Co-utilization of mixed sugars in an enzymatic fuel cell based on an in vitro enzymatic pathway

Accepted Manuscript Co-utilization of mixed sugars in an enzymatic fuel cell based on an in vitro enzymatic pathway Zhiguang Zhu, Chunling Ma, Y.-H. Percival Zhang PII:

S0013-4686(17)32445-3

DOI:

10.1016/j.electacta.2017.11.083

Reference:

EA 30673

To appear in:

Electrochimica Acta

Received Date: 21 April 2017 Revised Date:

10 November 2017

Accepted Date: 12 November 2017

Please cite this article as: Z. Zhu, C. Ma, Y.-H. Percival Zhang, Co-utilization of mixed sugars in an enzymatic fuel cell based on an in vitro enzymatic pathway, Electrochimica Acta (2018), doi: 10.1016/ j.electacta.2017.11.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Co-utilization of mixed sugars in an enzymatic fuel cell based on an in

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vitro enzymatic pathway Zhiguang Zhua,b*, Chunling Maa, Y.-H. Percival Zhanga,b,c a

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Science, 32 West 7th

c

Cell-Free Bioinnovations Inc., 3107 Alice Drive, Blacksburg, Virginia 24060, United States

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b

Biological Systems Engineering Department, Virginia Tech, 304 Seitz Hall, Blacksburg,

Virginia 24061, United States

Corresponding Author: Zhiguang Zhu ([email protected]), Tel: +86-022-24828797, Fax: +86-

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022-24828789

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*

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Avenue, Tianjin 300308, China

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Submitted to Electrochimica Acta

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Abstract The wide use of portable electronics urgently requires better batteries featuring high energy density, good safety, and biodegradability. Although sugar-powered enzymatic fuel cells (EFCs)

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could be next-generation, environmentally friendly, micropower sources, they suffer from incomplete oxidation of the sugar fuels and an inability to utilize mixed sugars, which causes the low efficiency of fuel utilization and limits the choice of fuels. In this study, we designed an in vitro 15-enzyme pathway that can co-utilize sucrose, glucose, and fructose in the anodic

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compartment of EFCs. The EFCs achieved Faraday efficiencies of approximately 95% for these three sugars, suggesting that the fuels were completely oxidized, and yielded a maximum power density of 0.80-1.08 mW cm-2. In addition, EFCs based on this versatile enzymatic pathway

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were capable of running on mixed sugars, such as commercial soft drinks. These results offer a possible solution for the extraction of the maximum energy stored in mixed sugar fuels and the achievement of high energy densities for EFCs; these EFCs offer good substrate flexibility and commercial potential.

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Keywords: Enzymatic fuel cell; In vitro enzymatic pathway; Complete oxidation; Co-utilization;

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Mixed sugars; Energy density

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1. Introduction

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Enzymatic fuel cells (EFCs) are electrochemical devices in which enzymes are employed as biocatalysts for the direct conversion of the chemical energy stored in a fuel into electricity [1, 2]. They have received much interest as a next-generation, environmentally friendly, micropower source or a living battery due to their high safety, biodegradability, biocompatibility, and modest

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reaction conditions [3]. Sugar as a fuel is widely available, renewable, and safe and can store a lot of chemical energy. If sugar can be completely oxidized in an EFC, it can produce a much higher energy density than that of a traditional Li-ion battery [4].

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However, in most cases, sugar-powered EFCs suffer from the incomplete oxidation of fuels because they only employ one redox enzyme at the anode for a single two-electron oxidation per molecule of glucose [5, 6]. In theory, one mole of hexose can generate 24 moles of electrons if completely oxidized. Inspired by nature, a few in vitro enzymatic pathways have been constructed in EFCs to mimic the complete catabolism of carbohydrates in living organisms for the complete oxidation of various fuels, including methanol [7], lactate [8], and maltodextrin [4].

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A six-enzyme pathway for the complete oxidation of glucose has been designed based on enzymes with promiscuous activities. However, claims of complete oxidation have been inconclusive because CO2 was also produced in the middle of the pathway [9]. The quantitative

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validation of the complete oxidation of glucose has not yet been accomplished either. Recently, a 12-enzyme pathway in an EFC was demonstrated to implement the complete oxidation of glucose with >95% Faraday efficiency, suggesting a high energy density for such glucose-

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powered EFCs [10].

In addition to glucose, several diverse sugars have been demonstrated to generate electricity in EFCs, from monosaccharides (e.g., glucose [11] and fructose [12]) to disaccharides (e.g., sucrose [13] and lactose [14]). These sugars were all used in a pure form in these previous studies. However, outside the laboratory, pure sugar is not always available for practical application, and EFC users may only have access to sugars in the complex form of biomass, soft drinks, or wastewater streams. Or in another example, users can only find a sucrose-rich fuel instead of high-fructose corn syrup-rich one. Under these circumstances, the co-utilization of mixed sugars in such complex fuels would allow great substrate flexibility and an increased energy utilization 3

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efficiency for EFCs. Unfortunately, so far, no study pertaining to the co-utilization of mixed sugars has been reported. In this study, we designed for the first time an in vitro ATP-free and CoA-free enzymatic pathway in EFCs capable of completely oxidizing fructose and sucrose on the anode with a

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nearly-theoretical Faraday efficiency. Such EFCs can also co-utilize glucose, fructose, and sucrose and run on complex mixed sugar samples, such as sugar-rich soft drinks, suggesting great substrate flexibility and a high fuel utilization efficiency.

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2. Experimental

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2.1. Chemicals

All chemicals were of reagent grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA), unless otherwise noted. The substance 9,10-anthraquinone-2,7-disulphonic acid (AQDS) used as the electron mediator in this work was purchased from Pfaltz & Bauer (Waterbury, CT, USA). Carbon paper (AvCarb MGL200) was purchased from Fuel Cell Earth (Stoneham, MA, USA). COOH-functionalized

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multiwall carbon nanotubes (CNTs) with an outer diameter of < 8 nm and a length of 10-30 µm were purchased from CheapTubes.com (Brattleboro, VA, USA). Membrane electrode assemblies (MEAs) consisting of the Nafion 212 membrane and a carbon cloth cathode modified with 0.5 mg cm-2 Pt were purchased from the Fuel Cell Store (San Diego, CA, USA). His-tagged proteins

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were purified by the Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA, USA). Regenerated amorphous cellulose (RAC) used for enzyme purification was prepared from Avicel

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PH105 (FMC, Philadelphia, PA, USA) via dissolution and regeneration, as described elsewhere [15]. Classic Coca-Cola, Arizona iced tea, and Minute Maid juice were bought from a Walmart store in Christiansburg, VA, USA.

2.2. Production and purification of recombinant enzymes All the enzymes used in this study are listed in Table 1. Recombinant protein expression was conducted in Escherichia coli BL21 Star (DE3) (Invitrogen, Carlsbad, CA, USA) in LuriaBertani (LB) medium with either 100 mg L-1 ampicillin or 50 mg L-1 kanamycin. The cultures

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were incubated at 37 °C until the absorbance at 600 nm reached 0.6–0.8, and 10-100 µM of isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression at 18 °C overnight. The cells were collected and lysed by ultrasonication. The target protein was purified by His-tag purification, CBM-intein cleavage, or heat treatment. The purity of a recombinant

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protein was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The activity of each enzyme was assayed as described elsewhere (Table 1).

Table 1. Information about the enzymes used in this study. Source / (KEGG ORF)

Purification

1

Sucrose (SP)

2.4.1.7

CBM/intein

5.3.1.5

Thermoanaerobacterium thermosaccharolyticum JW/SL-YS485 Streptomyces murinus

2

Xylose isomerase (XI)

3

Polyphosphate glucokinase (PPGK) Phosphoglucomutase (PGM) Glucose 6-phosphate dehydrogenase (G6PDH)

2.7.1.63

Tfu1811

5.4.2.2

Tk1108

1.1.1.49

Zymomonas mobilis

6

6-phosphogluconate dehydrogenase (6PGDH)

1.1.1.44

7

Ribose 5-phosphate isomerase (RPI) Ribulose 5-phosphate 3epimerase (RuPE) Transketolase (TK)

2.2.1.1

Transaldolase (TAL) Triosephosphate isomerase (TIM) Fructose 1,6bisphosphate aldolase (ALD) Fructose 1,6bisphosphatase (FBP) Phosphoglucose isomerase (PGI) Diaphorase (DI)

8 9 10 11 12

13 14 15

[16]

5

[17]

CBM/intein

0.10 ± 0.01 16±1

5

[18]

CBM/intein

2.0±0.1

5

[19]

His/NTA

120±8

5

[20]

Moth1283

His/NTA

2.8±0.2

5

[21]

5.3.1.6

Tm1080

Heat precipitation

60±4

1

[22]

5.1.3.1

Tm1718

Heat precipitation

0.8±0.0

1

[23]

Ttc1896

His/NTA

1.3±0.1

1

[24]

2.2.1.2 5.3.1.1

Tm0295 Ttc0581

His/NTA Heat precipitation

4.1±0.2 102±10

1 1

[25] [26]

4.1.2.13

Ttc1414

Heat precipitation

2.9±0.2

1

[24]

3.1.3.11

Tm1415

CBM/intein

3.0±0.1

1

[27]

5.3.1.9

Cthe0217

CBM/intein

201±15

1

[28]

1.6.99.3

GenBank accession# JQ040550

His/NTA

896±28

5

[29]

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5

Loading (U mL-1) 5

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4

phosphorylase

Sp. Act.* (U mg-1) 5.0±0.2

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E.C.

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Enzyme

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#

* The specific activity was measured at 23 °C.

5

Sigma (G4166)

Ref.

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2.3. Enzymatic fuel cell assembly

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For the complete oxidation experiments, an anode of CNT-casted carbon paper was submerged in the anolyte that contained non-immobilized enzymes, NAD+, an electron mediator, buffer, and the fuel. A commercial MEA consisting of the Nafion 212 membrane and a carbon cloth modified with 0.5 mg cm-2 Pt as an air-breathing cathode was used. The anode, membrane,

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and cathode were stacked in piles and used for the demonstration of power generation from pure and mixed sugars.

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2.4. Electrochemical measurement of EFCs

All electrochemical tests were performed using a 1000B Multi-Potentiostat (CH Instruments Inc., Austin, TX, USA) interfaced to a computer. The reactions were performed at 23 ˚C. To determine the Faraday efficiency, an amperometric measurement at 0 V was performed by monitoring the current generation as a function of the reaction time. The reaction was conducted

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in an “I-cell” configuration with the anolyte volume of 15 mL. The anolyte was flushed with nitrogen to remove the oxygen. Fifteen enzymes free in solution were included (Table 1), along with 100 mM HEPES buffer (pH 7.2) containing 8 mM NAD+, 10 mM MgCl2, 0.5 mM MnCl2, 10 mM AQDS, 20 mM potassium phosphate, 2 mM polyphosphate, 0.5 mM thiamine

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pyrophosphate, 50 mg/L kanamycin, 0.5 g L-1 sodium azide, 1 g L-1 bovine serum albumin, and 0.1% Triton X-100. An extremely low concentration of 0.2 mM of sugars was run out first to

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reach the equilibrium for all intermediate metabolites, before a solution of 2 mM of glucose, fructose, or sucrose was added. To partially oxidize sucrose to release 2, 4, or 8 electrons per sucrose molecule, the same conditions were used with a four-enzyme pathway (i.e., SP + XI + PPGK + G6PDH for 2 e- generation), a five-enzyme pathway (i.e., SP + XI + PPGK + G6PDH + 6PGDH for 4 e- generation), or a six-enzyme pathway (i.e., SP + XI + PPGK+ PGM + G6PDH + 6PGDH for 8 e- generation). The residual glucose was quantified using a HK/G6PDH glucose assay kit (Sigma-Aldrich, St. Louis, MO, USA). The residual sucrose and fructose concentrations were measured using the glucose assay kit supplemented with SP and XI, respectively. The Faraday efficiency was calculated using the equation below:

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Fsugar-current = Ctotal/(∆cglucose unit× V × n ×F) where Fsugar-current = the Faraday efficiency, Ctotal = the total charge generated (C), ∆cglucose

unit

=

cinitial - cremain (M), V = the reaction volume (L), n = n electrons generated per glucose unit

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consumed, and F = the Faraday constant = 96,485 C per mole of electrons. The charge extraction efficiency (η) was defined as the fraction of the number of electrons generated by an enzyme cocktail from a fuel (e.g., sucrose) divided by the theoretical number of electrons generated by the complete oxidation of that fuel (e.g., 48 per sucrose). The equation is

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shown below: η = nelec×Fsugar-current /nelec,theoretical

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where nelec = 2, 4, 8, or 48 depending on the different sets of enzymes used to oxidize the sucrose, F = the Faraday efficiency, and nelec,theoretical = 48 for sucrose or 24 for glucose and fructose.

The amperometric response for a sucrose-powered EFC in a stacked cell configuration was recorded with increasing concentrations of sucrose (0–64 mM). Reactions powered by 32 mM of sucrose, glucose, or fructose and mixed sugars were conducted under similar conditions. Linear

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sweep voltammetry was performed at a scan rate of 1 mV s-1, to plot power curves. The enzyme cocktails included a 15-enzyme pathway or a 13-enzyme pathway without PPGK and XI. Soft drinks were added to the anodic reaction in a 20% (v/v) loading. The final pH was 7 which did not affect the overall performance of the enzymatic pathway.

Their sugar content was

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determined by high-performance liquid chromatography (HPLC) using an Aminex HPX-87P column with a flow rate of 0.6 mL min-1. The stability test was performed based on the linear

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sweep voltammetry result at different days after the electrolyte was prepared. The enzyme cocktails included 15 enzymes and the sugar mixture included 32 mM of all three sugars.

3. Results

3.1. Enzymatic pathway design The in vitro enzymatic pathway on the anode was designed for the complete oxidation of sucrose, fructose, and glucose and was comprised of three functional modules (Scheme 1): (i) a sugar phosphorylation module; (ii) an electron generation module; and (iii) a glucose 6-

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phosphate (G6P) regeneration module. In module (i), sucrose is phosphorylated to glucose 1phosphate (G1P) and fructose by SP, fructose is converted to glucose by XI, and glucose is converted to G6P by PPGK. G1P is further converted to G6P by PGM. In module (ii), one G6P produces two molecules of reduced nicotinamide adenine dinucleotide (NADH), one ribulose 5-

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phosphate and one CO2 catalyzed by two cascade enzymes: G6PDH and 6PGDH. Two NADH molecules are oxidized to transfer four electrons to the anode through an electron transfer chain consisting of a mediator and DI. In module (iii), one molecule of the C5 compound ribulose 5phosphate is recycled back to 5/6 molecules of the C6 compound G6P through an 8-enzyme

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pathway. In sum, 24 electrons per hexose unit can be theoretically generated. Furthermore, this pathway does not require either ATP or CoA, which is too costly and unstable for EFCs. The

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gaseous product, CO2, can be easily released from the anode compartment. Except XI, the other 14 recombinant enzymes were all expressed in E. coli BL21(DE3) and purified to homogeneity.

3.2. Complete oxidation of sugars

The set-up of the fuel cell is shown (Scheme 2). Various charge extraction efficiencies of sucrose were obtained by applying different enzyme cocktails from the enzymatic pathway to

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release 2, 4, 8 or 48 electrons per sucrose molecule. The four-enzyme cocktail containing SP, XI, PPGX, and G6PDH can generate two electrons per sucrose because only one dehydrogenase is involved; if this pathway is supplemented with 6PGDH (i.e., the five-enzyme cocktail), it can generate four electrons per sucrose from the two dehydrogenases; the further addition of PGM

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(i.e., the six-enzyme cocktail), which helps utilize the G1P from sucrose, can generate eight electrons per sucrose; and finally, the 15-enzyme pathway can generate 48 electrons per sucrose

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with the participation of the G6P regeneration module. EFCs catalyzed by these four sets of enzymes generated peak current densities of 0.66, 0.68, 0.66 and 0.73 mA cm-2, respectively (Fig. 1A). The 15-enzyme pathway generated a current for a much longer duration than the other pathways, indicating that more electrons were extracted per hexose molecule. The cumulative charges for the four-, five-, six-, and 15-enzyme sets from sucrose consumed (1.50 mM) were 4.6, 10.4, 21.3 C and 101.3 C, respectively, representing 3.9%, 8.2%, 15.9%, and 94.9% of charge extraction efficiencies (Fig. 1B). The 15-enzyme pathway was designed to extract all charge potentials of glucose, fructose, and sucrose. When 2 mM glucose, fructose or sucrose was added, the anodic current curves peaked 8

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in 1-1.5 hours at 0.98, 0.84, and 0.73 mA cm-2, respectively (Fig. 1C). No currents were generated at hour 50 for glucose or fructose, while the sucrose solution generated currents until hour 120. The cumulative electric charges generated from glucose (1.54 mM), fructose (1.30 mM) or sucrose (1.50 mM) consumed were 51.8, 43.7, and 101.3 C, respectively, corresponding

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to Faraday efficiencies of 97.1%, 90.2%, and 94.9% (Fig. 1D). These quantitative results suggested that nearly 24 electrons per hexose can be produced regardless of the sugar type. The charge extraction efficiencies of glucose-, fructose-, and sucrose-powered EFCs are increased by more than one order of magnitude compared to those of EFCs that release two electrons per

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hexose.

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3.3. Co-utilization of pure sugars in EFCs

The EFC equipped with all 15 enzymes in the anodic compartment and a platinum-coated carbon cloth as the cathode was assembled in a stacked cell configuration. The effect of a sequentially added sucrose solution on the current density was investigated by amperometry to determine the optimal fuel load (Fig. 2A). The dependence of the current density on the sucrose concentration fit Michaelis-Menten-like kinetics (Fig. 2B), where the apparent Km was 3.46 mM

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and the Imax was 2.56 mA cm-2.

To increase the reaction rate and the power density of EFCs, the enzyme load was optimized based on our previous results [4, 10]. The enzymes responsible for electron generation were set at 5 U mL-1, and the other enzymes were set at 1 U mL-1. When 32 mM of pure sucrose, glucose,

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or fructose was added separately, the OCP increased from 0.36 to ~0.8 V for all three EFCs (Fig. 3A). Glucose was the best fuel in terms of the maximum power density (0.97 mW cm-2),

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possibly because it had the shortest enzyme pathway for electron generation. The maximum power densities for sucrose- or fructose-powered EFCs were 0.69 or 0.81 mW cm-2, respectively (Fig. 3B). An extremely low power of 0.02 mW cm-2 was generated when no fuel or no enzyme was added.

3.4. EFCs powered by mixed sugars A sugar mixture containing 32 mM of sucrose, glucose, and fructose was prepared and investigated electrochemically. The overall sugar content of 22 g L-1 in this mixture represented

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20% of the total amount of sugar in two soft drinks (~110 g L-1) based on a sugar content analysis via HPLC. All three sugars could be utilized in the EFC via our 15-enzyme pathway, yielding a maximum power density of 1.08 mW cm-2 (Fig. 4A). This result was ~10% higher than that from the glucose-powered EFC, possibly due to the increased concentration of available

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sugars. When PPGK and XI were not included, EFCs comprised of 13 enzymes could only utilize sucrose. In this case, the maximum power density was reduced to 0.62 mW cm-2, similar to that from a sucrose-only-powered EFC.

Furthermore, we tested whether a commercial soft drink could generate electricity in our EFC.

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Cola, iced tea, and orange juice were added at a 1:4 (vol./vol.) ratio of the sample to the enzymebuffer solution. A sugar content analysis indicated that cola and iced tea were rich in fructose

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(58~66%) followed by glucose (30~35%), while orange juice had a large amount of sucrose (55%). When the 15-enzyme pathway was employed to utilize all three simple sugars in the mixtures, cola, iced tea and juice yielded maximum power densities of 0.60, 0.27, and 0.14 mW cm-2, respectively (Fig. 4B). A performance comparison of EFCs powered by pure sugar and complex sugar mixtures obviously indicated that some unknown compounds in the latter may have negative effects on the enzyme cocktail, especially those in the iced tea and juice samples

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thereby decreasing the maximum power densities of those EFCs. In addition to these power

clock (Video 1).

4. Discussion

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density data, we also demonstrated that a series of two cola-powered EFCs could power a digital

Although a few in vitro enzymatic pathways used in EFCs aimed to oxidize various fuels

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(Table 2), evidence for the complete oxidation of these fuels is inconclusive or indirect, based on the formation of CO2 or increases in the power output with greater use of cascade oxidoreductases. Although quantitative Faraday efficiencies have been obtained for lactate and glucose, these values were too low to indicate the complete oxidation of the fuel. In this study, an extraordinarily high Faraday efficiency of ~95% was achieved via a 15-enzyme pathway, suggesting the most efficient utilization of three simple sugars in EFCs so far. As a result, the energy density for the classic cola containing 11% wt./wt. sugar was as high as 395 Ah kg-1. If dehydrated powdered sugar was used, an order of magnitude higher energy density could be

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reached. Compared with a previous soft drink-powered EFC that released only two electrons per hexose [30-32], this study featured the extraction of 24 electrons per hexose regardless of

Table 2. Comparison of various fuels completely oxidized in EFCs. Fuel name

Key enzymes in the pathway

Open circuit potential /V

Maximal power density / mW cm-

Theoretical electrons per unit molecule

Faraday efficiency

Ref.

6

n/a

[33]

12

n/a

[34]

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2

Methanol

Alcohol DH, Formate DH

DH,

0.80

0.68

Ethanol

Alcohol DH, Aldehyde DH, TCA cycle enzymes

0.82

1.01

Glycerol

PQQ-based DH, Oxalate oxidase

/

1.32

12

n/a

[35]

Pyruvate

Pyruvate enzymes

cycle

/

0.93

10

n/a

[36]

Lactate

Lactate DH, Pyruvate DH, TCA cycle enzymes

/

/

12

70%

[8]

Maltodex trin

Glucan phosphorylase, Glucose6-phosphate DH, 6Phosphogluconate DH, PPP enzymes

0.70

0.40

24

92%

[4]

Glucose

PQQ-based DH, Oxalate oxidase

/

/

24

63%

[37]

Glucose

Polyphosphate glucokinase, Glucose-6-phosphate DH, 6Phosphogluconate DH, PPP enzymes

0.75

0.97

24

97%

[10]

Fructose

Xylose isomerase, Polyphosphate glucokinase, Glucose-6-phosphate DH, 6-Phosphogluconate DH, PPP enzymes

0.76

0.81

24

90%

This study

Sucrose

Sucrose phosphorylase, Xylose isomerase, Polyphosphate glucokinase, Glucose-6-phosphate DH, 6-Phosphogluconate DH,

0.80

0.69

48

95%

This study

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TCA

Aldolase,

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DH,

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Aldehyde

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glucose, fructose, sucrose, or a mixture.

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PPP enzymes

This study is the first in which three sugars – sucrose, glucose and fructose –simultaneously were co-utilized in EFCs based on an in vitro enzymatic pathway. If not only pure sugars but

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mixed or unpurified sugars can be used, the availability of the fuel for EFCs would be greatly widened, which is especially important for the successful commercialization of EFCs. In the future, more inexpensive mixed sugar fuels such as pretreated or hydrolyzed biomass and fermentation waste streams will be considered for powering EFCs, although possible inhibitory

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effects on the enzymatic pathways from the fuels need to be resolved. This study additionally shows the advantage of easy regulation for an in vitro enzymatic pathway. By introducing more

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enzyme components or modules, an enzymatic pathway can be engineered to have improved functions and catalyze multiple substrates.

Because the issues of mixed sugar utilization and the complete oxidation of hexoses were addressed in this study, future R&D on EFCs should focus on the development of a powerful cathode biocatalyst, increasing the power density, and prolonging the lifetime of EFCs. EFCs with current densities of 100 mA cm-2 or higher can be expected [38] if a combination of newly

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developed high electrical conductivity and high surface area nanomaterials, such as graphene foams [39] and carbonized nanofiber aerogels [40, 41], are used as electrodes, better electron mediators and more active enzymes or enzymatic complexes [42] are introduced, and enzyme loads predicted by in silicon models [43] are optimized. In terms of stability, the current version

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of the EFC can only last for several days (Fig. 5). It can be found that after 3 days, the performance drops greatly, which may be attributed to the loss of enzyme activity and

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degradation of the cofactors. To further increase the stability of the EFCs to months or longer, the use of only thermoenzymes and engineered enzymes would be introduced. As cofactordependent enzymatic electrocatalysis is often limited by cofactor diffusion and regeneration, more efforts should be made to construct artificial enzyme-cofactor conjugates, develop nanomaterial-facilitated cofactor or mediator immobilization approaches, and so on, in order to increase the electron transfer rate in EFCs. Native cofactors such as NAD+ could also be replaced with a biomimic with improved stability and reduced cost, and the cofactor preference of two redox enzymes could be engineered [44-46].

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5. Conclusions In summary, EFCs that used a 15-enzyme pathway extracted all the charge potential of sucrose, glucose, and fructose and achieved a high energy storage density. These EFCs could co-

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utilize the three sugars as well as the sugar mixtures in various soft drinks. Such EFCs, featuring great fuel flexibility, high safety, 100% biodegradability, and low-cost biocatalysts, could provide a variety of promising educational, outdoor, military, and healthcare applications in the

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near future.

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Acknowledgements This research was supported by the Tianjin Institute of Industrial Biotechnology (Grant 11ZCKFSY07800 and ZDRW-ZS-2016-3), 100 Talents Program of the Chinese Academy of Sciences, Cell-Free Bioinnovations Inc. (SBIR II Award IIP-1353266), and Biological Systems

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Engineering Department of Virginia Tech.

References

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[1] M.J. Cooney, V. Svoboda, C. Lau, G. Martin, S.D. Minteer, Enzyme catalysed biofuel cells, Energy Environ. Sci., 1 (2008) 320. [2] I. Willner, B. Willner, E. Katz, Biomolecule–nanoparticle hybrid systems for bioelectronic applications, Bioelectrochemistry, 70 (2007) 2. [3] E. Katz, K. MacVittie, Implanted biofuel cells operating in vivo - methods, applications and perspectives - feature article, Energy Environ. Sci., 6 (2013) 2791. [4] Z.G. Zhu, T.K. Tam, F.F. Sun, C. You, Y.H.P. Zhang, A high-energy-density sugar biobattery based on a synthetic enzymatic pathway, Nat. Commun., 5 (2014) 3026. [5] I. Wheeldon, The potential of multi-enzyme pathways to create novel anodes for enzymatic biofuel cells, Biofuels, 4 (2013) 463. [6] Z.G. Zhu, Y.R. Wang, S.D. Minteer, Y.H.P. Zhang, Maltodextrin-powered enzymatic fuel cell through a non-natural enzymatic pathway, J Power Sources, 196 (2011) 7505. [7] Y.H. Kim, E. Campbell, J. Yu, S.D. Minteer, S. Banta, Complete Oxidation of Methanol in Biobattery Devices Using a Hydrogel Created from Three Modified Dehydrogenases, Angew. Chem. Int. Ed., 52 (2013) 1437. [8] S. Tsujimura, J. Fukuda, O. Shirai, K. Kano, H. Sakai, Y. Tokita, T. Hatazawa, Microcoulometric study of bioelectrochemical reaction coupled with TCA cycle, Biosens. Bioelectron., 34 (2012) 244. [9] S. Xu, S.D. Minteer, Enzymatic Biofuel Cell for Oxidation of Glucose to CO2, ACS Catal., 1 (2011) 91. [10] Z. Zhu, Y.H.P. Zhang, In vitro metabolic engineering of bioelectricity generation by the complete oxidation of glucose, Metab. Eng., 39 (2017) 110. [11] A. Zebda, C. Gondran, A. Le Goff, M. Holzinger, P. Cinquin, S. Cosnier, Mediatorless high-power glucose biofuel cells based on compressed carbon nanotube-enzyme electrodes, Nat. Commun., 2 (2011) 370. [12] Y. Kamitaka, S. Tsujimura, N. Setoyama, T. Kajino, K. Kano, Fructose/dioxygen biofuel cell based on direct electron transfer-type bioelectrocatalysis, Phys. Chem. Chem. Phys., 9 (2007) 1793. [13] D.P. Hickey, F. Giroud, D.W. Schmidtke, D.T. Glatzhofer, S.D. Minteer, Enzyme Cascade for Catalyzing Sucrose Oxidation in a Biofuel Cell, ACS Catal., 3 (2013) 2729. [14] V. Krikstolaityte, P. Lamberg, M.D. Toscano, M. Silow, O. Eicher-Lorka, A. Ramanavicius, G. Niaura, L. Abariute, T. Ruzgas, S. Shleev, Mediatorless Carbohydrate/Oxygen Biofuel Cells with Improved Cellobiose Dehydrogenase Based Bioanode, Fuel Cells, 14 (2014) 792.

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[15] Y.-H.P. Zhang, J.-B. Cui, L.R. Lynd, L.R. Kuang, A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidences from enzymatic hydrolysis and supramolecular structure, Biomacromolecules, 7 (2006) 644. [16] P. Qi, C. You, Y.H.P. Zhang, One-Pot Enzymatic Conversion of Sucrose to Synthetic Amylose by using Enzyme Cascades, ACS Catal., 4 (2014) 1311. [17] J.-E. Kim, Y.-H.P. Zhang, Biosynthesis of D-xylulose 5-phosphate from D-xylose and polyphosphate through a minimized two-enzyme cascade, Biotechnol. Bioeng., 113 (2016) 275. [18] H.H. Liao, S. Myung, Y.-H.P. Zhang, One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP Appl. Microbiol. Biotechnol., 93 (2012) 1109. [19] Y. Wang, Y.H.P. Zhang, A highly active phosphoglucomutase from Clostridium thermocellum: cloning, purification, characterization and enhanced thermostability, J Appl. Microbiol., 108 (2010) 39. [20] A.J. Anderson, E.A. Dawes, Regulation of glucose 6-phosphate dehydrogenase in Zymomonas mobilis CP4, FEMS Microbiol. Lett., 27 (1985) 23. [21] Y.R. Wang, Y.H.P. Zhang, Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration, Microb. Cell Fact., 8 (2009) 30. [22] F.F. Sun, X.Z. Zhang, S. Myung, Y.-H.P. Zhang Thermophilic Thermotoga maritima ribose-5-phosphate isomerase RpiB: Optimized heat treatment purification and basic characterization Protein Expr. Purif., 82 (2012) 302. [23] J.S. Martín del Campo, J. Rollin, S. Myung, Y. Chun, S. Chandrayan, R. Patiño, M.W.W. Adams, Y.-H.P. Zhang, High-Yield Production of Dihydrogen from Xylose by Using a Synthetic Enzyme Cascade in a Cell-Free System, Angew. Chem. Int. Ed., 52 (2013) 4587. [24] S. Myung, J. Rollin, C. You, F. Sun, S. Chandrayan, M.W.W. Adams, Y.H.P. Zhang, In vitro metabolic engineering of hydrogen production at theoretical yield from sucrose, Metab. Eng., 24 (2014) 70. [25] S.Y. Huang, Y.H.P. Zhang, J.J. Zhong, A thermostable recombinant transaldolase with high activity over a broad pH range, Appl. Microbiol. Biotechnol. , 93 (2012) 2403. [26] Y. Wang, W. Huang, N. Sathitsuksanoh, Z. Zhu, Y.-H.P. Zhang, Biohydrogenation from biomass sugar mediated by in vitro synthetic enzymatic pathways, Chem. Biol., 18 (2011) 372. [27] S. Myung, Y.R. Wang, Y.-H.P. Zhang, Fructose-1,6-bisphosphatase from a hyperthermophilic bacterium Thermotoga maritima: Characterization, metabolite stability and its implications, Proc. Biochem., 45 (2010) 1882. [28] S. Myung, X.Z. Zhang, Y.H.P. Zhang, Ultra-Stable Phosphoglucose Isomerase Through Immobilization of Cellulose-Binding Module-Tagged Thermophilic Enzyme on Low-Cost HighCapacity Cellulosic Adsorbent, Biotechnol. Prog., 27 (2011) 969. [29] Z.G. Zhu, F.F. Sun, X.Z. Zhang, Y.H.P. Zhang, Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases, Biosens. Bioelectron., 36 (2012) 110. [30] Y. Handa, K. Yamagiwa, Y. Ikeda, Y. Yanagisawa, S. Watanabe, N. Yabuuchi, S. Komaba, Fabrication of Carbon-Felt-Based Multi-Enzyme Immobilized Anodes to Oxidize Sucrose for Biofuel Cells, ChemPhysChem, 15 (2014) 2145. [31] Y. Liu, S. Dong, A biofuel cell harvesting energy from glucose–air and fruit juice–air, Biosens. Bioelectron., 23 (2007) 593.

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[32] D. Wen, X. Xu, S. Dong, A single-walled carbon nanohorn-based miniature glucose/air biofuel cell for harvesting energy from soft drinks, Energy Environ. Sci., 4 (2011) 1358. [33] G.T.R. Palmore, H. Bertschy, S.H. Bergens, G.M. Whitesides, A methanol/dioxygen biofuel cell that uses NAD(+)-dependent dehydrogenases as catalysts: application of an electroenzymatic method to regenerate nicotinamide adenine dinucleotide at low overpotentials, J Electroanal Chem, 443 (1998) 155. [34] D. Sokic-Lazic, S.D. Minteer, Citric acid cycle biomimic on a carbon electrode, Biosens. Bioelectron., 24 (2008) 939. [35] R.L. Arechederra, S.D. Minteer, Complete Oxidation of Glycerol in an Enzymatic Biofuel Cell, Fuel Cells, 9 (2009) 63. [36] D. Sokic-Lazic, S.D. Minteer, Pyruvate/Air Enzymatic Biofuel Cell Capable of Complete Oxidation, Electrochem. Solid-State Lett., 12 (2009) F26. [37] S. Xu, S.D. Minteer, Characterizing Efficiency of Multi-Enzyme Cascade-Based Biofuel Cells by Product Analysis, ECS Electrochem. Letters, 3 (2014) H24. [38] S. Tsujimura, K. Murata, W. Akatsuka, Exceptionally High Glucose Current on a Hierarchically Structured Porous Carbon Electrode with “Wired” Flavin Adenine DinucleotideDependent Glucose Dehydrogenase, J. Am. Chem. Soc., 136 (2014) 14432. [39] Z. Chen, W. Ren, L. Gao, B. Liu, S. Pei, H.-M. Cheng, Three-dimensional flexible and conductive interconnected graphene networks grown by chemical vapour deposition, Nat. Mater., 10 (2011) 424. [40] Z.-Y. Wu, C. Li, H.-W. Liang, J.-F. Chen, S.-H. Yu, Ultralight, Flexible, and Fire-Resistant Carbon Nanofiber Aerogels from Bacterial Cellulose, Angew. Chem. Int. Ed., 52 (2013) 2925. [41] S. Chen, G. He, Q. Liu, F. Harnisch, Y. Zhou, Y. Chen, M. Hanif, S. Wang, X. Peng, H. Hou, U. Schroder, Layered corrugated electrode macrostructures boost microbial bioelectrocatalysis, Energy Environ. Sci., 5 (2012) 9769. [42] C. You, S. Myung, Y.-H.P. Zhang, Facilitated substrate channeling in a self-assembled trifunctional enzyme complex, Angew. Chem. Int. Ed., 51 (2012) 8787. [43] I. Ardao, A.-P. Zeng, In silico evaluation of a complex multi-enzymatic system using onepot and modular approaches: Application to the high-yield production of hydrogen from a synthetic metabolic pathway, Chem. Eng. Sci., 87 (2013) 183. [44] L. Zhang, J. Yuan, Y.F. Xu, Y.H.P. Zhang, X.H. Qian, New artificial fluoro-cofactor of hydride transfer with novel fluorescence assay for redox biocatalysis, Chem. Commun., 52 (2016) 6471. [45] E. Campbell, M. Meredith, S.D. Minteer, S. Banta, Enzymatic biofuel cells utilizing a biomimetic cofactor, Chem. Commun., 48 (2012) 1898. [46] C.E. Paul, I.W.C.E. Arends, F. Hollmann, Is simpler better? synthetic nicotinamide cofactor analogues for redox chemistry, ACS Catal., 4 (2014) 788.

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Figure captions Scheme 1. Schematic of the in vitro 15-enzyme pathway in the anode of the EFC. The enzymes are SP, sucrose phosphorylase; XI, xylose isomerase, PPGK, polyphosphate glucokinase, PGM, phosphoglucomutase;

G6PDH,

glucose

6-phosphate

dehydrogenase;

6PGDH,

6-

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phosphogluconate dehydrogenase; RPI, ribose-5-phosphate isomerase; RuPE, ribulose 5phosphate 3-epimerase; TK, transketolase; TAL, transaldolase; TIM, triose phosphate isomerase; ALD, aldolase; FBP, fructose 1,6-bisphosphatase; PGI, phosphoglucose isomerase; and DI, diaphorase. The metabolites are glucose 1-phosphate (G1P), glucose 6-phosphate (G6P), 6-

5-phosphate

(X5P),

sedoheptulose

7-phosphate

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phosphogluconate (6PG), and ribulose 5-phosphate (Ru5P), ribose 5-phosphate (R5P), xylulose (S7P),

erythrose

4-phosphate

(E4P),

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glyceraldehyde 3-phosphate (G3P), dihydroxyacetone 3-phosphate (DHAP), fructose 1,6bisphosphate (F16P), fructose 6-phosphate (F6P). Pi, inorganic phosphate; and (Pi)n, polyphosphate. Scheme 2. Schematic of the EFC used.

Figure 1. The oxidation of three sugars with different charge extraction efficiencies via the

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enzymatic pathways. (A) Profiles for current generation from the partial oxidation of sucrose using different enzyme pathways to release two, four, eight, or 48 electrons per unit sucrose. (B) Charge extraction efficiency for the partial oxidation of sucrose. (C) Profiles for current generation from the complete oxidation of sucrose, glucose, or fructose. (D) Charge extraction

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efficiency for complete oxidation of sucrose, glucose, and fructose. Figure 2. Fuel load optimization for the sucrose-powered EFC. (A) Amperometric response for

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the EFC with various concentrations of sucrose (0–64 mM). (B) Calibration curve of the current density versus the sucrose concentration. Figure 3. Demonstration of pure sugar-powered EFCs. Profiles of (A) open circuit potential or (B) power density versus current density. Figure 4. Demonstration of pure sugar-mixture-powered or soft drink-powered EFCs. (A) Profiles of the power density versus the current density for mixed sugar-powered EFCs containing 32 mM of sucrose, glucose, and fructose. The 15-enzyme pathway was utilized to oxidize all three simple sugars (black). Alternatively, PPGK and XI were not supplemented, so

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only sucrose was utilized (red). (B) Profiles of the power density versus current density for juice, iced tea, and cola. The fuel load was 20% (v/v). The 15-enzyme pathway was used in this case. Figure 5. Stability of pure sugar-mixture-powered EFCs.

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https://www.youtube.com/watch?v=yp1TYqrwLhs

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Video 1. Demonstration of two cola-powered EFCs that could provide power for a digital clock.

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