Coupling Enzymes and Inorganic Piezoelectric Materials for Electricity

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Coupling enzymes and inorganic piezoelectricmaterials for electricity production from renewable fuels Susana Velasco-Lozano, Mato Knez, and Fernando López-Gallego ACS Appl. Energy Mater., Just Accepted Manuscript • DOI: 10.1021/acsaem.7b00328 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 27, 2018

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ACS Applied Energy Materials

Coupling enzymes and inorganic piezoelectric materials for electricity production from renewable fuels Susana Velasco-Lozanoa,d, Mato Knezb,c and Fernando López-Gallegoa,c,d,e*

a

Heterogeneous biocatalysis group, CIC biomaGUNE, Edificio Empresarial "C", Paseo de Miramón

182, 20014, Donostia, Spain. b

Nanomaterials Group, CIC nanoGUNE, Tolosa Hiribidea, 76, 20018 Donostia, Spain.

c

IKERBASQUE, Basque Foundation for Science, María Díaz Haroko Kalea, 3, 48013, Bilbao, Spain

d

Heterogeneous Biocatalysis Laboratory. University of Zaragoza (iQSCH-CSIC), C/Pedro Cerbuna,

12, Zaragoza, 50009, Spain e

ARAID foundation, Av. de Ranillas 1-D, planta 2ª, oficina B, 50018, Zaragoza, Spain

*Corresponding author. (F. López-Gallego) Tel: +34 943003500 Ext 309, Fax: +34 943003501 E-mail addresses: (F. López-Gallego) [email protected]

KEYWORDS: generators, oxidase, catalase, electricity, energy harvesting

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ABSTRACT Sustainable electricity generation is one of the major current challenges for our society. In this context, the evolution of nanomaterials and nanotechnologies has enabled the fabrication of microscopic devices to produce clean energy from a great variety of renewable sources. To expand the possibilities of energy generation, we have designed and fabricated bio-inorganic generators capable to produce electricity by conversion of chemical energy from renewable fuel sources. Unlike traditional generators, the systems described herein produce mechanical energy through enzyme driven gas-production which generates vibration and pressure that is thus converted into electricity by the action of a piezoelectric component properly integrated into the device. Our generators are able to produce an electric output from different renewable sources like glucose, ethanol and amino acids, attaining energy outputs around 250 nJ•cm-2 and reaching maximum opencircuit voltages of up to 1 V. In addition, the produced energy can be easily regulated by adjusting both enzyme and fuel concentration which can tune the electrical output according to the application. The system described herein proposes a new concept for selfsufficient energy harvesting that bridges biocatalysis and piezoelectricity, where the energy production is based on the piezoelectric effect triggered by enzymatic action rather than on the enzyme-driven electron transfer that governs biofuel-cells. Although the electric output is too low yet to be considered an alternative for energy production, this technology opens the door to power small devices. We envision the utilization of this technology in such remote locations where mechanical energy is lacking but there are chemical energy reservoirs.


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1. INTRODUCTION Concerned by the global warming and the energy crisis, the scientific community seeks the development of clean and self-sufficient systems capable of meeting the energy requirements of our current and future society. The development of energy producing systems from alternative sources has been facilitated by the nanotechnology revolution.1 Nanomaterial-based electricity generators2 are among the most successful examples able to produce up to 1 µJ•cm-2 from mechanical energy.3 This technology furnishes a new generation of energy harvesting self-powered micro devices as implantable biosensors, micro-electro mechanical systems, nanorobots, environmental sensors and personal electronics. For applications in remote inaccessible locations for humans, these devices require an autonomous, independent, continuous and sustainable energy supply instead of using rechargeable batteries that require human intervention and limit their miniaturization.4 In this field, piezoelectric nanogenerators have emerged as an efficient technology to harvest the ubiquitous and abundant environmental mechanical energy.5 In this context, piezoelectric nanomaterials have been widely applied for the development and design of electronic devices and biosensors6-7 using mechanical stimuli as energy source. In piezoelectric materials, surface electric charges are generated when they are submitted to mechanical forces (pressure or vibration) producing a voltage that can be easily measured in an open circuit or harvested and stored in electrical capacitors.8 Several crystalline inorganic materials such as ZnO, CdS, GaN, lead zirconate titanate (PZT), LiNbO3 and BaTiO3 have shown piezoelectric properties.9-10 Although piezoelectric materials are widely exploited for acoustic and analytical applications,11 the first nanogenerator was reported in 2006 by Prof. Wang´s group, this system was based on ptype zinc oxide nanowires (ZnONW) that were deformed through mechanical stimuli in order to produce electricity.12 Since then, a great variety of nanogenerators with a wide 3 ACS Paragon Plus Environment


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diversity of architectures has been designed to harvest mechanical energy from the environment and convert it into electrical energy.13 However, most of these devices need mechanical energy sources whose supply is often discontinuous and hardly tunable, limiting their standalone applications. An ideal nanogenerator should exclusively rely on autonomous mechanical energy sources, which should be continuously and precisely controlled. Additionally, the energy generation must be environmentally sustainable to achieve high energy conversions producing neither toxic wastes nor greenhouse gases. The transformation of free chemical energy into kinetic energy (movement) is a routine task excellently performed by nature,14 since most living organisms require such transformation in order to survive. The idea of powering living systems by transforming chemical energy into mechanical has inspired the scientific community to fuel artificial devices. In particular, enzyme driven bubbling production has been exploited to propel nano- and micromaterials as swimmer sensors to increase their detection efficiency for artificial purposes.15-16 Under this scheme, gas-producing enzymes (like catalase and urease) have been coupled to different nanomaterials in order to achieve self-propelled particles with autonomous motion at particular environments.17-20 The use of H2O2 dominates the pool of fuels for enzyme driven propulsion because catalase is a well-known enzyme that disproportionates H2O2 into H2O and O2; the latter ultimately forms bubbles that propel the particles. Such propulsion power intrinsically triggers a mechanical stimulus that can also be exploited in clean energy generation since the reaction products are innocuous and H2O2 is gaining momentum as energy storage molecule produced by artificial photosynthesis using light and water.21 In this pioneering work, we present a self-sufficient bio-inorganic generator capable to harvest chemical energy from different renewable chemical sources to produce electricity. To this aim, biological machineries (enzymes) that convert renewable chemical fuels


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(chemical energy) into mechanical stimuli (i.e. pressure or mechanical vibration) have been coupled with piezoelectric composites that harvest such mechanical energy through material deformation (bending or compression), generating electricity. The idea of enzymatically transforming chemical energy into electricity gave rise to enzyme and microbial fuel-cells in the early 60´s.22-23 Although this technology is very mature, it suffers electron transfer issues (most of the time mediators are required)24 and is restricted to few chemical fuels, overall for the cathodic reaction (oxygen-breathing dependence). Alternatively, we herein arise with a new concept where the energy generation occurs by cooperation of biocatalysis and piezoelectricity to sequentially transform chemical energy into mechanical one that is eventually converted into electricity. Herein, electron transfer from enzymes to electrodes is not required, and we can find a vast number of gasproducing enzymatic systems (i.e oxidase-catalase pairs) which potentially broaden the repertoire of chemical fuels (sugars, alcohols, aminoacids…).

2. EXPERIMENTAL SECTION 2.1. Materials Reagents and substrates such as hydrogen peroxide, glucose, 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid (ABTS), D-alanine, L-phenylalanine were purchased from Sigma-Aldrich (St. Louis, MO, USA). As well as the enzymes catalase (CAT) from bovine liver, glucose oxidase (GOX) from Aspergillus niger, alcohol oxidase (AOX) from Hansenula sp., D-amino oxidase (DAO) from Trigonopsis variabilis and L-amino acid oxidase (LAO) from Crotalus adamanteus Type I and Horse radish peroxidase (HRP). The piezoelectric material (PZT disc) (diaphragm, 6.3 kHz, 1 KΩ, 0.01 µF, 20 mm x 0.42 mm, cat. 7BB-20-6l0, Murata) was acquired from Farnell Element14 Components (Barcelona, Spain). 5 ACS Paragon Plus Environment


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2.2. Enzymatic activity measurements All enzymatic activities were spectrophotometrically measured in UV-VIS 96-well plates using a VarioskanTM Flash Multimode Reader (Thermo Scientific). The catalase activity was measured by monitoring the absorbance decrease at 240 nm of 35 mM hydrogen peroxide in 50 mM sodium phosphate buffer at pH 7.5 and 25ºC. One unit of catalase activity was defined as the amount of enzyme required for the hydrolysis of 1 µmol of H2O2 per minute at the assayed conditions. The catalytic activities of different oxidases (GOX, AOX, DAO and LAO) were determined by monitoring the timely increased absorbance at 414 nm corresponding to the ABTS (1 mg•mL-1) oxidation catalyzed by HRP (1 mg•mL-1) that uses the H2O2 concomitantly produced by the corresponding oxidase during the substrate oxidation (300 mM glucose or 300 mM ethanol or 13 mM L-phenylalanine or 300 mM Dalanine) in 100 mM sodium phosphate buffer at pH 7 and 25ºC. One unit of GOX, AOX, DAO or LAO activity was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per minute at the assayed conditions. 2.3. Protein quantification Protein measurements were carried out according to Bradford’s methodology.25 Briefly, 200 µL of Bradford mixture were mixed with 5 µL of properly diluted enzyme solution in a 96well plate and incubated for 5 min at 25ºC. Afterwards, the absorbance was measured at 595 nm using a UV-Vis VarioskanTM Flash Multimode Reader (Thermo Scientific). Bovine serum albumin (Cat. P0834, Sigma) was used as standard pattern to estimate the protein content at the same assayed conditions.


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2.4. Electric energy output measurements 2.4.1. Closed system bio-inorganic generator (CBIG) Catalase solution (88 µg•mL-1) in 50 mM sodium phosphate buffer at pH 7.5 was placed inside a Teflon sealed chamber (2 cm internal diameter and 2.5 cm internal height) integrating a valve and coupled with a PZT disc (1.25 cm2 active piezoelectric surface) properly connected to an oscilloscope (Siglent, model SHS806) allowed recording the open-circuit voltage versus time (Fig. S3.A, Supporting Information). Once the output voltage reached 0 V (equilibration time  1 min), H2O2 (100-200 mM) was added through the valve which was immediately closed after the substrate supply. The system was maintained closed during the following 4 minutes and then the valve was opened. The voltage was recorded until the system reached 0 V. The produced energy was calculated according to equation 1 by integrating the curve voltage vs time after the substrate addition with the valve closed (chamber pressurization) and after opening the valve (chamber depressurization).

2.4.2. Open system bio-inorganic generator (OBIG) A PZT disc was coupled with a plastic column of different sizes (0.33 cm2 active piezoelectric surface). The PZT disc was connected to an oscilloscope (Siglent model SHS806) to monitor the open-circuit voltage vs time (Fig. S3.B, Supporting Information). An enzyme solution that contained only CAT or a mix of CAT and one oxidase in 50 mM sodium phosphate buffer at pH 7.5 was placed inside the column. The reaction was triggered by the addition of the substrate, H2O2 (10-600 mM). Besides the H2O2, the added substrate was varied (glucose, ethanol, L-phenylalanine or D-alanine, at the indicated concentration) depending on the oxidase utilized. Before triggering the reaction, the system was equilibrated until the Volt signal reached 0 V. 7 ACS Paragon Plus Environment


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OBIG resistance was measured by recording the maximum open-circuit resistance vs time reached when the system was working with an enzyme solution that contained CAT in 88 µg•mL-1 sodium phosphate buffer at pH 7.5. The reaction was triggered by the addition of 600 mM H2O2 and the system was connected to an oscilloscope (Siglent model SHS806) in resistance mode.

3. RESULTS AND DISCUSSION 3.1. Design and fabrication of bio-inorganic generators Renewable chemicals are excellent sources of energy that can be harvested for sustainable electricity production. We envision the conversion of chemical energy into electricity by efficiently coupling existing biocatalysts (enzymes) with piezoelectric materials. To prove this concept, we have designed and fabricated different bio-inorganic generators by coupling a commercially available CAT from bovine liver with a commercially available inorganic piezoelectric material (PZT disc) composed of lead zirconate titanate separated by bottom-copper and top-silver electrodes. In this system, the enzyme efficiently converts a potentially renewable fuel such as H2O2 into water and molecular oxygen that forms bubbles in solution26. In parallel, the enzyme driven bubbling and gasification stimulates the piezoelectric material transforming the mechanical stimuli into electricity. To prove this concept, we developed two different types of bio-inorganic generators that selectively harvest the two mechanical stimuli resulting from the H2O2 disproportionation ; 1) the pressure that increases due to the oxygen accumulated in the gas phase and 2) the vibrational motions caused by the bubbling. To harvest the pressure increasing, we fabricated a closed system bio-inorganic generator (CBIG) formed by a 5 mL sealed


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chamber with a PZT disc on top. In this setup, the reaction media never contact with the PZT disc (Fig. 1A) because there is a headspace between the working solution and the piezoelectric material. Therefore, the produced oxygen accumulates in that headspace increasing the pressure inside the chamber, which stimulates the piezoelectric material and triggers electric energy output (Fig. 1B). As alternative, we also fabricated an open system bio-inorganic generator (OBIG) that integrates the same PZT disc at the bottom of a 4 mL open column. In this set-up, the reaction media directly contacts with the surface of the PZT disc (Fig. 1C).

A

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Figure 1. Architecture of bio-inorganic generators. A: Closed system bio-inorganic generator (CBIG), cross section view. B) CBIG’s representative electric energy output response. Timing: 0-1 min: system stabilization, 1-1.1 min: substrate administration (green); 1.1-1.8 min: valve closure; 1.8-4 min: gas-produced pressurizing effect (red); 4 min: valve opening; and 4-5 min: gas-released depressurizing effect (blue). C: Open system bio-inorganic generator (OBIG). D: OBIG’s representative electric energy output response. Timing: 0-1 min: system stabilization, 1 min: substrate addition and 1-1.7 min: vibrational effect driven by bubbles (blue).



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Hence, the enzyme driven bubbling is traduced into a vibration stimuli on the surface of the piezoelectric material that triggers its charge rearrangement, giving rise to electrical output (Fig. 1D). Since the OBIG system was in contact with the H2O2 solution, the top-silver electrode was damaged and the performance of the system was unreliable. To overcome this issue, we deposited a silicon dioxide protective layer (5 nm) above this electrode in order to prevent the silver oxidation and spam OBIG working half life time. The SiO2 coating allows maintaining the same initial electric power output response after more than 20 uses.

3.2. Performance of Closed system bio-inorganic generator (pressure response): In the CBIG architecture (Fig. 1A), the reaction mixture never contacts with the piezoelectric material placed at the top of the sealed chamber. Once we add the substrate fuel (min 1, Fig. 1B, green zone), CAT starts oxidisizing H2O2 until the valve is closed, then under sealed conditions the enzymatically produced molecular oxygen increases the gas pressure at the head space (Fig. 1B, red zone). After several minutes of operation, we depressurize the chamber by opening the valve which releases the accumulated gas during the enzymatic reaction (Fig. 1B, blue zone). To quantify the electrical output obtained with both pressurization and depressurization events, we recorded the open-circuit voltage versus time and calculated the energy of the electrical outputs according to equation 1.27

Eq. 1 Where E is the generated electrical energy, V is the generated voltage from the start (t1) to the end (t2) of a cycle at a constant resistance load (R). R value was fixed to 60 MΩ (experimentally determined at the maximum produced voltage by the enzymes). Interestingly, the obtained 0.18 and 0.20 nJ•cm-2 for the pressurizing and depressurizing cycles, respectively, indicate that the compression (Fig. 1B, red zone) and decompression (Fig. 1B, blue zone) of the PZT disc require the same energy, although the maximum 10 ACS Paragon Plus Environment

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voltage produced during the decompression was higher than during the compression. Differences between total energy and maximum voltages might rely on the kinetics of the pressurizing and depressurizing mechanisms. While the enzyme-driven pressurizing requires longer times to reach the maximum pressure inside the sealed chamber, the manual depressurizing is extremely fast. Hence, depressurizing produces the same energy than pressurizing but in shorter time, which explains why the maximum voltage obtained during the pressure release is higher than during the pressure accumulation. Then, we evaluated the effect of the amount of fuel (H2O2) on the electric energy output of CBIG. We observed that larger working volumes and higher H2O2 concentrations generated higher energy outputs (Figure 2A). Expectedly, when the system was fueled with more H2O2, it produced more gas that was concentrated inside smaller head space volumes. Consequently, the system creates higher oxygen pressures in the top-gas phase of the CBIG system that are translated into higher energy productions. The same experiments lacking either the enzyme or the substrate resulted in no electric output, which means that the electric energy is directly and exclusively related to harvesting the mechanical energy produced by the enzymatic disproportionation of hydrogen peroxide. To study the operational window of CBIG systems, we evaluated the system response when working at sequential pressurizing/depressurizing cycles after a single fuel addition in the first cycle (Figure 2B). Performing 1 minute pressurizing/depressurizing cycles, we detected an electric output production after 10 cycles using 44 μg•mL-1 CAT; this result indicates that during 10 minutes the enzyme is continuously working although the amount of fuel inside the chamber is lower after each cycle. However, using 88 μg•mL-1 CAT under the same pressurizing/depressurizing conditions, the system generated electric outputs during only 5 cycles; the more enzyme inside the chamber, the fuel is faster consumed. When the reaction rate was higher (high enzyme concentrations), the total energy after 5



cycles was 361 pJ•cm-2 with a maximum voltage of 88 mV after the first cycle, while slower reactions produced the same energy but during 10 cycles, achieving half of the maximum voltage after the first cycle (46 mV, Fig. 2B). Therefore, the sum of energy obtained after each pressurization/depressurization cycle is proportional to the fuel concentration regardless the reaction rate, however the maximum voltage depends on the reaction rate. Hence, we can modulate the duration and the intensity of energy output obtained with CBIG just by adjusting the enzyme loading and/or the fuel concentration in the working chamber.

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Valve openings Figure 2. Electric energy output of CBIG. A) Effect of substrate concentration and reaction volume on the produced energy (bars) and maximum produced voltage (circles). H2O2 100 mM (gray) and 200 mM (white). All values correspond to chamber depressurization. In all cases the reaction mixture was composed by CAT -1 (88 µg•mL ) and H2O2 in phosphate buffer 50 mM pH 7.5 at 25ºC. B) Effect of catalase concentration. In all cases reaction mixture was composed by 5 mL of 100 mM H 2O2 in phosphate buffer 50 mM pH 7.5 at 25ºC. Energy values correspond to the produced energy when the chamber was repeatedly depressurized every minute (valve opening).

In the light of these results, the CBIG system harvests the pressure stimuli generated by an enzymatic reaction that converts a chemical fuel into gas. Furthermore, this system can easily tune the electric output by controlling the valve opening, the working volume and both fuel and enzyme concentrations. These two features make this system very attractive for further applications in self-powered devices where variable energy demands are required.


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3.3. Performance of the open system bio-inorganic generator (vibration response): Beside pressure stimuli, we also exploited vibrational motions as mechanical stimuli. To this aim, we designed an open bio-inorganic generator (OBIG) able to harvest vibrational mechanical

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disproportionation catalyzed by CAT (Fig. 1C). Here, the mechanical stimulus lasts until the enzyme has consumed all the fuel (peroxide), yielding a voltage output along the reaction and defining so one operation cycle (Fig. 1D). To evaluate the performance of OBIG, we firstly tested different enzyme and fuel concentrations (Fig 3). Like the CBIG system, OBIG generates a higher electric energy when using higher enzyme concentrations (Fig. 3A). Furthermore, the bio-inorganic generator achieved the highest electric energy outputs at 400-600 mM of H2O2 and 88 µg•mL-1 CAT (Fig. 3B). This behavior indicates that the bubbling generation rate driven by CAT relies on fuel (substrate) concentration and thus determines the magnitude of the electrical outputs. Hence, the PZT disc harvests higher energy when bubbles are formed faster, creating a stronger mechanical stimulus that is also translated into a higher maximum voltage.

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Figure 3. Effect of CAT (A) and fuel concentration (B) on the harvested energy (black squares) and maximum voltage (white squares) using OBIG system. Reaction conditions: A) The working solution contains CAT (at different concentrations), 50 mM H2O2 in 50 mM phosphate buffer at pH 7.5 and 25ºC. B) The working -1 solution contains 88 µg•mL CAT, H2O2 (different concentrations) in 50 mM phosphate buffer at pH 7.5 and 25ºC.



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3.4. Productivity of open and close bio-inorganic generators To adequately evaluate and compare the performance of both bio-inorganic generators (closed and open systems), we defined a new parameter named as generator’s productivity according to Eq 2. Eq. 2 This parameter reflects the efficiency of one bio-inorganic generator to harvest chemical energy from fuels and convert it into electric energy. Table 1 shows that OBIG transforms chemical energy into electric energy up to 816 times more efficiently than CBIG under the same fuel concentration (200 mM H2O2). This difference points out that the mechanical stimuli triggered by pressure increasing due to oxygen accumulation in the head space is weaker than the mechanical stimuli caused by vibrational motions promoted by the enzyme driven bubbling. This effect may be also related to the inherent working mechanism of PZT generator, which prefers periodical deformation occurred in the open system, rather than the unidirectional pressure generated in the closed system. Remarkably, OBIG achieves a maximum productivity of 1.37 µJ•mmol-1•cm-2 when the system is working at fuel saturation conditions.

Table 1. Performance of bio-inorganic generators System H2O2 Working volume (mM) (mL) CBIG 100 3.5 200 3.5 100 5.0 200 5.0 OBIG 100 0.4 200 0.4 400 0.4 600 0.4

Energy (nJ•cm-2) 0.21 ± 0.02 0.67 ± 0.25 0.80 ± 0.03 1.43 ± 0.21 25 ± 1.8 94 ± 8.7 220 ± 14.9 265 ± 26.2

a

Productivitya (nJ•mmol-1•cm-2) 0.6 ± 0.27 1.0 ± 0.36 1.6 ± 0.05 1.4 ± 0.21 620 ± 44 1170 ± 109 1372 ± 93 1106 ± 109 2

Productivity is calculated as the electric energy (nJ) per mmol of H2O2 per area (cm ). All reaction were -1 carried out with 88 µg x mL CAT at 25º.


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Like for the closed system, we studied the effect of the working volume on the performance of the closed system. OBIG operated at different working volumes but it was less productive with higher volumes because the harvested energy remained constant at working volumes higher than 1 mL (Fig. 4). This plateau in energy harvesting was not observed in the CBIG system (Fig. 2A). In the light of these results, we suggest that the differential effect of working volume on the performance of the OBIG and CBIG systems relies on the different nature of the mechanical stimuli; pressure or vibration. In the case of CBIG, the energy harvested is enhanced by a high accumulation of enzymatically produced oxygen in smaller head spaces, which significantly increases the gas pressure inside the closed chamber and provokes a higher mechanical deformation of the PZT disc. In this scenario, high reaction volumes generate and concentrate more oxygen in smaller head spaces due to the chamber volume limitation. In contrast, OBIG harvests the energy through local contacts between the gas bubbles and the PZT-disc surface creating the vibration stimulus that triggers the piezoelectric effect. In the OBIG system, the stimulation area of PZT remains constant despite using taller chambers that accommodate larger working volumes; this setup explains the saturation in energy harvesting found for the OBIG system. Consequently, the productivity of the open system decays at larger volumes because the total amount of fuel in the open chamber is higher while the harvested energy remains constant. This study demonstrates that the concentration of fuel is more effective than the total amount of fuel to more efficiently harvest and convert the chemical energy into electricity. In our hands, OBIG system optimally operated with 200 µL of working volume containing 600 mM H 2O2 and 88 µg•mL-1 CAT generating 265 nJ•cm-2 with an open-circuit maximum voltage of 0.9 V and a productivity of 1.11 µJ•mmol-1•cm-2. This electrical output is translated into 4.4 nW•cm-2; a figure one order of magnitude lower than the power densities required to power some of the state-of-the-art nanosensors (83 nW•cm-2).28 Besides, the maximum electric output of OBIG



falls in the range of the energy densities reported for nanogenerators based on ZnO NW and PZT ceramics which can generate 1-1000 nJ•cm-2.3 Despite the low produced electric energy (3 order of magnitude lower than state-of-the-art enzyme fuel cells), we are introducing a new concept to convert chemical energy into electricity by coupling enzymes and piezoelectric materials. At this starting point, the technology herein described works with a commercial piezoelectric device which is not designed ad hoc to maximize gas bubbling harvesting. We are thus envisioning future piezoelectric materials and enzyme complexes specifically designed to maximize electrical energy production of these bio-inorganic generators. When one looks at mature technologies like enzyme fuel cells, one realizes that three decades of intense research were needed to improve their performance from µW•cm -2 to mW•cm-2.29 Likewise, the nanogenerators based on piezoelectric materials, which were firstly described in 2006, have been optimized in the last decade to produce 1-100 mW•cm-2.13 Therefore, these pioneering bio-inorganic generators can follow an optimization pathway to generate electric outputs comparable with enzyme fuel cells or nanogenerators that harvest environmental

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3.5. Production of electric energy from different renewable fuels In nature we can find a great variety of enzymatic systems that produce other gases beyond oxygen. Inspired by nature, we thus exploited the OBIG system using other renewable liquid fuels that can be readily transformed into gases by enzymatic action. Initially we evaluated CO2-producing enzymes but unfortunately we did not detect electrical outputs because the gas-bubbling was insufficient to stimulate the piezoelectric response of the PZT disc (Table S1, Supporting Information). The ineffective CO2-bubbling conducted by the enzymes is likely due to the low Henry constant of CO2 (KH = 29 L x atm x mol-1)30 that requires too high CO2 pressures to form bubbles.31 On the contrary, the O2-producing system based on CAT is highly efficient because KH(O2) is one order of magnitude higher than KH(CO2). Taking into account the physicochemical properties of the gases, we further investigated bi-enzymatic systems coupling a catalase with a large family of enzymes; the flavin dependent oxidases that generate H2O2 as by-product when oxidizing a broad diversity of substrates such sugars, aminoacids, alcohols, etc. That H 2O2 is concurrently disproportionate by CAT to generate oxygen bubbles that trigger the vibration stimulus (Scheme 1).

A

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Scheme 1. A) Oxygen and hydrogen peroxide production from glucose by coupling an oxidase (OX) and a catalase (CAT). B) Gas production from renewable fuels catalyzed by bi-enzymatic systems composed by CAT and oxidase.



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To prove this concept, we firstly utilized the well-known bi-enzymatic system composed by a glucose oxidase (GOX) and CAT.32 Herein, GOX catalyzes the glucose oxidation into gluconate with the concomitant production of H2O2 which is ultimately disproportionated into O2 and H2O by CAT (Scheme 1). Unfortunately, the OBIG integrating GOX and CAT and fed with only glucose failed in the production of electricity. We suggest that this failure is due to the fact that flavin-dependent oxidases require stoichiometric amounts of oxygen as electron acceptor to catalyze the oxidation of the substrate, limiting the fast generation and high accumulation of H2O2 needed for an efficient bubbling that triggers an effective piezoelectric response. To overcome this issue underlying the oxidase family, we added initial small amounts of H2O2 to trigger the oxidase action with the aim of pairing the H2O2 generation and gas-bubble formation rates. The oxygen replenishment performed by catalase is described in several works.33-34 In order to demonstrate this effect in our system, we measured the displaced gas volume when catalase/GOX decomposes 50 mM of H 2O2 in presence and absence of 300 mM of glucose. The displaced volume was 3.7 times higher when glucose was added to the system (0.14 mL and 0.56 mL with or without glucose, respectively), thus demonstrating higher oxygen production. When we compared the performance of the OBIG system run by either CAT or the CAT/GOX pair, the bi-enzymatic system using glucose and H2O2 as fuels generated up to 6 times higher electric energy than the single CAT (Fig. 5A). Likewise, the open-circuit peak voltage was 2 times higher when OBIG is driven by the CAT/GOX pair, although the extent of the improvement in maximum voltage was lower than in the harvested energy (Fig. 5A). Moreover, the productivity was twice higher when OBIG incorporates GOX/CAT pair than when using only CAT despite the addition of 100 mM of glucose (Fig. 5A). To optimize the OBIG system operated with the GOX/CAT pair, we studied the effect of CAT concentration and the GOX/CAT activity ratio on the electric output. As well as using single CAT, the


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OBIG operated with the bi-enzyme system worked more efficiently at high CAT concentrations since the H2O2 disproportionation was faster, speeding up the releasing of molecular oxygen that supplies the GOX to oxidize the glucose and produces more peroxide. The minimal CAT concentration to harvest the maximum energy (25 nJ•cm-2) under these conditions is 18 µg•mL-1 CAT, although the maximum voltage peak was obtained with at least 88 µg•mL-1 CAT (Fig. S1, Supporting Information). On the other hand, the higher GOX/CAT mass ratio under the optimal CAT concentration (88 µg•mL-1) enhanced the glucose oxidation that accumulated faster and more H2O2 in the working solution, which boosted the CAT action to generate a more powerful mechanical stimulus that ends up in higher electricity production (Fig. 5B). Finally, we studied the effect of glucose concentration on the OBIG performance. We found longer periods of electricity generation at higher glucose concentrations (Fig. S4, Supporting Information), harvesting more energy up to reaching a maximum plateau at 68 mM glucose (Fig. 5C). At higher glucose concentrations, GOX is practically working under saturation conditions (substrate concentration 3 times higher than KMGlucose)35 harvesting the same electric energy but the system’s productivity significantly decays because of the large excess of fuel (Fig. 5C). Therefore, under the optimal conditions with only 50 mM H2O2, the OBIG system driven by GOX/CAT pair generated up to 26 nJ•cm-2 and a voltage peak of 0.159 V with a maximum productivity of 443 nJ•mmol-1•cm-2. These values point out that the use of glucose as co-fuel improves the harvested energy, the maximum voltage and the system productivity 6.3, 2.3 and 2.1 times, respectively, compared to the systems fueled only with H2O2 under the same conditions.



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0

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Figure 5. GOX/CAT bi-enzymatic system coupled to OBIG. A: OBIG performance when the system is composed only by CAT (black) and by the two enzymes GOX/CAT (light gray). In all cases the reaction -1 -1 mixture was composed by CAT (88 µg•mL ) or GOX/CAT (GOX at 0.75 mg•mL ), 100 mM glucose and 50 mM H2O2 in 50 mM phosphate buffer pH 7.5 at 25ºC. B: Effect of different GOX/CAT activity ratios in the produced energy (black squares) and maximum voltage (white squares) by the GOX/CAT-OBIG. In all cases -1 -1 the reaction mixture composed by CAT (88 µg•mL ), GOX (0-750 µg•mL ), 50 mM H2O2 and 100 mM glucose in 50 mM phosphate buffer pH 7.5 at 25ºC. C: Effect of glucose concentration in the produced energy (black squares) and productivity (black triangles) by the GOX/CAT-OBIG. In all cases the reaction mixture -1 -1 was composed by CAT (88 µg•mL ) and GOX (0.38 mg•mL ), glucose (as indicated) and 50 mM H2O2 in 50 mM phosphate buffer pH 7.5 at 25ºC.

These excellent results encourage us to broaden OBIG’s workability towards other liquid renewable fuels employing different oxidase/catalase pairs. For this purpose, we investigated other bi-enzyme system beyond the GOX/CAT pair: alcohol oxidase/catalase (AOX/CAT), D-amino acid oxidase/catalase (DAO/CAT) and L-amino acid oxidase/catalase (LAO/CAT) that utilize ethanol, D-alanine and L-phenylalanine as co-fuels, respectively. Like the GOX/CAT pair, the higher mass ratios of AOX/CAT maximized the electric outputs of OBIG (Fig. S2, supporting information). Therefore, we compared the harvesting efficiency of OBIG driven by the four different catalase/oxidase pairs under the same fuel concentration. All the tested catalase/oxidase systems harvested more energy and generated higher maximum voltages than the system using only CAT (Fig. 6). Nevertheless, all of them required the initial addition of H2O2 to trigger the oxidase activity. Interestingly, the DAO/CAT pair showed the highest electric energy production and productivity among all the bi-enzyme systems herein tested. The excellent performance of the DAO/CAT pair may rely on the high inhibition constant of DAO towards H2O2 compared to the other three oxidases. Moreover, DAO presents a relatively low KM towards oxygen and D-alanine (co-fuel) compared to the other oxidases tested in


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this study (Table S2 Supplementary Information). Therefore, the excellent affinity of DAO for its natural substrates and the low inhibition by hydrogen peroxide make this enzyme an excellent catalyst to convert bio-based materials such as amino acids into H2O2, which is further converted to molecular oxygen creating a vibrational energy that triggers the piezoelectric effect which ultimately produces the electric output. High productivities were also obtained with the OBIG driven by LAO, however the output energy was substantially lower than with other oxidase/catalase pairs due to the low solubility of the co-fuel (Lphenylalanine). All these systems demonstrate the functionality and versatility of the bioinorganic generators to transform chemical energy from different renewable sources into electrical outputs.

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Enzymatic system Figure 6. Different oxidase/catalase pairs coupled to OBIG system. The produced energy (light gray bars) -1 and productivity (dark gray bars). In all cases the reaction mixture was composed by CAT (88 µg•mL ) and an oxidase (for GOX, AOX and DAO and activity ratio catalase/oxidase = 46:1; and for LAO an activity ratio of 4934:1), 100 mM glucose or 100 mM ethanol or 100 mM D-alanine or 13 mM L-phenylalanine, for GOX, AOX, DAO or LAO, respectively; and 50 mM H2O2 in 50 mM phosphate buffer pH 7.5 at 25ºC.

4. CONCLUSIONS We have designed and fabricated bio-inorganic generators capable of producing electricity from renewable chemical sources. These systems are based on biocatalytic systems that



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covert chemical energy into mechanical energy, coupled to a piezoelectric material that harvests that mechanical energy to ultimately produce an electric output. This system needs to be further optimized to generate higher electrical output in order to power state-ofart biosensors. The architecture of these bio-inorganic generators is easily adaptable to different renewable fuel sources by a suitable selection of the enzymatic system. Coupling flavin oxidases and catalases have been shown most efficient to maximize the energy production and voltage. Interestingly, the generated electric outputs can be simply regulated by properly controlling the enzyme and fuel concentrations in the working solutions. Nowadays, several nanogenerators have been described; however, all of them are based on harvesting mechanical stimuli from the environment to produce electric energy, but none incorporates biological machineries as mechanical stimuli generators. To the best of our knowledge, this is the first work where bio-inorganic generators autonomously harvest chemical energy from renewable sources in situ generating a mechanical energy that is further converted into electric outputs. These promising results open a new field of opportunities for coupling enzymes with piezoelectric materials for the sustainable electricity production. Besides, we foresee the expansion of this concept to more complex multi-enzyme system to produce other types of gas that provide more vigorous mechanical stimuli. Similarly, this concept may be translated to other piezoelectric materials with better properties in mechanical energy harvesting and finally integrate these architectures into flow systems for repeated and continuous operations, aiming at extending their uses at particular industrial applications.

COMPETING INTERESTS The authors declare not competing financial interests.


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SUPPORTING INFORMATION Supporting information related to this document contains: Table S1 with a list of the assessment gas-producing enzymes triggering bubbling effect. Table S2 with kinetic parameters of different oxidases. Figure S1: Effect of different CAT concentrations in the produced energy and maximum voltage by OBIG when the system is composed only by CAT and by the two enzymes CAT/GOX. Figure S2: Effect of different enzymatic activity ratios in the produced energy output by the CAT/AOX-OBIG. Figure S3. Bio-inorganic generators connected to an oscilloscope. Figure S4. Effect of glucose concentration in the produced energy by the GOX/CAT-OBIG.

ACKNOWLEDGEMENTS

We would like to thank Marie-Curie Actions (NANOBIENER project), IKERBASQUE foundation for funding Dr. F. López-Gallego and the support of COST Action CM1303 Systems Biocatalysis. We also acknowledge to Dr. Pedro Ramos (CIC biomaGUNE) and Dr. Luis Yate (XPS platform, CIC biomaGUNE) for the fabrication of the Teflon based CBIG chamber and the deposition of different metal layers on the top of the PZT disc, respectively. We also acknowledge to HERGAR foundation for the founding.

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Coupling Enzymes and Inorganic Piezoelectric Materials for Electricity

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