Conversion of Ethanol to 2-Ethylhexenal at Ambient Conditions Using

Research Article Cite This: ACS Sustainable Chem. Eng. 2017, 5, 10483-10489

pubs.acs.org/journal/ascecg

Conversion of Ethanol to 2‑Ethylhexenal at Ambient Conditions Using Tandem, Biphasic Catalysis Glenn R. Hafenstine,† Alexander W. Harris,† Ke Ma,† Jennifer N. Cha,*,†,‡ and Andrew P. Goodwin*,†,‡ †

Department of Chemical and Biological Engineering and ‡Materials Science and Engineering Program, University of Colorado Boulder, 3145 Colorado Avenue, 596 UCB, Boulder, Colorado 80303, United States

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S Supporting Information *

ABSTRACT: Ethanol is a ubiquitous fermentation product well-tolerated by microbes, but purification from growth media requires multiple distillations or other energy intensive processes. Converting such metabolites to larger, hydrophobic products would both yield higher energy products and facilitate separation. Here, we demonstrate the conversion of C2 ethanol to C8 2-ethylhexenal via a sequential oxidation−aldol−hydrogenation−aldol process with solar energy as the only required input. Photocatalysis was utilized to drive enzymatic oxidation of ethanol, while biphasic media in conjunction with aldol coupling and Pd assisted hydrogenation kept the oxidation and reduction processes physically and chemically separated. Using this process, 2-ethylhexenal was produced from ethanol in both buffer and diluted yeast media. KEYWORDS: Heterogeneous catalysis, Enzyme catalysis, Photocatalysis, Prganocatalysis, Green chemistry, Nonequilibrium processes

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INTRODUCTION Increasing strain on global energy reserves has driven exploration of alternative sources of usable fuels for transportation, heating, or other applications. Single-celled organisms are potential sources of specific chemicals derived from renewable feedstocks such as sugar.1−8 However, obtaining the desired product from the growth media requires a trade-off between yield, utility, and energy consumed in separation processes. For example, algae are able to generate long chain fatty acids,9−11 but the lysis required for product extraction limits their use in a continuous batch process. High heating value, hydrophilic metabolites such as butanol have inherent cell cytotoxicity; for example, the upper limit for butanol production via acetone−butanol−ethanol (ABE) fermentation of Clostridium acetobutylicum is about 150 mM.12,13 Shorter chain alcohols such as ethanol are well tolerated by a variety of yeast strains at very high concentrations (∼10% w/w),14 but ethanol has a lower heating value.15,16 In addition, because of their unfavorable partitioning into organic solvents, shorter chain alcohol metabolites must also be distilled from growth media, which greatly reduces or even negates the energy stored in the metabolites. One possible solution is to convert a high yielding, low molecular weight alcohol metabolite such as ethanol to an aqueous-immiscible product that can be easily extracted from the growth media. For example, we recently demonstrated the successful transformation of C4 butanol (BuOH) to C8 2-ethylhexenal (2-EH) via a tandem enzymemediated oxidation and organocatalytic aldol condensation. However, converting C2 molecules to C8 molecules requires © 2017 American Chemical Society

multiple aldol condensations, which in turn requires hydrogenating the unsaturated aldehyde formed upon aldol coupling to a saturated aldehyde. In addition, oxidation of the alcohol and subsequent hydrogenation reactions must also occur in the same reaction mixtures without mutual interference. Here, we demonstrate a biphasic reaction scheme where specific catalysts are sequestered in each phase to transform C2 ethanol to C8 2-ethylhexenal (2-EH) using five sequential reactions (Scheme 1). In this sequence, ethanol (EtOH) is continuously oxidized to acetaldehyde (AA) using alcohol dehydrogenase (ADH). A Pt-decorated CdS nanorod (Pt@ CdS) photocatalyst regenerates the NAD+ cofactor under solar illumination, driving the reaction forward while at the same time generating H2.17−27 Next, the AA is converted to the unsaturated C4 crotonaldehyde (CA) through aqueous aldol condensation by the organocatalyst β-alanine.28−37 The longer C4 molecule can then partially partition to an immiscible oil phase, where it is hydrogenated to butyraldehyde (BA) using the Pt@CdS-generated H2 and Pd nanoparticles (PdNPs). By partitioning back to the aqueous phase, the BA then can undergo a second aldol condensation reaction to produce the C8 2-ethylhexenal. Overall, the reaction sequence takes advantage of the increasing partitioning of the growing carbon chain to the oil phase to promote forward over backward reaction. We show that each reaction can not only run Received: July 23, 2017 Revised: September 8, 2017 Published: October 4, 2017 10483

DOI: 10.1021/acssuschemeng.7b02487 ACS Sustainable Chem. Eng. 2017, 5, 10483−10489

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ACS Sustainable Chemistry & Engineering

Scheme 1. Overall Reaction Scheme for Multicatalytic Conversion of Ethanol (EtOH) to 2-Ethylhexenal (2-EH)a

a

Measured turnover frequencies are given next to each catalyst. bubbling with argon. Then, cadmium oxide (210 mg) and ODPA (1.06 g) were mixed in TOPO (2.75 g) and evacuated for 1 h at 120 °C. The solution was heated to 320 °C under argon and maintained for 15 min to allow for cadmium complexation. The reaction mixture was then cooled to 120 °C and evacuated again for 1.5 h to remove trace amounts of water. Next, the solution was reheated to 320 °C and TOP (2 g) was added to act as a stabilizing ligand. TOPS (1.95 g) was then injected, and the reaction was allowed to proceed at 315 °C for 85 min to form nanorods (NRs). The NRs were washed with equal amounts of 2-propanol and octanoic acid before being dispersed in toluene. Pt nanoparticles were photodeposited onto the CdS NRs using the process developed by Dukovic et al.19 Briefly, 33 nM CdS NRs, 13.3 mM (1,5-cyclooctadiene)dimethylplatinum(II), and 50 μL triethanolamine were mixed in 600 μL of toluene and purged with argon for 30 min. The solution was then irradiated with a 9 W LED lamp emitting 460 nm light for approximately 30 min until a slight brown color developed. The solution was stored at 4 °C in toluene until immediately before use. The catalysts were transferred into H2O by ligand exchange with 25 μL of 2-mercaptoethanol in 1 mL of H2O for 20 min, and impurities were removed by microcentrifugation with 30k MWCO filters (Pall Life Sciences) by three washes of 500 μL of H2O at 800 rcf. Partition Coefficient Measurements. Solutions containing AA, CA, BA, or 2-EH (5 mM) in 570 μL of 1 M PB (pH 8.8) and 30 μL of GT were stirred at room temperature for 3 h. The solutions were chilled for 5 min at 4 °C, centrifuged for 5 min at 200 rcf, and then extracted with CDCl3 (525 μL) containing 0.03% (v/v) tetramethylsilane (TMS) and assessed by 1H NMR spectroscopy. Product concentrations were determined by comparing peak integrations of aldehyde peaks (9.83 ppm for AA, 9.51 ppm for CA, 9.78 ppm for BA, and 9.38 ppm 2-EH) with TMS (0.13 ppm). The measured concentrations were normalized to an average value of 100% recovery by minimizing the root-mean-square error between all measurements, and the normalization factors were utilized for correcting concentrations of extractions from reaction mixtures (Supporting Information, Figure S2). Hydrogenation Tests. Solutions containing CA (5 mM) in 570 μL of 1 M PB (pH 8.8), 30 μL of organic solvent (GT, octane, or toluene), and 0.6 mg of PdNPs (Pd-TOP, Pd-Hex, or Pd-11/MUA) were injected with a steady pressure of hydrogen gas from a balloon. The reactions proceeded at room temperature for 3 h before chilling for 5 min at 4 °C, centrifuging for 5 min at 200 rcf, and extracting with CDCl3 (525 μL) containing 0.03% (v/v) TMS and assessing by 1H NMR spectroscopy. Product concentrations were determined by comparison with TMS standard peak integrations and adjusted by the partition coefficient values described above. Aldol Condensation Tests. Solutions containing β-alanine (550 mM) and AA or BA (5 mM) in 570 μL of 1 M PB (pH 8.8) and 30 μL of organic cosolvent (GT, octane, or toluene) were stirred at room temperature for 3 h. The reactions were chilled for 5 min at 4 °C, centrifuged for 5 min at 200 rcf, and then extracted with CDCl3 (525 μL) containing 0.03% (v/v) TMS and assessed by 1H NMR spectroscopy. Product concentrations were determined by comparison

concurrently with others but that sequential reactions can help to shift equilibria in a desired direction, thereby increasing conversion of the initial starting metabolite.

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EXPERIMENTAL SECTION

Materials. Alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, β-nicotinamide adenine dinucleotide sodium salt (NAD+), phosphate buffer solution, β-alanine, triethanolamine, 2-mercaptoethanol, deuterated chloroform (CDCl3) containing trimethylsilane (TMS), octadecylphosphonic acid (ODPA), trioctylphosphine oxide (TOPO), trioctylphoshine (TOP), octanoic acid, crotonaldehyde (CA), and butyraldehyde (BA) were purchased from Sigma-Aldrich. Acetaldehyde (AA), n-octane, and palladium acetylacetonate were purchased from Acros Organics. (1,5-Cyclooctadiene)dimethylplatinum(II), cadmium oxide, glycerol tributyrate (GT), and sulfur were purchased from Alfa Aesar. β-Nicotinamide adenine dinucleotide disodium salt hydrate (NADH) and 2-ethylhexenal (2EH) were purchased from TCI Chemicals. Butanol (BuOH), 2propanol, acetone, dextrose, and toluene were purchased from Fisher Scientific. Yeast nitrogen base (w/o amino acids and ammonium sulfate) was purchased from Beckton, Dickinson, and Company. Active dry yeast was purchased from ACH Food Companies, Inc. Ethanol (EtOH) was purchased from Decon Laboratories, Inc. Deuterium oxide (D2O) and sodium 2,2-dimethyl-2-silapentane-5sulfonate (DSS) were purchased from Cambridge Isotope Laboratories, Inc. Synthesis of Palladium Nanoparticles. Monodisperse palladium nanocrystals were produced as previously reported by Kim et al.38 through a thermal decomposition of a Pd−trioctylphosphine complex. First, 0.4 g of palladium acetylacetonate and 4 mL of trioctylphosphine (TOP) were combined under argon with bath sonication to form an orange solution. A 150 mL round-bottom flask was purged with argon before the addition of 36 mL of TOP. The Pd-TOP mixture was added to the flask, the argon stream was removed, and the reaction was slowly heated to 300 °C. The temperature was maintained for 30 min as the color changed to black before cooling back to room temperature. The palladium nanoparticles (PdNPs) were then precipitated with centrifugation at 400 rcf and washed three times with 50 mL of ethanol. For ligand exchanges, the procedure outlined by Woerhle et al.39 for attachment of thiolated ligands was followed. The as-synthesized TOP functionalized nanoparticles (Pd-TOP) were dispersed in dichloromethane (DCM) and stirred at room temperature. To prepare 1hexanethiol functionalized particles, 20 mg of 1-hexanethiol was added to 20 mg of Pd/TOP and mixed for 16 h. The particles were then washed three times with DCM and centrifuged down at 500 rcf. For the water-soluble 11-mercaptoundecanoic acid functionalized particles, 20 mg of 11-mercaptoundecanoic acid in 2 mL of H2O was added to 20 mg of Pd/TOP in 2 mL of DCM. The biphasic mixture was stirred for 16 h, and excess ligands were removed from the water phase by centrifuging at 500 rcf and washing three times with H2O. Synthesis of Photocatalyst. Photocatalytic CdS nanocrystals were produced as previously reported by Robinson et al.18 (Sample A synthesis procedure: Briefly, TOPS was produced by mixing TOP with an equimolar amount of sulfur for 48 h at room temperature while 10484


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ACS Sustainable Chemistry & Engineering with TMS standard peak integrations and adjusted by the partition coefficient values described above. Enzyme Kinetics Measurements. A biphasic solution containing 560 μL of 1 M phosphate (pH 8.8), 30 μL of organic cosolvent, 0.15 mM NAD+, and 50 mM EtOH was added to a cuvette. The UV−vis spectrophotometer was zeroed at 339 nm using this solution, and then 0.1 units of ADH in 10 μL of phosphate buffer was added. The absorbance of the solution at 339 nm was measured periodically for 30 min while agitating between measurements to determine the conversion of NAD+ into NADH due to enzyme activity. Photocatalysis Tests. For NADH oxidation tests, 570 μL of 1 M PB (pH 8.8), 30 μL of GT, Pt@CdS (25 nM), 0.6 mg of Pd-Hex, and NADH (15 mM) were added to a sealed vial. The solution was degassed with argon for 1 h before adding 10 mM CA and irradiating under the solar simulator with periodic GC measurements of the gas in the headspace. After 3 h, the reactions were chilled for 5 min at 4 °C, centrifuged for 5 min at 200 rcf, and then extracted with CDCl3 (525 uL) containing 0.03% (v/v) TMS and assessed by 1H NMR spectroscopy. Product concentrations were determined by comparison with TMS standard peak integrations and adjusted by the partition coefficient values described above. Similarly, for the composite reaction tests, 495 μL of 1 M PB (pH 8.8), 30 μL of GT, Pt@CdS (25 nM) NAD+ (3 or 15 mM), β-alanine (550 mM, when applicable), and 0.6 mg of Pd-Hex (when applicable) were added to a vial. The solution was degassed with argon for 1 h. Alcohol dehydrogenase (100 units in 75 μL of phosphate buffer) and EtOH (50 mM) were injected into the vial, and the reaction was stored in the dark for 15 min to allow for induction of NADH. The solution was then irradiated by the solar simulator with periodic GC measurements of the gas in the headspace. After 3 h, the reactions were chilled for 5 min at 4 °C, centrifuged for 5 min at 200 rcf, and then extracted with CDCl3 (525 μL) containing 0.03% (v/v) TMS and assessed by 1H NMR spectroscopy. Product concentrations were determined by comparison with TMS standard peak integrations and adjusted by the partition coefficient values described above. For the yeast media tests, cultures were prepared in a minimal media containing 6.7 g of yeast nitrogen base and 40 g of dextrose per L of H2O. To this, approximately 10 mg of active dry yeast powder was added and the microbes were grown at 37 °C for 2 days, at which point the cells were pelleted down at 15000 rcf for 10 min. Then 450 μL of this solution was mixed with 50 μL of D2O containing 100 mM sodium 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) and assessed by 1 H NMR under parameters for water suppression to determine the concentration of EtOH (approximately 320 mM). For the composite reaction tests, 120 μL of this culture media, 375 μL of 1 M PB (pH 8.8), 30 μL of GT, Pt@CdS (25 nM) NAD+ (15 mM), β-alanine (550 mM), and 0.6 mg of Pd-Hex were added to a vial. The solution was degassed with argon for only 15 min to reduce evaporative losses of EtOH. Alcohol dehydrogenase (100 units in 75 μL of phosphate buffer) was injected into the vial, and the reaction was stored in the dark for 15 min to allow for induction of NADH. The solution was then irradiated by the solar simulator with periodic GC measurements of the gas in the headspace. After 3 h, the reactions were chilled for 5 min at 4 °C, centrifuged for 5 min at 200 rcf, and then extracted with CDCl3 (525 μL) containing 0.03% (v/v) TMS and assessed by 1H NMR spectroscopy. Product concentrations were determined by comparison with TMS standard peak integrations and adjusted by the partition coefficient values described above.

stirred for 30 min before cooling and washing with dichloromethane. The resulting TOP-capped spherical nanocrystals were approximately 3.5 nm in diameter (Figure 1a). Next, the

Figure 1. (a) Transmission electron microscopy (TEM) image of the as-synthesized PdNPs. Scale bar = 100 nm. (b) Image of PdNPs in biphasic mixtures of 1 M PB (pH 8.8) and GT (5% v/v; top phase). (c,d) Hydrogenation of CA in (c) different mixtures of PB and organic cosolvents (5% v/v) containing Pd-Hex NPs and (d) with different Pd capping ligands in GT unless noted. All reactions were run for 3 h using 1 mg/mL PdNPs at room temperature. Error bars represent one standard deviation from triplicate measurements.

PdNPs and the effect of their capping ligands on hydrogenating the unsaturated aldehyde crotonaldehyde was evaluated. To do this, the PdNPs were either used as synthesized (Pd-TOP), or their ligands were exchanged with either 1-hexanethiol (PdHex) or 11-mercaptoundecanoic acid (Pd-11/MUA) as described by Woehrle et al.39 Figure 1b shows how the distribution of the PdNPs between the aqueous and the organic phase glycerol tributyrate (GT) varies for the three ligands due to the changing hydrophilicity. As expected, the Pd-11/MUA appeared well dispersed in the aqueous phase, while the Pd-Hex and Pd-TOP were clearly not miscible with water and preferred the GT phase. The PdNPs were then tested for CA hydrogenation in mixtures of phosphate buffer (PB) and two water-immiscible solvents: GT and n-octane. Briefly, 10 mM CA in 1 M PB at pH 8.8 was mixed with 5% (v/v) of the organic solvent and the differently coated PdNPs at 1 mg/mL. This fraction of organic solvent was chosen to maximize the sequestration of hydrogen in the liquid phase while minimizing interference of a nondeuterated compound during the NMR measurement. The reactions were then agitated under a H2-filled balloon at near ambient pressure for 3 h. The mixture was extracted with CDCl3 containing tetramethylsilane (TMS) as an internal standard for 1H NMR spectroscopy. Because of the imperfect extractions of small molecules from the aqueous phase caused by factors such as hydrate formation,43 the amount of BA product was calculated using partition coefficient standards (Supporting Information, Figure S2). As shown in Figure 1c, the hexanethiol modified Pd (Pd-Hex) yielded quantitative conversion of CA into the saturated product BA in biphasic solutions of PB and either GT or n-octane (triplicate measurements). The hydrophobic ligands Pd-TOP and Pd-

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RESULTS AND DISCUSSION Selective hydrogenation of the aldol products is vital for accomplishing the reaction sequence depicted in Scheme 1. For this, PdNPs were first evaluated as hydrogenation catalysts due to their high catalytic activity, facile synthesis, variety of potential ligands,38 and known selectivity for alkene hydrogenation at mild temperatures to avoid exhaustive hydrogenation.40−42 Briefly, a solution of palladium acetylacetonate and trioctylphosphine (TOP) was slowly heated to 300 °C and 10485


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ACS Sustainable Chemistry & Engineering Hex showed complete conversion of CA after 3 h using GT as the organic solvent while the more hydrophilic ligand of Pd-11/ MUA converted only 3 ± 1% of CA with a biphasic mixture and only 2 ± 1% in PB alone (Figure 1d). Higher conversions from the hydrophobic ligand-capped NPs (Pd-TOP and PdHex) were attributed to the higher solubility of H2 in organic solvents over the aqueous phase.44 Because EtOH will be converted to 2-EH in a closed system (i.e., no added H2), an unsaturated aldehyde (Scheme 1) must be hydrogenated by the H2 generated in situ, as opposed to hydrogen that has been externally supplied. In our previous study, Pt@CdS NPs were used to regenerate the NAD+ cofactor for the enzyme alcohol dehydrogenase (ADH) to continuously oxidize the starting alcohol substrate. This reaction also produced hydrogen gas.37 To test if the H2 produced in situ could hydrogenate CA to BA, 25 nM Pt@ CdS was mixed with varying concentrations of NADH and 1 mg/mL Pd-Hex in 1 M PB at pH 8.8. The solutions were degassed with Ar for 1 h before adding 10 mM CA and 5% (v/ v) GT and photoirradiated with a solar simulator at 1 sun for 3 h. H2 formation was measured by GC analysis of gas aliquots, which were periodically removed from the headspace. As shown in Figure 2a, H2 was produced quickly, suggesting facile

Figure 3. Production of CA (a) and 2-EH (b) from β-alanine catalyzed condensation of AA or BA in the presence of 1 M PB and glycerol tributyrate (GT) or octane (5% v/v) at room temperature for 3 h. Error bars represent one standard deviation from triplicate measurements.

(Supporting Information, Figure S6). For BA condensation to 2-EH, conversions were higher: 18.2 ± 1.8% and 36.6 ± 1.5% for GT and octane, respectively (Figure 3b). The increased conversion seen in octane may be due to a higher partitioning of the more hydrophobic products (CA and 2-EH) into the less polar solvent (octane), shifting equilibrium to obtain higher yields. Converting C2 EtOH to C4 BA in one pot requires that three reactions must operate simultaneously: (a) the ADH must continuously oxidize EtOH to AA in biphasic media, (b) the Pt@CdS nanorod catalysts must convert the ADH cofactor NADH to NAD+ and also generate H2, and (c) CA must be hydrogenated under these conditions. Because successful aldol coupling and hydrogenation of CA to BA was seen in biphasic media, we next tested the activity of the ADH enzyme for EtOH oxidation to AA in a mix of PB and GT. Literature precedence has shown that ADH activity is preserved in biphasic media,45−47 and this phenomenon was confirmed here. Briefly, 50 mM EtOH was added to a solution of 0.15 mM NAD+ and 0.1 units48 of ADH in 1 M PB at pH 8.8, with or without 5% (v/v) GT (for definition of ADH unit, see Supporting Information). The amount of NADH produced by this method was similar regardless of whether or not GT was added (Supporting Information, Figure S8). Having confirmed that the ADH remains active in a mixture of PB and GT, we next studied the conversion of C2 ethanol to C4 CA via sequential oxidation−aldol reactions. For this, a mixture of 25 nM Pt@CdS and 3 mM NAD+ in 1 M PB at pH 8.8 was degassed with Ar for 1 h before adding 100 units of ADH, 5% (v/v) GT, and 50 mM EtOH. After 15 min in the dark to oxidize EtOH and accumulate NADH, the solution was irradiated with a solar simulator at 1 sun for 3 h. Samples were periodically removed from the headspace to measure the amount of H2 formed by Pt@CdS oxidation of NADH back to NAD+ (Figure 4a). After CDCl3 extraction, 1H NMR showed that 2.1 ± 0.7 mM AA was formed, while in the absence of photoirradiation, little to no H2 and only 0.3 mM AA were measured (Figure 4b). To study the effect of the aldol organocatalyst β-alanine, the Pt@CdS, ADH, NAD+, and EtOH reactions were then mixed with 550 mM β-alanine in 1 M PB with 5% (v/v) GT at pH 8.8. After degassing, photoirradiation (during which time H2 was measured periodically), and CDCl3 extraction, 1H NMR spectroscopy showed 2.1 ± 0.4 mM AA and 0.6 ± 0.1 mM CA had formed. Furthermore, the addition of β-alanine organocatalyst increased H2 production after 3 h from 38 ± 1 μmol to 47 ± 2 μmol, indicating that the presence of the organocatalyst shifted the reaction equilibrium toward increased ethanol oxidation. Lastly, in the complete absence of

Figure 2. (a) Production of H2 from Pt@CdS with varied concentrations of NADH acting as a hole-scavenger. (b) % Conversion of CA to BA in the presence of palladium catalyst. Error bars indicate one standard deviation from triplicate measurements.

oxidation of NADH, with increasing amounts of H 2 corresponding to increasing concentrations of the cofactor. The 15 mM NADH sample reached equilibrium after only 1 h, after which the H2 concentration slowly decreased because of an imperfect seal in the reaction vessel. With higher NADH starting concentrations, more than 2 h were required to approach completion. Next, the solutions were extracted with CDCl3 and 1H NMR spectra were taken to measure CA to BA conversion. Starting with 15 mM NADH and 25 nM Pt@CdS, a 27 ± 4% CA to BA conversion was measured which increased to 67 ± 4% and 79 ± 7% when using 50 mM and 75 mM NADH, respectively (Figure 2b). Next, because two different aldol condensations are required to convert C2 EtOH to C8 2-EH (Scheme 1)), the activity of the aldol organocatalyst β-alanine was evaluated for conversion of AA to CA and BA to 2-EH in a mixture of PB and organic solvent. For this, each aldol substrate (AA or BA) was dissolved to 5 mM in 1 M PB at pH 8.8 containing 5% (v/v) organic solvent along with 550 mM β-alanine.36 The reactions were agitated for 3 h before extraction with CDCl3/TMS and evaluated by 1H NMR spectroscopy. As shown in Figure 3a, the samples containing GT and octane showed similar AA to CA conversions of 10.6 ± 1.7% and 12.4 ± 6.3%, respectively. Products of multiple aldol reactions such as 2,4-hexadienal and 2,4,6-octatrienal were not seen under these conditions 10486


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biomass or microbial fermentation products, we next studied the catalytic conversion of alcohols produced by yeast. To study this, the yeast species Saccharomyces cerevisiae was first cultured to produce ethanol with yields approaching ∼1.1 M in a synthetic yeast minimal media (Supporting Information, Figure S11). Next, because we found significant inhibition of ADH activity by the culture media (Supporting Information, Figure S12), the yeast produced ethanol was instead diluted with PB to ∼180 mM. Similar to the previous tests, 25 nM Pt@CdS, 15 mM NAD+, 550 mM β-alanine, and 1 mg/mL Pd-Hex were dispersed in a mixture of 20% (v/v) yeast culture media (containing 1.08 M EtOH) and 80% (v/v) 1 M PB at pH 8.8. The solution was degassed for only 15 min with argon to reduce evaporative losses of EtOH before the injection of 100 units of ADH and 5% (v/v) GT. Again, the reaction was kept for 15 min in the dark, followed by 3 h irradiation under solar light with periodic GC measurements to monitor H2 formation (Figure 6a). After 3 h, the solutions were extracted with CDCl3

Figure 4. (a) Production of H2 from Pt@CdS with and without βalanine organocatalyst. (b) Generation of AA and CA from EtOH in the presence and absence of aldol catalyst, ADH, or light. Error bars represent one standard deviation from triplicate measurements.

ADH, only 7 μmol H2 was produced. This small amount of H2 was presumably due to water or ligand oxidation on Pt@CdS during solar illumination. Having optimized and determined the yields at each step (Pt@CdS assisted ADH oxidation of EtOH to AA, H2 generation by Pt@CdS, aldol reactivities of AA to CA and BA to 2-EH, and hydrogenation of CA to BA), we next ran a full, closed reaction of C2 EtOH to C8 2-EH. A mixture of 25 nM Pt@CdS, 15 mM NAD+, 550 mM β-alanine, and 1 mg/mL Pd-Hex in 1 M PB at pH 8.8 was degassed with argon for 1 h before adding 100 units of ADH, 5% (v/v) GT, and 50 mM EtOH. The solution was then stored in the dark for 15 min to allow NADH to accumulate, followed by irradiating for 3 h with periodic sampling of the headspace. As shown in Figure 5a, a

Figure 6. (a) H2 production and (b) product yields after 3 h of irradiation of a mixture of EtOH, ADH, NAD+, β-alanine, Pt@CdS, and Pd-Hex with EtOH obtained from diluted yeast culture media. All error bars indicate one SD from triplicate measurements.

and yielded maximum values of 59 ± 5 μmol H2 and organic product yields of 0.2 ± 0.02 mM 2-EH, 0.4 ± 0.1 mM BA, 0.4 ± 0.1 mM CA, and 2.4 ± 0.3 mM AA (Figure 6b). These results show that microbial products can be successfully converted from ethanol to 2-EH, allowing facile separation of products from media as well as producing more energy intensive materials with only solar energy as the input.

Figure 5. (a) H2 production and (b) product yields after 3 h of irradiation of a mixture of EtOH, ADH, NAD+, β-alanine, Pt@CdS, and Pd-Hex with pure EtOH. All error bars indicate one standard deviation from triplicate measurements.

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CONCLUSION In this work, a one-pot system was developed to perform aldol, enzymatic, and heterogeneous catalysis simultaneously for lightdriven upgrading from C2 ethanol to C8 2-ethylhexenal without additional reagents or energy sources. This conversion was carried out through a sequential oxidation−aldol−hydrogenation−aldol sequence, which was facilitated by sequestering hydrophobic-functionalized Pd nanocrystals for hydrogenation inside the oil phase of a biphasic mixture. These hydrogenation catalysts were combined with Pt@CdS photocatalysts for regenerating the enzymatic cofactor NAD+ and producing H2, which was subsequently utilized for reduction of the unsaturated intermediates to promote further carbon−carbon coupling. Finally, this system was applied to the upgrading of yeast-produced EtOH to show the conversion of microbederived C2 alcohol to C8 product in mild, aqueous conditions with concurrent separation out of the water phase. Thus, using only solar light as an energy source, a common metabolite both upgraded to a higher molecule and extracted from water.

steady production of H2 was measured by GC, with a maximum yield of 55 ± 14 μmol. After 3 h, the entire reaction solution was extracted with CDCl3 and evaluated by 1H NMR to show 0.4 ± 0.1 mM of 2-EH along with 0.6 ± 0.1 mM BA, 0.8 ± 0.3 mM CA, and 3.2 ± 1.4 mM AA (Figure 5b), which corresponded to an EtOH conversion of 7.6 mM. The saturated C8 aldehyde (2-ethylhexanal) was not detected in these tests (Supporting Information, Figure S10), possibly due to low 2-EH concentration. No cross-aldol products of reactions between AA and BA were seen (Supporting Information, Figure S10), which is consistent with literature precedence for organocatalysis of aldol condensation.29 Control reactions of no Pt@CdS, no β-alanine, or no light yielded 2.2, 5.9, and 1.6 mM yields of AA with no higher-order products. Overall, these results showed the upgrading of C2 alcohol to a C8 product under mild conditions with solar light as the only input. However, to more closely approximate conversion of 10487


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(6) Wu, L.; Moteki, T.; Gokhale, A. A.; Flaherty, D. W.; Toste, F. D. Production of Fuels and Chemicals from Biomass: Condensation Reactions and Beyond. Chem. 2016, 1 (1), 32−58. (7) Deneyer, A.; Renders, T.; Van Aelst, J.; Van den Bosch, S.; Gabriëls, D.; Sels, B. F. Alkane production from biomass: chemo-, bioand integrated catalytic approaches. Curr. Opin. Chem. Biol. 2015, 29, 40−48. (8) Schwartz, T. J.; O’Neill, B. J.; Shanks, B. H.; Dumesic, J. A. Bridging the Chemical and Biological Catalysis Gap: Challenges and Outlooks for Producing Sustainable Chemicals. ACS Catal. 2014, 4 (6), 2060−2069. (9) Wargacki, A. J.; Leonard, E.; Win, M. N.; Regitsky, D. D.; Santos, C. N. S.; Kim, P. B.; Cooper, S. R.; Raisner, R. M.; Herman, A.; Sivitz, A. B.; et al. An Engineered Microbial Platform for Direct Biofuel Production from Brown Macroalgae. Science 2012, 335 (6066), 308− 313. (10) Scott, S. A.; Davey, M. P.; Dennis, J. S.; Horst, I.; Howe, C. J.; Lea-Smith, D. J.; Smith, A. G. Biodiesel from algae: challenges and prospects. Curr. Opin. Biotechnol. 2010, 21 (3), 277−286. (11) Wijffels, R. H.; Barbosa, M. J. An Outlook on Microalgal Biofuels. Science 2010, 329 (5993), 796−799. (12) Evans, P. J.; Wang, H. Y. Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants. Appl. Environ. Microbiol. 1988, 54 (7), 1662−1667. (13) Qureshi, N.; Blaschek, H. P. Production of Acetone Butanol Ethanol (ABE) by a Hyper-Producing Mutant Strain of Clostridium beijerinckii BA101 and Recovery by Pervaporation. Biotechnol. Prog. 1999, 15 (4), 594−602. (14) Henderson, C. M.; Lozada-Contreras, M.; Jiranek, V.; Longo, M. L.; Block, D. E. Ethanol Production and Maximum Cell Growth Are Highly Correlated with Membrane Lipid Composition during Fermentation as Determined by Lipidomic Analysis of 22 Saccharomyces cerevisiae Strains. Appl. Environ. Microbiol. 2013, 79 (1), 91− 104. (15) Green, E. M. Fermentative production of butanolthe industrial perspective. Curr. Opin. Biotechnol. 2011, 22 (3), 337−343. (16) Friedl, A. Downstream process options for the ABE fermentation. FEMS Microbiol. Lett. 2016, 363 (9), fnw073. (17) Kalyanasundaram, K.; Borgarello, E.; Duonghong, D.; Grätzel, M. Cleavage of Water by Visible-Light Irradiation of Colloidal CdS Solutions; Inhibition of Photocorrosion by RuO2. Angew. Chem., Int. Ed. Engl. 1981, 20 (11), 987−988. (18) Robinson, R. D.; Sadtler, B.; Demchenko, D. O.; Erdonmez, C. K.; Wang, L.-W.; Alivisatos, A. P. Spontaneous Superlattice Formation in Nanorods Through Partial Cation Exchange. Science 2007, 317 (5836), 355−358. (19) Dukovic, G.; Merkle, M. G.; Nelson, J. H.; Hughes, S. M.; Alivisatos, A. P. Photodeposition of Pt on Colloidal CdS and CdSe/ CdS Semiconductor Nanostructures. Adv. Mater. 2008, 20 (22), 4306−4311. (20) Berr, M.; Vaneski, A.; Susha, A. S.; Rodríguez-Fernández, J.; Döblinger, M.; Jäckel, F.; Rogach, A. L.; Feldmann, J. Colloidal CdS nanorods decorated with subnanometer sized Pt clusters for photocatalytic hydrogen generation. Appl. Phys. Lett. 2010, 97 (9), 093108. (21) Simon, T.; Bouchonville, N.; Berr, M. J.; Vaneski, A.; Adrović, A.; Volbers, D.; Wyrwich, R.; Döblinger, M.; Susha, A. S.; Rogach, A. L.; et al. Redox shuttle mechanism enhances photocatalytic H2 generation on Ni-decorated CdS nanorods. Nat. Mater. 2014, 13 (11), 1013−1018. (22) Yehezkeli, O.; de Oliveira, D. R. B.; Cha, J. N. Electrostatically Assembled CdS-Co 3 O 4 Nanostructures for Photo-assisted Water Oxidation and Photocatalytic Reduction of Dye Molecules. Small 2015, 11 (6), 668−674. (23) Walter, M. G.; Warren, E. L.; McKone, J. R.; Boettcher, S. W.; Mi, Q.; Santori, E. A.; Lewis, N. S. Solar Water Splitting Cells. Chem. Rev. 2010, 110 (11), 6446−6473.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02487. Gas chromatograph calibration curve, calculation of partition coefficient values, 1H NMR spectra of extracted products, and enzyme inhibition testing results (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*A.P.G.: phone 3034923573; E-mail, andrew.goodwin@ colorado.edu. *J.N.C.: E-mail, [email protected]. ORCID

Jennifer N. Cha: 0000-0002-2840-1653 Andrew P. Goodwin: 0000-0002-7284-4005 Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Omer Yehezkeli and Dr. Dylan Domaille for helpful discussions, Dr. Liangcan He for help with TEM, and Prof. John Falconer for use of his solar simulator and photocatalysis-GC setup. Research was supported by NIH (DP2EB020401) and by the U.S. Dept of Energy (DOE), Office of Science, Basic Energy Sciences (BES) under award no. DE-SC0006398.

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ABBREVIATIONS ABE, acetone−butanol−ethanol mixture; BuOH, n-butanol; 2EH, 2-ethylhexenal; EtOH, ethanol; AA, acetaldehyde; ADH, alcohol dehydrogenase; Pt@CdS, platinum-seeded cadmium sulfide nanorods; CA, crotonaldehyde; BA, butyraldehyde; NPs, nanoparticles; GT, glycerol tributyrate; NAD+, βnicotinamide adenine dinucleotide; NADH, β-nicotinamide adenine dinucleotide (reduced); TOP, trioctylphosphine; PdNPs, palladium nanoparticles; Pd002DTOP, PdNPs capped with TOP ligands; Pd-Hex, PdNPs capped with 1-hexanethiol ligands; Pd-11/MUA, PdNPs capped with 11-mercaptoundecanoic acid ligands; PB, phosphate buffer; TMS, tetramethylsilane

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REFERENCES

(1) Latif, H.; Zeidan, A. A.; Nielsen, A. T.; Zengler, K. Trash to treasure: production of biofuels and commodity chemicals via syngas fermenting microorganisms. Curr. Opin. Biotechnol. 2014, 27, 79−87. (2) Peralta-Yahya, P. P.; Zhang, F.; del Cardayre, S. B.; Keasling, J. D. Microbial engineering for the production of advanced biofuels. Nature 2012, 488 (7411), 320−328. (3) Lennen, R. M.; Pfleger, B. F. Microbial production of fatty acidderived fuels and chemicals. Curr. Opin. Biotechnol. 2013, 24 (6), 1044−1053. (4) Stephanopoulos, G. Challenges in Engineering Microbes for Biofuels Production. Science 2007, 315 (5813), 801−804. (5) Bond-Watts, B. B.; Bellerose, R. J.; Chang, M. C. Y. Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat. Chem. Biol. 2011, 7 (4), 222−227. 10488


Research Article

ACS Sustainable Chemistry & Engineering (24) Amirav, L.; Alivisatos, A. P. Photocatalytic Hydrogen Production with Tunable Nanorod Heterostructures. J. Phys. Chem. Lett. 2010, 1 (7), 1051−1054. (25) Kalisman, P.; Kauffmann, Y.; Amirav, L. Photochemical oxidation on nanorod photocatalysts. J. Mater. Chem. A 2015, 3 (7), 3261−3265. (26) Kalisman, P.; Nakibli, Y.; Amirav, L. Perfect Photon-toHydrogen Conversion Efficiency. Nano Lett. 2016, 16 (3), 1776− 1781. (27) Ma, K.; Yehezkeli, O.; Domaille, D. W.; Funke, H. H.; Cha, J. N. Enhanced Hydrogen Production from DNA-Assembled Z-Scheme TiO 2 -CdS Photocatalyst Systems. Angew. Chem., Int. Ed. 2015, 54 (39), 11490−11494. (28) List, B.; Lerner, R. A.; Barbas, C. F. Proline-Catalyzed Direct Asymmetric Aldol Reactions. J. Am. Chem. Soc. 2000, 122 (10), 2395− 2396. (29) Nozière, B.; Córdova, A. A Kinetic and Mechanistic Study of the Amino Acid Catalyzed Aldol Condensation of Acetaldehyde in Aqueous and Salt Solutions. J. Phys. Chem. A 2008, 112 (13), 2827−2837. (30) Lipshutz, B. H.; Ghorai, S. Organocatalysis in Water at Room Temperature with In-Flask Catalyst Recycling. Org. Lett. 2012, 14 (1), 422−425. (31) Watanabe, Y.; Sawada, K.; Hayashi, M. A green method for the self-aldol condensation of aldehydes using lysine. Green Chem. 2010, 12 (3), 384. (32) Sirasani, G.; Tong, L.; Balskus, E. P. A Biocompatible Alkene Hydrogenation Merges Organic Synthesis with Microbial Metabolism. Angew. Chem., Int. Ed. 2014, 53 (30), 7785−7788. (33) Yang, H.; Fu, L.; Wei, L.; Liang, J.; Binks, B. P. Compartmentalization of Incompatible Reagents within Pickering Emulsion Droplets for One-Pot Cascade Reactions. J. Am. Chem. Soc. 2015, 137 (3), 1362−1371. (34) Wei, L.; Zhang, M.; Zhang, X.; Xin, H.; Yang, H. Pickering Emulsion as an Efficient Platform for Enzymatic Reactions without Stirring. ACS Sustainable Chem. Eng. 2016, 4 (12), 6838−6843. (35) Matthiesen, J. E.; Carraher, J. M.; Vasiliu, M.; Dixon, D. A.; Tessonnier, J.-P. Electrochemical Conversion of Muconic Acid to Biobased Diacid Monomers. ACS Sustainable Chem. Eng. 2016, 4 (6), 3575−3585. (36) Domaille, D. W.; Hafenstine, G. R.; Greer, M. A.; Goodwin, A. P.; Cha, J. N. Catalytic Upgrading in Bacteria-Compatible Conditions via a Biocompatible Aldol Condensation. ACS Sustainable Chem. Eng. 2016, 4 (3), 671−675. (37) Hafenstine, G. R.; Ma, K.; Harris, A. W.; Yehezkeli, O.; Park, E.; Domaille, D. W.; Cha, J. N.; Goodwin, A. P. Multicatalytic, LightDriven Upgrading of Butanol to 2-Ethylhexenal and Hydrogen under Mild Aqueous Conditions. ACS Catal. 2017, 7 (1), 568−572. (38) Kim, S.-W.; Park, J.; Jang, Y.; Chung, Y.; Hwang, S.; Hyeon, T.; Kim, Y. W. Synthesis of Monodisperse Palladium Nanoparticles. Nano Lett. 2003, 3 (9), 1289−1291. (39) Woehrle, G. H.; Brown, L. O.; Hutchison, J. E. ThiolFunctionalized, 1.5-nm Gold Nanoparticles through Ligand Exchange Reactions: Scope and Mechanism of Ligand Exchange. J. Am. Chem. Soc. 2005, 127 (7), 2172−2183. (40) Claus, P. Selective hydrogenation of a′,β-unsaturated aldehydes and other C = O and C = C bonds containing compounds. Top. Catal. 1998, 5 (1), 51−62. (41) Thomson, S.; Hoffmann, C.; Ruthe, S.; Schmidt, H.-W.; Schüth, F. The development of a high throughput reactor for the catalytic screening of three phase reactions. Appl. Catal., A 2001, 220 (1−2), 253−264. (42) Ammari, F. An emergent catalytic material: Pt/ZnO catalyst for selective hydrogenation of crotonaldehyde. J. Catal. 2004, 221 (1), 32−42. (43) Wiberg, K. B.; Morgan, K. M.; Maltz, H. Thermochemistry of Carbonyl Reactions. 6. A Study of Hydration Equilibria. J. Am. Chem. Soc. 1994, 116 (24), 11067−11077.

(44) Purwanto; Deshpande, R. M.; Chaudhari, R. V.; Delmas, H. Solubility of Hydrogen, Carbon Monoxide, and 1-Octene in Various Solvents and Solvent Mixtures. J. Chem. Eng. Data 1996, 41 (6), 1414−1417. (45) Leskovac, V.; Trivic, S.; Pericin, D. The three zinc-containing alcohol dehydrogenases from baker’s yeast, Saccharomyces cerevisiae. FEMS Yeast Res. 2002, 2 (4), 481−494. (46) Eckstein, M. F.; Peters, M.; Lembrecht, J.; Spiess, A. C.; Greiner, L. Maximise Equilibrium Conversion in Biphasic Catalysed Reactions: Mathematical Description and Practical Guideline. Adv. Synth. Catal. 2006, 348 (12−13), 1591−1596. (47) Dickenson, C. J.; Dickinson, F. M. A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates. Biochem. J. 1975, 147 (2), 303−311. (48) Racker, E. Crystalline Alcohol Dehydrogenase From Bakers’ Yeast. J. Biol. Chem. 1950, 184, 313−320.

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Conversion of Ethanol to 2-Ethylhexenal at Ambient Conditions Using

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