Sustainable Bioelectrosynthesis of the Bioplastic Polyhydroxybutyrate

Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

pubs.acs.org/journal/ascecg

Sustainable Bioelectrosynthesis of the Bioplastic Polyhydroxybutyrate: Overcoming Substrate Requirement for NADH Regeneration Bassam Alkotaini,† Sofiene Abdellaoui,† Kamrul Hasan,† Matteo Grattieri,† Timothy Quah,† Rong Cai,† Mengwei Yuan,† and Shelley D. Minteer*,† †

Department of Chemistry, University of Utah, 315 S. 1400 E. Room 2020, Salt Lake City, Utah 84112, United States S Supporting Information *

ABSTRACT: One of the main limitations to achieve sustainable synthesis of polyhydroxybutyrate (PHB) is the cost of NADH regeneration, as it requires a side enzymatic reaction usually including a NAD-dependent dehydrogenase enzyme with its substrate or other photo- and electrochemical approaches that create unwanted byproducts and the enzymatically inactive dimer NAD2. Herein, a bioelectrocatalytic method combining both enzymatic and electrochemical approaches was used to regenerate enzymatically active NADH. The method employed a modified glassy carbon electrode that possesses both NADH regeneration and acetoacetyl-CoA (AcAcCoA) reduction features. The modified electrode exhibited an apparent Michaelis constant (KM) value of 814 ± 11 μM and a maximum current density (jmax) of 27.9 ± 1.3 μA cm−2 for NAD+ reduction and a KM value of 47 ± 2 μM and jmax of 0.97 ± 0.03 μA cm−2 for AcAcCoA reduction. The modified electrode was subsequently employed in the bioelectrosynthesis of the bioplastic PHB and yielded 1.6 mg in a 5 mL reaction mixture, indicating that the NADH was regenerated at least 8 times during the 16 h reaction. KEYWORDS: Sustainable bioplastic, Bioelectrosynthesis, in vitro, Polyhydroxybutyrate, NADH regeneration

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INTRODUCTION Polyhydroxyalkanoates (PHAs) are biocompatible and sustainable biodegradable polyesters that are produced by a variety of microorganisms.1 Polyhydroxybutyrate (PHB) is the most common type of PHA, which is accumulated in some microbial cells under nitrogen nutrient-limited conditions as carbon and energy stocks.2 Due to its biodegradability, biocompatibility, and thermal processability, PHB has gained a commercial value comparable with that of petroleum-derived plastics for a sustainable future.3 According to a report published by the European Bioplastics Association (http://www.europeanbioplastics.org) in 2017, the global production capacities of biodegradable plastics are growing steadily from around 0.9 million tons in 2016 to almost 1.3 million tons in 2021, in which PHB is expected to quadruple by 2021 compared to 2016. PHB is currently produced by global companies such as Biomer (Krailling, Germany), Metabolix (MA, United States), Kaneka (Takasago, Japan), and Bio-on (Bologna, Italy).4 In the past decade, the production of PHB has been widely investigated through the accumulation of the biobased plastic inside the cell of several microorganisms such as Cupriavidus necator, Haloferax mediterranei, Bacillus megaterium, and recombinant Escherichia coli strains.5 In general, the large scale production of PHB is carried out by a fermentation process using different bacterial cells (in vivo). However, the commercial viability of PHB undoubtedly depends on the cost of the production process presented in relatively cheap © XXXX American Chemical Society

substrates for the polymer, PHB yield on the substrate, and the efficiency of product formulation in downstream processing.6,7 On the basis of these factors, the cost of production can range from $4−16 per kg; however, the price should be reduced to $3−5 per kg to be commercially feasible.8 Therefore, focus has been on development of efficient production and downstream processes to achieve competitive PHA price compared with petroleum-derived plastics.9 Researchers have reported different ways to reduce the cost of the PHB production such as using low cost substrates, including whey, starch, algae, and sugar cane,10,11,5,12 and efficient substrate hydrolysis process.13,14 Other attempts investigated the in vitro production as a potential low cost procedure, because it simplifies the downstream processing, as well as shortens the process time to approximately 12 h compared to the in vivo microbial fermentation that requires at least 48 h.14,15 Acetyl-CoA is the starting intermediate of the tricarboxylic acid (TCA) cycle and is considered as the key precursor for PHB biosynthetic pathway.16,17 β-Ketothiolase (phaA) catalyzes the reversible condensation reaction of two molecules of acetyl-CoA to form acetoacetyl-CoA (AcAcCoA), which is further reduced by NADH-dependent acetoacetyl-CoA reductase (phaB) to 3-hydroxybutyryl-CoA (3HBCoA), the Received: November 22, 2017 Revised: February 18, 2018 Published: February 19, 2018 A

DOI: 10.1021/acssuschemeng.7b04392 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

NADH as opposed to the formation of irreversible enzymatically inactive dimerization product NAD2 obtained by the electrochemical methods.26,27 In the present study, phaA, phaB, and phaC were expressed, purified, and assayed. A glassy carbon (GC) electrode was modified with a redox polymer, N-benzyl-N′-propyl-4,4′bipyridinium-modified linear polyethylenimine, benzylpropylviologen (BPV-LPEI) cross-linked with a diaphorase (DH) for the regeneration of NADH. On top of that, an additional layer of LPEI-based polymer, octyl-linear polyethylenimine (C8LPEI), and phaB were immobilized onto the same electrode for the reduction of AcAcCoA into 3HBCoA by the regenerated NADH. The kinetics of the modified electrode were evaluated for NADH regeneration and AcAcCoA reduction, and the electrode was then employed in the synthesis of PHB in the presence of acetyl-CoA synthase (Acs), phaA, and phaC in the reaction mixture. Herein, we reported the first study that introduced the bioelectrocatalytic regeneration of NADH accompanied by the reduction of AcAcCoA for the in vitro conversion of acetate into PHB, as illustrated in (Figure 2).

subunit of PHB. Finally, PHB is produced by a irreversible polymerization reaction of 3HBCoA molecules with PHA synthase (phaC) releasing CoA (Figure 1).18,19

Figure 1. Biosynthesis pathway of acetyl-CoA to PHB.

On the other hand, the in vitro PHB synthesis pathway requires supply of exogenous NADH rather than being in vivo regenerated by other metabolic pathways such as the pentose phosphate pathway and the TCA cycle.20 Considering the low stability and the high market price of NADH (1 g of NADH is estimated to be $20−2521), addition of NADH is relatively expensive for the PHB production and thus increases the production cost. Recent studies have investigated the production of PHB through the regeneration of NADH using a side enzymatic reaction involving glucose or lactate dehydrogenases and glucose or lactate as electron donors. The reaction produces gluconolactone and releases two electrons for the reduction of NAD+ to NADH.15,22 However, employing substrates such as glucose or lactate lead to unprofitable cost-intensive production processes because those substrates are listed as food-based materials.4,23 Therefore, it is essential to employ an alternative to these substrates as an electron donor that does not compete with the foodbased materials for the reduction of NAD+ to achieve a sustainable production process of PHB based on a renewable energy source. Alternative approaches for NADH regeneration such as chemical and photochemical processes have been described to require complicated procedures and usually yield unwanted byproducts.24,25 Consequently, a bioelectrocatalytic approach is a promising strategy for NADH regeneration which has significantly enhanced the production of active isomers of

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EXPERIMENTAL SECTION

Chemicals and Strains. PHB was purchased from Sigma-Aldrich. NADH/NAD+, and E. coli One Shot TOP 10 and One Shot BL21(DE3)pLysS were purchased from Invitrogen by Thermo Fisher Scientific, United States. Diaphorase (DH, EC 1.6.5.2) was purchased from Asahi Kasei, Japan. The enzyme was cloned from B. megaterium and expressed in E. coli. Acs was purchased from Sigma-Aldrich. Sodium phosphate dibasic anhydrous and citric acid monohydrate were purchased from Fisher Scientific. Ethylene glycol diglycidyl ether (EGDGE) was purchased from Polysciences, Inc., United States. GC electrodes (3 mm in diameter) and saturated calomel reference electrodes (SCE) were purchased from CH Instruments. BPV-LPEI and C8-LPEI were previously synthesized in our laboratory by literature protocols.26 All solutions and buffers were prepared using deionized water produced by a Millipore Type 1 (Ultrapure) Milli-Q system (18.2 ΩM cm). Cloning, Expression, and Purification of Enzymes. The genes encoding phaA, phaB, and phaC from C. necator ATCC 17699 were synthesized by Integrated DNA Technologies synthesis service for expression in E. coli BL21. To obtain a biological active phaC gene product, the third nucleotide of the second codon was changed from GCG(Ala) to GCT(Ala).18 The designated genes were inserted in a linearized pET28a using Gibson assembly kit. The constructed plasmids were transformed into E. coli BL21 (DE3) and grown on

Figure 2. Representation of the bioelectrocatalytic conversion of acetate (underlined) into PHB. The reaction is mediated by four enzymatic reactions and additional one on the modified electrode for the NADH regeneration. Acs: acetyl-CoA synthase, phaA: β-ketothiolase, phaB: acetoacetyl-CoA reductase, phaC: PHB synthase, DH: diaphorase. B


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reduction of NAD+. The current was recorded every 0.1 s, and NAD+ or NADH was injected when a stable current was observed.26 Kinetics of NADH-Dependent phaB. For the analysis of the kinetic parameters KM and Vmax, the assay was run in triplicate with constant AcAcCoA concentration of 100 μM and NADH concentrations that varied from 0 to 1200 μM. The decrease in absorbance at 340 nm was recorded, and the reaction velocities were calculated as described in the assay section. The KM and Vmax were obtained using Michaelis−Menten equation. On the other hand, the electrochemical kinetics were measured at constant polarization of −0.63 V in the presence of 100 μM AcAcCoA. After current stabilization, NADH was injected at different rates. The KM, Vmax, and jmax were obtained by measuring the stabilized current density produced after each injection, and the data were plotted using the Michaelis−Menten equation. Bioelectrosynthesis of PHB. The bioelectrocatalytic in vitro synthesis of PHB was performed at constant polarization of −0.63 V vs SCE for 16 h at room temperature. The reaction mixture contained 100 mM citrate phosphate buffer (7.0 pH), 60 mM sodium acetate, 60 mM ATP, 1 mM CoA, 0.4 mM NADH, 5 mM MgCl2, 130 U of phaA, 10 U of phaC, and the modified electrode. The reaction was initiated by the addition of 0.4 U of Acs after current stabilization, and the developments of the current were recorded every 0.1 s. Three controls were employed: the first one was performed using a modified electrode with denatured DH in the reaction mixture, and the second control included the modified electrode in a reaction mixture without sodium acetate. The third control was performed using the modified electrode in the reaction mixture without applied potential. At 4 h intervals, a total of 3 mL was withdrawn to measure the optical density at 600 nm and then returned back to the reaction. By measuring the OD600 of standard PHB concentrations ranging from 0.1 to 0.6 mg/mL, the amount of the synthesized PHB during the reaction were determined based on their OD600. At the end of the reaction, the PHB pellets were harvested, lyophilized, and extracted with D-chloroform (99.8%) at 60 °C for 3 h. Part of the organic fraction was then dried and analyzed by measuring the dry weight and using FT-IR; meanwhile, the other part was analyzed with 1H NMR.

LB media containing kanamycin. The expression of the enzymes was induced at an OD600 of about 0.6 by adding isopropyl β-D-1thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM and further incubating the samples at 20 °C for 24 h. The cells were harvested by centrifugation and resuspended in a lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4). The cells were disrupted by the microfluidizer method (3× passages ∼18 000 psi). The histidine-tagged enzymes were purified with a HisTrap-HP (1 × 5 mL) column (GE Healthcare) in a FPLC system (Ä KTA, Sweden) according to the protocols of the manufacturer. The histidine-tagged enzymes were eluted with elution buffer (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, pH 7.4). The purified enzymes were pooled and concentrated using 10 kDa MWCO centrifugation tubes, and the resulted phaA and phaB fractions were dialyzed overnight against 20 mM Tris-HCI (pH 8.0) containing 5 mM EDTA, lyophilized, and stored at −20 °C. Meanwhile phaC fraction was dialyzed overnight against 10 mM sodium phosphate (pH 7.2) containing 5% (v/v) glycerol, lyophilized, and stored at −80 °C for future use.21 The protein concentration was determined using Pierce BCA protein assay kit (Life Technologies, Carlbad, CA). Enzymes Activity Assays. phaA was assayed by the thiolysis reaction of AcAcCoA. The reaction mixture contained 100 mM citrate phosphate buffer (7.0 pH), 50 mM MgCl2, 0.06 mM AcAcCoA, 0.06 mM CoA. The reaction was initiated by the addition of phaA, and the rate of AcAcCoA decrease was measured at 303 nm using a spectrophotometer (Hitachi, Tokyo). The concentration of AcAcCoA was measured at a molar absorption coefficient of 12.9 mM−1 cm−1. One unit (U) was defined as the amount of enzyme that catalyzed the conversion of 1 μmol of AcAcCoA per min.19 Enzyme phaB reductase was assayed by NADH reduction. The reaction mixture contained 100 mM citrate phosphate buffer (7.0 pH), 0.05 mM AcAcCoA, 0.2 mM NADH, and 2 μg of enzyme. The reaction was initiated by the addition of phaB, and the rate of NADH decrease was measured at 340 nm using a spectrophotometer (Hitachi, Tokyo). The concentration of NADH was determined at a molar absorption coefficient of 6300 M−1 cm−1. One unit (U) was defined as the amount of enzyme that catalyzed the conversion of 1 μmol of NADH per min.19 Enzyme phaC was assayed by measuring the CoA released after the incubation of the enzyme with the reaction mixture (100 mM citrate phosphate buffer (7.0 pH), 1 mM 3HBCoA, 5% (v/v) glycerol, and 6 μg of the enzyme). The reaction was initiated by addition of the phaC and was incubated for 20 min prior to termination by precipitating protein using acetone. The mixture was centrifuged to remove the pellets, and the amount of CoA was measured in the supernatant using Coenzyme A (CoA) Assay Kit (Sigma-Aldrich). One unit (U) was defined as the amount of enzyme that generate 1 μmol of CoA per min.18 Electrode Surface Modification. Electrode surface (BPV-LPEI/ DH/C8-LPEI/phaB) was prepared as described previously.26 Briefly, an aliquot of 3 μL of a mixture containing 21 μL of 10 mg/mL BPVLPEI, 9 μL of 10 mg/mL DH, and 1.125 μL of (10% v/v) EGDGE, was drop coated on a GC electrode. The electrode was then dried overnight under airflow at room temperature. On the following day, an additional layer of C8-LPEI/phaB was drop coated over the first one by dropping 3 μL of a mixture containing 21 μL of 10 mg/mL C8LPEI, 9 μL of 10 mg/mL phaB, and 0.316 μL of 62.5 mM glutaraldehyde. The electrode was then dried for 2 h under airflow at room temperature and was stored at 4 °C until use. Electrochemical Method. Electrochemical measurements were conducted using a CH Instrument model 660e potentiostat anaerobically at room temperature. The experiments were run in 100 mM phosphate/citrate buffer, pH 7 at room temperature using a glassy carbon as a working electrode, platinum mesh as a counter electrode, and SCE as a reference electrode. The prepared electrode was allowed to stabilize by scanning the potential between −0.7 and −0.3 V for 10 cycles with a scan rate of 5 mV s−1. Because the redox polymer (BPVLPEI) has a formal redox potential of approximately −0.55 V, the applied constant potential was selected to be −0.63 V versus SCE to ensure a sufficient overpotential to drive the reaction into the

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RESULTS AND DISCUSSION Expression and Kinetics of the Enzymes. Three genes encoding phaA, phaB, and phaC were cloned from C. necator and expressed in E. coli. The specific activities of the purified phaA, PhaB, and PhaC were 22.1, 16.2, and 24.6 U/mg of protein, respectively. A recent study demonstrated the bioelectrocatalytic conversion of NAD+/NADH using a GC modified electrode with BPV-LPEI and DH.26 It was shown that this bioelectrode can catalyze both reduction and oxidation of NAD+ and NADH at moderate potentials of −0.63 and −0.38 V, respectively, vs SCE (Figure 3).26 Herein, the modified electrode was prepared as described previously,26 in which a second layer of LPEI-based polymer C8-LPEI and phaB was added over the first one to design a GC electrode (BPVLPEI/DH/C8-LPEI/phaB) able to catalyze the conversion of NAD+ into NADH coupled with the production of 3HBCoA from AcAcCoA (Figure 2). The kinetics of the modified electrode (BPV-LPEI/DH/C8LPEI/phaB) were evaluated for the reduction of NAD+ and the reduction of AcAcCoA into 3HBCoA associated with the NADH regeneration. The continuous injection of NAD+ or NADH resulted in reductive current generation due to the ongoing reduction of NAD+ or reduction of AcAcCoA, over applying a steady potential value of −0.63 V vs SCE, as shown in (Figure 4). The faradaic efficiency for NADH regeneration through the modified electrode was determined as 52 ± 3%. The current density signals were fitted to traditional Michaelis− Menten plot, yielding apparent Michaelis constant (KM) value of 814 ± 11 μM and a maximum current density (jmax) of 27.9 C


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Figure 3. Representative cyclic voltammograms demonstrating the bioelectrocatalytic oxidation and reduction of NADH and NAD+ by the modified electrode. Cyclic voltammetry was performed at 5 mV s−1 in phosphate-citrate buffer solution (pH 7.0, 0.1 M) in the absence (black line) or presence (red line) of 5 mM NADH or 5 mM NAD+ (blue line).

Figure 5. Results of amperometric steady-state experiments for (a) reduction of NAD+ into NADH on the modified electrode BPV-LPEI/ DH/C8-LPEI/denatured phaB and (b) reduction of acetoacetyl-CoA into 3-hydroxybutyryl-CoA with continuous injection of NADH by the modified electrode BPV-LPEI/DH/C8-LPEI/phaB. Control experiment was performed with denatured diaphorase and phaB. The amperometric steady-state experiments were performed in phosphatecitrate buffer solution (pH 7.0, 0.1 M) using constant reduction potential at −0.63 V vs SCE. The Michaelis−Menten equation, V y = K max+xx , was fitted to NAD+ and acetoacetyl-CoA reduction data. m

Error bars represent standard deviation (n = 3).

accompanied by NADH oxidation into NAD+; BPV-LPEI and DH layer on the modified electrode then regenerated NADH from the oxidized NAD+ to be reused by C8-LPEI and phaB layer for another AcAcCoA reduction. Then, PHB synthesis reaction took place through the polymerization of 3HBCoA into the PHB polymer (Figure 2). The regeneration of NADH, in the proposed method, is mediated by modified electrode (BPV-LPEI/DH/C8-LPEI/phaB). The constant potential supplied electrons as a cofactor needed for DH to complete the reduction of NAD+ into NADH, in which these electrons are transferred between the electrode and DH through the redox polymer BPV-LPEI, as illustrated in Figure 2. As the incubation continued, the current density increased due to the bioelectrocatalytic regeneration of NADH from the produced NAD+ (Figure 6), and the reaction mixture became cloudy due to the precipitated PHB granules. All controls did not exhibit increments in the current density due to the inactivated DH in the first control, and the absence of the modified electrode and the applied potential in the second and third controls, respectively. The amount of the synthesized PHB with the bioelectrocatalytic regeneration of NADH and the first and third controls are shown in Figure S3, Supporting Information. At the end of the incubation, PHB granules were harvested by centrifugation and washed twice with distilled water prior to

Figure 4. Amperometric steady-state i−t curves for the injection of NAD+ without the presence of AcAcCoA (red) and NADH in the presence of AcAcCoA (blue). Control experiment was performed with denatured DH and NAD+ injection (black). The amperometric steadystate i−t curves were performed in phosphate-citrate buffer solution (pH 7.0, 0.1 M) using constant reduction potential at −0.63 V vs SCE.

± 1.3 μA cm−2 for NAD+ reduction (Figure 5a) and a KM value of 47 ± 2 μM and jmax of 0.97 ± 0.03 μA cm−2 for AcAcCoA reduction (Figure 5b). The KM values are relatively closed to those obtained using phaB and AcAcCoA in solution (Figure S2, Supporting Information). The controls were performed using denatured DH or phaB in which there was no activity recorded (Figure 5). In Vitro Bioelectrosynthesis of PHB. The in vitro synthesis of PHB by electrolysis at −0.63 V vs SCE was carried out in 5 mL, and the reaction was monitored through measuring the current density for 16 h. The reaction was initiated by the addition of Acs, causing two molecules of acetate to convert into two molecules of acetyl-CoA. These molecules were condensed by phaA into one AcAcCoA, which was further reduced by the C8-LPEI and phaB layer on the modified electrode into 3HBCoA. The AcAcCoA reduction was D


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Figure 6. Amperometric steady-state i−t curves for the injection of Acs in the presence (blue) and absence (red) of sodium acetate. The experiments were performed in an anaerobic environment with stirring in phosphate−citrate buffer solution (pH 7.0, 0.1 M) using constant reduction potential at −0.63 V vs SCE. The final formed PHB is shown on the right in the presence (A) and absence (B) of sodium acetate.

at 2915 and 2955 cm−1 are due to the C−H stretching bond in the PHB structure. The band found at 1460 cm−1 corresponded to the asymmetrical deformation of the CH2 groups, while the one found at 1377 cm−1 represents the equivalent for CH3 groups, presenting the chemical group of PHB.29 Unlike the substrate-required NADH regeneration methods, the bioelectrocatalytic method does not require chemicals as electron donors for the NAD+ reduction because a reductive current is applied to supply electrons. Table 1 summarizes and compares the most common methods used for the in vitro synthesis of PHB. In our proposed method, the regeneration of NADH is mediated by constant potential and is not accompanied by substrate consumption such as all modes operated for the in vitro synthesis of PHB. To scale up the technology, different aspects need to be considered. First, enhancing the lifetime of the systems can reduce the cost related with the utilization of pure enzymes. Good stability performance has been achieved in different reports for LPEI immobilized enzymes with lifetimes over 20 days.31,32 A second aspect is the cost related to the electrical energy supply. By coupling the developed systems with a renewable power source, it would be possible to develop a selfsustained system. As an example, microbial fuel cells or enzymatic fuel cells where microorganisms or enzymes are interfaced with electrode surfaces to convert the chemical energy present in organic compounds into electrical energy have been utilized as a power supply for different systems.33,34 A third important aspect would be the optimization of the Coulombic efficiency for the process. Different strategies can be used for this purpose such as enzyme engineering. It is evident that the scale up of the technology will represent a scientific and technological challenge. However, the achievement of more sustainable production of chemicals is a critical goal for the scientific community and for the development of a green economy. This report represents the first step toward sustainable production of PHA. For continuous bioelectrosynthesis production, immobilization of enzymes Acs, phaA, and phaB on the electrode surface could be considered to enable easy separation of the enzymes and PHB. This supports the reusability of the enzymes as well as decreases the diffusion of the enzymes’ substrates into the reaction medium. Unlike these

lyophilization. To obtain a pure PHB without phaC in the fraction, the lyophilized pellets were extracted with Dchloroform and were then dried to obtain 1.63 ± 0.17 mg of pure PHB from the bioelectrocatalytic reaction mixture. On the other hand, there was no PHB detected in the second control run due to the absence of sodium acetate, while both first and third controls yielded only 0.45 ± 0.10 mg. Moreover, the released CoA from both phaA and phaC were recycled by the conversion of acetate into acetyl-CoA with Acs to avoid the very effective competitive role of CoA on the polymerization reaction by phaC.28 Considering that the reaction started with 0.4 mM of NADH and yielded 1.63 mg of PHB, the cofactor NADH was recycled at least 8 times during the 16 h incubation period. The presence of PHB was confirmed by analyzing the purified fraction with FT-IR (Figure 7) and 1H NMR (Figure S4, Supporting Information). The analysis indicated that the product was PHB, in which the functional chemical bonds in the monomer are shown in (Figure 7). The spectrum revealed peaks found at 1738 and 1190 cm−1, corresponding to the stretching of the CO and C−O of the ester group, respectively. Meanwhile, the transmittance regions appeared

Figure 7. FT-IR Spectroscopy of bioelectro-synthesized PHB (red) and standard PHB (blue) showing the corresponding chemical bonds. E


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According to the latest study conducted by the United States Energy Information Administration, the average price of electricity to customers in November 2017 was 10.38 cents per kilowatt/hour. According to United States Department of Agriculture, the cost of glucose in January 2018 was $500 per ton.

30 CoA, ATP, or NADH are not required for the reaction. The study was conducted using an expensive prepared 3-HB-CoA; $20 per 1 mg. The process forms a thin film of PHB; however, the accumulation of PHB led to the phaC layer becoming buried in its own product, thus impairng the diffusion of 3-HB-CoA to the active site of phaC and decreasing the amount of PHB. surface-initiated polymerization

enzymes, phaC polymerizes 3HB-CoA into PHB to produce PHB polymer chain. As far the accumulation of the PHB polymer goes, the phaC is becoming buried in its own product, making the recovery of the enzyme difficult. Further studies to understand how the PHB chain termination takes place are needed to develop phaC and PHB polymer recovery plans. Herein, this study considered recycling expensive cofactors NADH and CoA. Future plans will include enzyme to catalyze the recycling of ATP; e.g., polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK). This system uses polyphosphate as a cheaper phosphate donor compared with acetyl phosphate or creatine phosphate.35 This study reported the first PHB in vitro sustainable synthesis using bioelectrocatalytic method from acetate as a relatively inexpensive substrate. The conversion rate of acetate into PHB, however, is relatively low, probably limited by the low NADH regeneration rate that led to low AcAcCoA reduction by the modified electrode.

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CONCLUSIONS Ecofriendly bioplastic has been intensively investigated and proposed as a potential candidate to substitute for petroleumderived plastics. However, the high production cost restricted the move toward a sustainable biodegradable plastic. In this study, we introduced electrochemistry as a potential sustainable inexpensive source of electrons to reduce NAD+ to NADH. Enzyme phaB together with C8-LPEI, DH, and BPV-LPEI were used to modify a GC electrode that exhibited a regular ability to regenerate NADH from the oxidized form NAD+. The NADH regeneration was accompanied by the reduction of AcAcCoA into 3HBCoA, which is the subunit of PHB. The modified electrode was then employed for the in vitro synthesis of PHB from acetate with the presence of Acs, phaA, and phaC in the reaction mixture. The nature of the PHB was confirmed by FTIR and 1H NMR. Data suggest that this approach has a significant implication for bioplastic production that overcomes the requirement of high cost steps involved in enzymatic reaction, e.g., glucose dehydrogenase and glucose. For future prospects, further electrode modification aspects are currently being investigated in our laboratory to enhance the rate of NADH as well as NADPH regeneration and thus increase the activity of phaB.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b04392. Control CVs, reduction and oxidation CVs, kinetics, and 1 H NMR figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Bassam Alkotaini: 0000-0002-9924-6400 Matteo Grattieri: 0000-0002-1795-3655 Shelley D. Minteer: 0000-0002-5788-2249 Notes

The authors declare no competing financial interest.

b

18 Acs for CoA regeneration. No ATP regeneration; while NADH is not required because the study used an expensive prepared 3-HB; $130 per 1 g. Although thermostable enzymes were used to overcome denaturation occurs during the reaction, the cost of temperature maintenance at 45 °C is a critical concern.

thermotolerant Acs, phaA, LA polymerizing enzyme immobilized phaC on surface thermotolerant synthesis

a

21 Glucose and Acs for NADH and CoA regeneration. No ATP regeneration. Gluconolactone, AMP, and PPi are accumulated in the reaction medium. The process has been studied through the past 20 years; however, scale-up is not proposed due to the relatively high cost of glucose for NADH regeneration. Glucose is used as an electron donor; the cost of NADH regeneration in 5 mL during 24 h is 50 cents.2 Acs, phaA, phaB, phaC, GDH enzyme-catalyzed synthesis

solution of 3HB-CoA flow on the surface

reference

this study

comments

Modified electrode and Acs for NADH and CoA regeneration. No ATP regeneration. Only AMP and PPi are accumulated in the reaction medium. Intensive studies and optimization are required prior to scaling-up the process. The applied current is the electron donor; the cost of the NADH regeneration in 5 mL during the 16 h reaction is below 0.002 cents.1 However, the cost of the current could be decreased in case of using a sustainable source such as microbial and enzymatic fuel cells.

acetate, CoA, ATP, electron, and NADH acetate, CoA, ATP, NADPH, and glucose acetate, CoA, ATP, and 3HB or LA

substrates enzymes

Acs, phaA, phaB, phaC, DH bioelectrosynthesis

PHB in vitro method

Table 1. Comparison between the Most Common Methods Used for the in Vitro Synthesis of PHB


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ACKNOWLEDGMENTS The authors would like to thank the Army Research Office (MURI grant #W911NF1410263) for funding this research.

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