49, 6668–6670. [CrossRef] [PubMed] Klemm, D.; Schumann, D.; Udhardt, U.; Marsch, S. Bacterial synthesized cellulose-artificial blood vessels for microsurgery. Prog. Polym. Sci. 2001, 26, 1561–1603. [CrossRef] Petersen, N.; Gatenholm, P. Bacterial cellulose-based materials and medical devices: Current state and perspectives. Appl. Microbiol. Biot. 2011, 91, 1277–1286. [CrossRef] [PubMed] Jonas, R.; Farah, L.F. Production and application of microbial cellulose. Polym. Degrad. Stab. 1998, 59, 101–106. [CrossRef] Liang, H.W.; Guan, Q.F.; Zhu, Z.; Song, L.T.; Yao, H.B.; Lei, X.; Yu, S.H. Highly conductive and stretchable conductors fabricated from bacterial cellulose. NPG Asia Mater. 2012, 4, e19. [CrossRef] Wang, B.; Li, X.; Luo, B.; Yang, J.; Wang, X.; Song, Q.; Chen, S.; Zhi, L. Pyrolyzed bacterial cellulose: A versatile support for lithium ion battery anode materials. Small 2013, 9, 2399–2404. [CrossRef] [PubMed] Wang, M.; Anoshkin, I.V.; Nasibulin, A.G.; Korhonen, J.T.; Jani, S.; Jaakko, P.; Kauppinen, E.I.; Ras, R.H.A.; Olli, I. Modifying native nanocellulose aerogels with carbon nanotubes for mechanoresponsive conductivity and pressure sensing. Adv. Mater. 2013, 25, 2428–2432. [CrossRef] [PubMed] Wan, Y.; Creber, K.A.M.; Peppley, B.; Bui, V.T. Chitosan-based solid electrolyte composite membranes I. Preparation and characterization. J. Membr. Sci. 2006, 280, 666–674. [CrossRef] Ouyang, W.; Sun, J.; Memon, J.; Wang, C.; Geng, J.; Huang, Y. Scalable preparation of three-dimensional porous structures of reduced graphene oxide/cellulose composites and their application in supercapacitors. Carbon 2013, 62, 501–509. [CrossRef] Feng, Y.; Zhang, X.; Shen, Y.; Yoshino, K.; Feng, W. A mechanically strong, flexible and conductive film based on bacterial cellulose/graphene nanocomposite. Carbohyd. Polym. 2012, 87, 644–649. [CrossRef] Oikawa, T.; Ohtori, T.M. Production of cellulose from d-mannitol by acetobacter xylinum ku-1. Biosci. Biotechnol. Biochem. 1995, 59, 331–332. [CrossRef] Kuo, C.H.; Chen, J.H.; Liou, B.K.; Lee, C.K. Utilization of acetate buffer to improve bacterial cellulose production by Gluconacetobacter xylinus. Food Hydrocoll. 2015, 53, 98–103. [CrossRef]


23.

24. 25.

26. 27. 28. 29.

30.

31.

32. 33.

10 of 10

Kiziltas, E.E.; Kiziltas, A.; Rhodes, K.; Emanetoglu, N.W.; Blumentritt, M.; Gardner, D.J. Electrically conductive nano graphite-filled bacterial cellulose composites. Carbohyd. Polym. 2016, 136, 1144–1151. [CrossRef] [PubMed] Czaja, W.; Romanovicz, D.; Brown, R.M. Structural investigations of microbial cellulose produced in stationary and agitated culture. Cellulose 2004, 11, 403–411. [CrossRef] Nandgaonkar, A.G.; Wang, Q.; Fu, K.; Krause, W.E.; Wei, Q.; Gorga, R.; Lucia, L.A. A one-pot biosynthesis of reduced graphene oxide (RGO)/bacterial cellulose (BC) nanocomposites. Green Chem. 2014, 16, 3195–3201. [CrossRef] Torres, F.G.; Commeaux, S.; Troncoso, O.P. Biocompatibility of bacterial cellulose based biomaterials. J. Funct. Biomater. 2012, 3, 864–878. [CrossRef] [PubMed] Parakalan, K.; Raed, H.; Mike, T. Modified cellulose morphologies and its composites; SEM and TEM analysis. Micron 2011, 42, 751–761. Yan, Z.Y.; Chen, S.Y.; Wang, H.P.; Wang, B.A.; Jiang, J.M. Biosynthesis of bacterial cellulose/multi-walled carbon nanotubes in agitated culture. Carbohyd. Polym. 2008, 74, 659–665. [CrossRef] Zhao, B.; Hu, H.; Yu, A.P.; Perea, D.; Haddon, R.C. Synthesis and characterization of water soluble single-walled carbon nanotube graft copolymers. J. Am. Chem. Soc. 2005, 127, 8197–8203. [CrossRef] [PubMed] Zhou, T.; Chen, D.; Jiu, J.; Nge, T.T.; Sugahara, T.; Nagao, S.; Koga, H.; Nogi, M.; Suganuma, K.; Wang, X.; et al. Electrically conductive bacterial cellulose composite membranes produced by the incorporation of graphite nanoplatelets in pristine bacterial cellulose membranes. Express Polym. Lett. 2013, 7, 756–766. [CrossRef] Rambo, C.R.; Recouvreux, D.O.S.; Carminatti, C.A.; Pitlovanciv, A.K.; Antonio, R.V.; Porto, L.M. Template assisted synthesis of porous nanofibrous cellulose membranes for tissue engineering. Mater. Sci. Eng. 2008, 28, 549–554. [CrossRef] Gavaghan, D.J.; Myland, J.C.; Oldham, K.B. The effect of periodic modulation on the aperiodic current in linear-scan and cyclic voltammetries. J. Electroanal. Chem. 2001, 516, 2–9. [CrossRef] Li, Y.; Wu, G.; Cong, R.; Qi, Z.; Yan, W.; Doherty, W.; Xie, K.; Wu, Y. Composite cathode based on doped vanadate enhanced with loaded metal nanoparticles for steam electrolysis. J. Power Sources 2014, 253, 349–359. [CrossRef] © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).