Using Reverse Osmosis Membranes to Couple Direct Ethanol Fuel

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Using Reverse Osmosis Membranes to Couple Direct Ethanol Fuel Cells with Ongoing Fermentations Justin P. Jahnke, Marcus S Benyamin, James Jeffery Sumner, and David M. Mackie Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b02915 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 5, 2016

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Industrial & Engineering Chemistry Research

Using Reverse Osmosis Membranes to Couple Direct Ethanol Fuel Cells with Ongoing Fermentations Justin P. Jahnke*, Marcus S. Benyamin, James J. Sumner, David M. Mackie U.S. Army Research Laboratory, 2800 Powder Mill Road, Adelphi, MD 20740, USA

KEYWORDS: separation, bio-hybrid fuel cell, acetic acid, amino acid, biofuel, diffusion, yeast

ABSTRACT Separations in biological systems remains a challenging problem, and can be particularly so in the case of biofuels, where purification can use a significant fraction of the energy content of the fuel. For small-molecule biofuels like ethanol, reverse osmosis (RO) membranes show promise as passive purifiers, in that they allow uncharged small molecules to pass through while blocking most other components of the growth medium. Here, we examine the use of RO membranes in developing bio-hybrid fuel cells, closely examining the case where a direct ethanol fuel cell (DEFC) is coupled with an ongoing yeast fermentation across an RO membrane. We show that, contrary to initial good performance, the acetic acid produced by the DEFC readily diffuses back

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across the RO membrane and kills the fermentation after a few days.

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We introduce an

amelioration chamber where the acetic acid is converted to acetate ions. The RO membrane rejects the acetate ions due to their charge, preventing acetic acid buildup in the fermentation. We also show that some small, charged components of the fermentation such as amino acids are imperfectly rejected by RO membranes. Because of the high sensitivity of DEFCs to low concentrations (10s of µM) of amino acids, even a very slow diffusion of amino acids across the RO membranes can limit bio-hybrid fuel cell lifetimes.

INTRODUCTION Extracting energy from biomass, and especially, deriving biofuels from biomass has become increasingly important. Ethanol has the best-established production and distribution,1-2 but a wide range of fuels has been generated from biomass, including methane, hydrogen, higher alcohols and long chain fatty acids.3-5 One challenge with any biofuel is separation and purification of the fuel after production, which can consume a large portion of the energy content of the fuel. For example, in ethanol production, distillation of the ethanol into a nearly pure product can consume a third of the energy content of the fuel.6 Further, although the energetic and economic costs of distillation are not the limiting factor in the use of bio-ethanol as a fuel, these costs are amplified as the scale decreases, such that the only economic means of distillation is in large, centralized plants, which increases transportation costs.6 The challenges of purification are amplified for biofuels that can only be produced by microorganisms at very low concentrations, such as those produced in acetone-butanol-ethanol fermentations.7 Developing better ways to purify biofuels therefore remains an important technological challenge.


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Fuels cells have the potential to partially bypass the separations challenge because they can often run on dilute fuel streams. For example, direct ethanol fuel cells (DEFCs) often run optimally on only a few percent ethanol in water. However, fuel cells are often readily poisoned by trace components in biological fermentations. Amino acids, for example, can be problematic due to their amine and thiol groups that readily bind to noble metal catalysts used in fuel cells.8-9 Even salts (especially cations) can damage the fuel cell due to their tendency to block the conduction channels of proton exchange membranes.10 While it is possible to run fuel cells directly on the fermentate if the conditions are carefully controlled,11 in general some purification of fermentate is necessary for use in a fuel cell. In particular, purification is an unavoidable issue when trying to optimize ethanol production rates or trying to produce ethanol from waste water.12-13 There is also the challenge that the fuel cell can produce chemicals toxic to the fermentation if the fuel is incompletely oxidized; existing room temperature DEFCs predominantly produce acetic acid rather than carbon dioxide.14-17 While acetic acid is not extremely toxic to organisms such as yeast, the rate of acetic acid production by the fuel cell makes long term simultaneous operation of fermentation next to the fuel cell anode impossible unless steps are taken to remove the acid.18 Separation membranes are a common method of purifying desired components and removing unwanted components in biological systems.19-22 Membranes can be combined with evaporation in pervaporation separations; these systems are capable of improving the efficiency of fuel purification at intermediate scales,6 and the development of new membranes that achieve higher separations is an active area of research.23-24 Alternatively, separation membranes can be used by themselves, with the type of membrane depending on the application. Large pore membranes (e.g., dialysis tubing) are sufficient if the goal is only to remove cell bodies, but most


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biomolecules are removed only with very small pore sizes (e.g., nanofiltration and reverse osmosis (RO) membranes).13, 25 Since the biological molecules that poison fuel cells are as small as amino acids, only small pore size membranes have been shown to improve the performance of fuel cells running on complex fermentation media,13, 25 with RO membranes being particularly effective. When considering separation membrane pore size, there is also a tradeoff of slower ethanol diffusion through small pore (e.g., RO) membranes, which can limit fuel cell performance.12 This slow ethanol diffusion can be improved by applying pressure20 or by adding glucose to balance the osmotic pressure.13 While a range of separation membranes have been shown be to be suitable for separating ethanol from fermented media, continuous operation of the fermentation with a fuel cell remains largely unexplored.

A major challenge is that

separation membranes generally have limited selectivity between the ethanol and the acetic acid. Since operation of the fuel cell requires ready ethanol diffusion across the separation membrane, acetic acid produced by the fuel cell will diffuse back into the fermentation, rapidly killing the yeast. Here we explore in detail the use of RO membranes for linking yeast fermentations and fuel cells. Many of the challenges are common to biofuel purification and separations in general, including incomplete rejection of the media, and cross-over of a fuel cell oxidation product (acetic acid) back through the separation membrane. When considering biofuels such as ethanol, it is especially desirable to avoid as much parasitic power loss as possible to avoid cannibalizing the power produced by the fuel cell. In the case of acetic acid, which is produced by the fuel cell and is toxic to yeast, we show that altering its charge provides a mechanism of controlling its diffusion across RO membranes. We show that limiting back diffusion of the acetic acid allows simultaneous operation of a fuel cell with an ongoing fermentation, in contrast to previous


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separation membrane systems where acetic acid rapidly stops the fermentation.

We also

examine the rejection by RO membranes of amino acids, which are normally present in fermentations and are extremely harmful to fuel cell performance. The amino acid permeability is shown to be small but nonetheless sufficient to cause significant decreases in fuel cell performance over a period of days to weeks, limiting the lifetime of bio-hybrid fuel cells.

EXPERIMENTAL Fermentation conditions The VL3 wine yeast strain (Zymaflore, Bordeaux, France) of Saccharomyces cerevisiae was used to ferment the growth medium YPD, a mixture of yeast extract (2%), bacteriological peptone (2%), and glucose (concentrations noted in text). YPD is a standard growth medium for yeast that ensures rapid ethanol production. It is a rich medium with a mix of carbohydrates, lipids, and digested proteins, ensuring that it contains the full range of molecules that are commonly found in growth media and waste water and that might poison the fuel cell.11, 16 In experiments where the fermentation was linked to the fuel cell using RO membranes, a 30 mL fermentation volume was used and the glucose concentration chosen to balance any osmotic pressure differential with the other chambers; additional glucose was added each day to replace glucose consumed by the yeast. For some experiments, acetic acid was added to this medium as noted in the text. Fourier Transform Infrared spectroscopy Fourier Transform Infrared (FTIR) spectroscopy was used to determine component concentrations, in the fuel cell and fermentations.

An FTIR spectrometer was used, either an


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Alpha (Bruker Optics Inc.: Billerica, MA, USA) or Nicolet 6700 (Thermo Fischer Scientific: Waltham, MA, USA), both with a diamond attenuated total reflection attachment.

The

compositions of mixtures were determined using reference spectra of components and a least squares fitting method developed in-house.26 Water backgrounds were collected prior to measurement, with 48 scans being typically run with a 50 µL sample volume. Based on analysis of known mixtures and comparisons to chromatography data, the solution concentrations determined using FTIR spectroscopy were found to have very low absolute errors (typically around 0.1%) so long as the major components of the solution are known.26 Fuel cell measurements Most fuel cell measurements were conducted with home built DEFCs made using commercial membrane electrode assemblies (FuelCellsEtc: College Station, TX, USA). 4 mg cm-2 platinumruthenium catalyst was used on the anode side and 4 mg cm-2 platinum black catalyst on the cathode side. The proton exchange membrane (PEM) was Nafion117 and the electrodes were carbon mesh. The active area was 1” x 1” (6.45 cm2). These fuel cells are referred to as the ‘large’ fuel cells in the text. A few measurements (such as the amino acid poisoning experiments) were conducted on less expensive pre-built (‘small’) fuel cells with a lower active area (obtained from fuelcellstore.com, SKU 1071041, H-Tec Ind., GmbH, single plate methanol/air PEMFC, 2.68 cm2 active area).

The fuel cell used is noted in the text.

Electrochemical measurements were performed with an eight channel VMP3 potentiostat (Biologic: Claix, France). The cathode was used as both the counter electrode and as a pseudoreference electrode.

Performance was characterized with chronoamperometric (I-t)

measurements poised at 200 mV to be consistent with previous measurements that had shown this voltage to produce near-peak power when the fuel cell is operating optimally; under non-


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optimal conditions the peak power may be produced at lower voltages, but this was not investigated here.11 Permeability measurements Permeability measurements were done with a Dow Filmtec Flat Sheet Membrane, RO, SW30HR, a commercial polyamide RO membrane with a slightly negative zeta potential across its operating regime.27 A 1” x 1” (6.45 cm2) piece was used to separate two 10 mL chambers that were filled with solutions as noted in the text. To verify the absence of leaks, tests were done with sugars (glucose and galactose). No sugar flux across the membranes was observed over periods of at least one month. The concentrations in the two chambers were monitored using FTIR as described above. The concentrations in the two chambers gradually equilibrate from diffusion across the RO membrane. The instantaneous net flux can be described with Fick’s first law: = ∆

(1)

where F is the flux, P is the permeability, and ΔC is the concentration difference between the two chambers. Integrating this equation allows the concentration in the chamber over time to be determined. Specifically, an exponential decay is obtained that depends on the permeability. If the two chambers are equal in volume ΔC: ∆ = −∆

where A is the area of the membrane and V is the chamber volume.

(2) From fitting the

concentration data to this equation the permeability was obtained. Bio-hybrid fuel cells


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Bio-hybrid fuel cells were set up with a 30 mL fermentation separated from a 10 mL chamber adjacent to the fuel cell anode by a 1” x 1” RO membrane (similar to the membrane used in the permeability measurements). Glucose, which does not readily diffuse across the RO membranes, was used to balance the osmotic pressures. In some cases, as noted in the text, a third chamber was added between the fuel cell and the fermentation. This chamber was also 10 mL in volume and was separated from the fermentation and fuel cell by two 1” x 1” RO membranes.

RESULTS AND DISCUSSION Reverse Osmosis (RO) membranes are known to reject charged species and larger molecules, but to be permeable to small, uncharged molecules such as water, ethanol (used by the direct ethanol fuel cell (DEFC)) and acetic acid (a waste product of the DEFC).20-21 The permeability of the RO membranes to ethanol and acetic acid was characterized as shown in Figure 1. Figure 1A shows the concentration of ethanol in two chambers that were initially loaded either with a 4.6 M ethanol solution or with deionized water. The dotted lines show fits based on Fick’s law (see Equations 1 and 2) that enable the ethanol permeability to be determined; the permeability of ethanol was calculated to be 1.3 mmol d-1 cm-2 M-1. Figure 1B shows a similar experiment for acetic acid, except in this case both chambers were initially loaded with 1.7 M acetic acid. Since no buffer was used and since acetic acid is a weak acid, the acetic acid exists as a protonated (uncharged) species and therefore will readily cross the RO membrane. The acetic acid in the two chambers was distinguished by using perdeuterated acetic acid (d-acetic) in one of the chambers.

As with the ethanol, acetic acid readily crosses the RO membrane, with a

permeability of 1.08 mmol d-1 cm-2 M-1. Interestingly, the small change in molecular weight causes the deuterated acetic acid to diffuse slightly more slowly, with a permeability of only of


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1.03 mmol d-1 cm-2 M-1. The rapid movement of both acetic acid and ethanol across RO membranes is not surprising; RO membranes are known to have poor rejection of low molecular weight uncharged molecules.28 The particular RO membrane used here (a Dow Filmtec SW30HR membrane) has been observed to have incomplete rejection of ethanol and isopropanol (molecular weight of 60, similar to acetic acid) while having good rejections of glucose (molecular weight of 180).29 In contrast, ions are readily rejected by RO membranes including the one used here,28 and if acetic acid can be converted to the acetate ion it will no longer readily diffuse across an RO membrane, due to its negative charge.

Acetic acid can be readily

neutralized to acetate by adding a buffer or a base. Figure 1C shows the cross-over of sodium acetate for the RO membrane; the diffusion of the sodium acetate across the membrane is roughly 50 times lower than acetic acid (permeability of 0.054 vs 1.08 mmol d-1 cm-2 M-1). In summary, because ethanol and acetic acid have similar permeabilities for RO membranes, not only will ethanol readily diffuse from the fermentation to the fuel cell, but acetic acid will also diffuse from the fuel cell to the fermentation — unless it is deprotonated with a base to form an acetate salt.


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Figure 1. Diffusion of ethanol (A), acetic acid (B), and sodium acetate (C) across RO membranes. The initial concentrations (4.6 M for ethanol, 1.7 M for acetic acid and 1.2 M for sodium acetate) are normalized to 1. The chamber names refer to the species initially loaded into the chambers. Fits obtained using Fick’s first law are shown as dashed and solid lines. If acetic acid from the fuel cell is not prevented from diffusing to the fermentation, the fuel cell and fermentation cannot be operated simultaneously due to the yeast’s sensitivity to acetic acid. It is well known that acetic acid is toxic to yeast at low concentrations, but the toxicity depends on growth conditions and the strain of yeast used.30-32 To characterize the toxicity for our strain (VL3) and growth conditions (YPD), acetic acid was added at a variety of concentrations as


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shown in Figure 2. At 50 mM acetic acid, the fermentation is slightly slowed, while at 67 mM acetic acid it is slowed immensely. No growth at all was observed at 83 mM acetic acid. These concentrations are towards the lower range of acetic acid tolerances observed in yeast and could probably be improved modestly by using a different strain of yeast or a growth medium with greater buffering capacity.31,

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However, as discussed below, under optimal operating

conditions 0.7 mmol cm-2 d-1 of acetic acid can be produced by the fuel cell. Because of the similar permeability of ethanol and acetic acid, there will inevitability be a large amount of acetic acid cross-over with an appropriately sized RO membrane. For a reasonably sized fermentation (roughly 5 mL fermentation volume per cm2 of fuel cell area), the acetic acid concentration would increase 140 mM per day, a concentration that is almost inevitably toxic to yeast.31-32 Therefore, a single RO membrane can be used to extract ethanol from a completed fermentation for use in a fuel cell, but a fuel cell run with an ongoing fermentation will quickly overwhelm and kill the fermentation with acetic acid.


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Figure 2. Glucose (A) and ethanol (B) concentrations for fermentations grown with acetic acid at various concentrations: 0 mM (blue, circles), 17 mM (red, squares), 33 mM (purples, diamonds), 50 mM (orange, triangles), 67 mM (green, circles) or 83 mM (black, stars). As little as 50 mM acetic acid is enough to retard the fermentation and no growth is observed at 83 mM acetic acid. Since acetate has a much lower permeability, converting the acetic acid to acetate ions should allow the RO membrane to effectively prevent acetic acid backflow into the fermentation. In principle, acetic acid could be converted to acetate directly within the anode compartment, but since most salts and bases inhibit fuel cell performance, more flexibility can be obtained by adding a third chamber, between the fuel cell and fermentation, where the acetic acid is converted to acetate. This alternative bio-hybrid fuel cell setup with three chambers separated by RO membranes is shown in Figure 3.

The fermentation and fuel cell chambers are now

separated by an amelioration chamber where acetic acid is converted to acetate. Since acetate ions do not readily cross over into the fermentation chamber, acetate can build up without killing the yeast. Using the data in Figures 1 and 2, it is possible to calculate the expected fluxes and molar concentrations in the device. The ethanol and acetic acid fluxes (represented by the blue arrows) are 0.7 mmol cm-2 d-1. These fluxes assume no ethanol or acetic acid crosses through the fuel cell; in practice at least a small fraction of both will cross through, depending on concentration. Loss of acetic acid is beneficial as it will extend the lifetime of the amelioration chamber but any losses of unreacted ethanol result a loss in energy generation. Assuming the ethanol flux is completely converted to acetic acid by the fuel cell, the current can be calculated using Faraday’s constant with 4 electrons produced per mole of ethanol. A current density of 3 mA cm-2 is expected, equivalent to about 19 mA for the 6.45 cm2 fuel cells used here. The


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predicted ethanol concentration at the fuel cell (1.1 M) is near the optimal concentration for the fuel cell and will allow the fuel cell to produce the desired current density (see Figure S1 for current density as a function of ethanol concentration for the fuel cells used here). Because of the need for a concentration gradient to drive ethanol flux across the two RO membranes, the ethanol concentrations in the amelioration and fermentation compartments are higher (1.6 and 2 M, respectively). An ethanol concentration of 2 M is readily achieved by the strain of yeast used here, although, as shown in Figure 2, ethanol production has started to slow at this concentration. From Figure 2, the ethanol production at 2 M ethanol is approximately 0.15 mmol mL-1 d-1, so a relatively small fermentation is sufficient to operate the fuel cell (4.7 mL/cm2 or 30 mL for the fuel cells used here).

Figure 3. A schematic diagram for a three-chamber bio-hybrid fuel cell where the middle chamber converts acetic acid to acetate to prevent acetic acid from building up in the fermentation and killing the yeast. The concentrations of ethanol and acetic acid are shown for a flux of 0.7 mmol cm-2 d-1 of ethanol through the bio-hybrid fuel cell (blue arrows). This will produce a current density of approximately 3 mA cm-2.


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In the amelioration chamber, acetic acid produced by the fuel cell is converted into acetate salts that no longer pass through the RO membranes. While a wide range of salts and bases could in principle be used, sodium bicarbonate was chosen to demonstrate the principle of operating the three chamber bio-hybrid fuel cell. Because it reacts with acetic acid to form sodium acetate, water and carbon dioxide, the ion concentration in the chamber remains constant, preventing differences in osmotic pressure across the RO membrane. As shown in Figure S2, sodium bicarbonate does not readily pass through RO membranes. The amount of acetic acid that can be remediated depends on the size of the amelioration chamber and the bicarbonate concentration. If 1 M sodium bicarbonate is used, and the amelioration chamber sized similarly to the fermentation chamber (30 mL), the chamber is expected to be able to remediate acetic acid for at least seven days. While the use of sodium bicarbonate will keep the osmotic pressure constant in the amelioration chamber, it is important to balance this osmotic pressure in the other two chambers to ensure water does not accumulate in the amelioration chamber. In the fermentation, a wide range of salts or sugars could be used to balance the osmotic pressure, while in the fuel cell chamber, sugars or acids can both be used without impairing the fuel cell performance. Figure 4 shows the ability of a third chamber to ensure that almost none of the acetic acid produced by the fuel cell enters the fermentation chamber until the sodium bicarbonate is exhausted is shown in Figure 4. Figure 4A shows the current produced by a bio-hybrid fuel cell where the fuel cell chamber has been preloaded with 1.7 M ethanol, near the optimal ethanol concentration for the fuel cell. The currents are initially high (>25 mA) but gradually decrease due to the declining ethanol concentration (right axis). The shaded region shows one standard deviation away from the mean current. Acetic acid also begins to accumulate; the acetic acid produced does not match the ethanol consumed due to acetic acid and ethanol passing through


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the RO membrane and the proton exchange membrane of the fuel cell. By day 3 almost all of the ethanol has been converted to acetic acid. Figure 4B shows the concentrations of the various components of the amelioration chamber. For this test, a 200 mM bicarbonate solution was used with a 10 mL remediation chamber as this volume and concentration is sufficient to show the benefits of the third chamber. Higher sodium bicarbonate concentrations and larger volumes could be used to extend the amelioration lifetime. The sodium bicarbonate concentration declines steadily, reaching zero sometime between days 2 and 3. After the bicarbonate is exhausted, acetic acid begins to enter the fermentation (shown in Figure 4C), building up at a rate of roughly 0.03 M per day. By day 7, the concentration has reached an inhibitory level and the fermentation has stopped producing ethanol. In the two runs done, acetic acid enters the fermentation at only slightly different rates but this difference is enough to allow one of the fermentations to produce ethanol slightly longer (~12-24 h) than the other. This produces a large variability in the final ethanol concentrations but otherwise the runs are highly reproducible. In a similar experiment with the 2-chamber bio-hybrid fuel cell, acetic acid begins entering the fermentation almost immediately after it is produced by the fuel cell (See Figure S3). An acetic acid concentration in the fermentation chamber of 0.05 M was observed after 24 h, enough to inhibit ethanol production within the fermentation. A small amount of ethanol is observed in the fermentation, but this likely arises from ethanol at the fuel cell chamber diffusing into the fermentation chamber. These results clearly demonstrate that converting acetic acid to acetate is an effective method of limiting its diffusion. Periodic flushing of the middle chamber of the bio-hybrid fuel cell with fresh sodium bicarbonate could extend the fermentation lifetime indefinitely.


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Figure 4. (A) Current from a three-chamber bio-hybrid fuel cell (left axis, large fuel cell used, active area = 6.45 cm2) where the fuel cell chamber has been loaded with 1.7 M ethanol. The shaded region is one standard deviation away from the mean current. At day 3, a fuel cell became disconnected from the potentiostat, resulting in a current stoppage. Concentrations of ethanol and acetic acid in the fuel cell chamber are shown (right axis) (B) Concentration of sodium bicarbonate, acetic acid, acetate (left axis), and ethanol (right axis) in the amelioration chamber. (C) Concentration of ethanol, glucose (left axis), and acetic acid (right axis) in the fermentation chamber. Acetic acid enters the fermentation when bicarbonate is exhausted, ultimately stopping the fermentations by day 7. A subtle challenge with running bio-hybrid fuel cells with rich media is the incomplete rejection of amino acids by the RO membrane. Single amino acids and short chains of amino acids (peptides) are present in most rich media and furthermore are slowly produced by yeast themselves. The presence of the carboxylic acid and amine groups ensures that amino acids are


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normally charged and this results in a very low rate of amino acid flux through the membrane, even though amino acids are generally small molecules. However, as Figure 4A shows for two amino acids (cysteine and arginine), the flux is not quite zero. The cysteine concentrations are shown in black and the arginine concentrations are shown in red. Over several weeks, the amino acids slowly cross over the RO membrane and the concentrations begin to equilibrate; this process is slower for the arginine than for the cysteine, likely due to an additional positive charge, although its higher molecular weight may also contribute. From the data, permeabilities of 200 and 70 μmol d-1 cm-2 M-1 were obtained for cysteine and arginine, respectively.

Figure 5. (A) Diffusion across the RO membranes of two amino acids (cysteine and arginine, initial concentrations are 1.1 M and 0.8 M, respectively, but normalized to 1). Arginine diffuses


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more slowly, likely due to its additional positive charge, but both amino acids have some diffusion through the RO membrane. (B) Currents produced by a small fuel cell (active area = 2.68 cm2) with 3% ethanol and with either no amino acids added (blue, solid line), or with small amounts of cysteine (dashed, 10 µM yellow, 20 µM orange, 40 µM red) or glycine (100 µM, dotted black) added. Both amino acids decrease the current, with a larger decrease observed with cysteine, likely due to its thiol group. (C) Current (blue line, left axis) and ethanol concentration (red squares, right axis) of a large fuel cell (active area = 6.45 cm2) in a 2-chamber bio-hybrid fuel cell. Initially low currents are produced due to the low ethanol concentration but over time the current decreases despite an increasing ethanol concentration. While the diffusion rate of amino acids relative to uncharged small molecules such as ethanol is very slow, even a slow diffusion is problematic because of the high sensitivity of the fuel cells to very low amino acids concentrations (10s of µM). Two amino acids, cysteine and glycine, were added to fuel cells to demonstrate their negative effects. Glycine was chosen because it is the simplest amino acid and should be representative of the negative effects amino acids have regardless of functional group. In particular, all amino acids have an amine group which is normally positively charged and may have a negative effect on fuel cell performance similar to other cations.10-11 Cysteine has a thiol group that may be especially harmful to the platinumruthenium catalyst of the fuel cell.8-9 As shown in Figure 4B, both amino acids have some negative effects on fuel cell currents, with the cysteine having a larger effect, as expected. The solid blue line shows current produced by a fuel cell running on 3% ethanol without any amino acids added while the dashed and dotted lines show the currents obtained with the same fuel cell when cysteine and glycine are added, respectively. At 10 and 20 µM cysteine, the effect is modest (~10-20% current decrease) while at 40 µM cysteine, the current has decreased by a


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third. In contrast, even at 100 µM the glycine only has a slight negative effect on the fuel cell current. These results suggest that thiol groups of cysteine have a significant harmful effect in addition to a more modest negative effect that amino acids in general have. In general, rich media have high amino acid concentrations (~100s of µM) and the imperfect rejection of amino acids by RO membranes shown in Figure 4B implies that the amino acid concentrations at the anode could reach inhibitory concentrations in days. Large decreases in fuel cell performance over a period of days can be observed if an ongoing fermentation in a rich medium is separated from the fuel cell only by an RO membrane. (This setup only has 2 chambers rather than 3 chambers as in Figure 4.) Figure 5C shows the current of a bio-hybrid fuel cell running on ethanol produced by a yeast fermentation (blue line) and the ethanol concentration in the fuel cell chamber (red line). The fermentation was allowed to run for 2 days before the fuel cell was started. The fermentation and the fuel cell were in contact via the RO membrane for these 2 days. Initially the currents are very low, roughly 1.5 mA, but this is readily explained by the low ethanol concentration (0.1 M) in the fuel cell chamber. Since the fermentation has only had 2 days to produce ethanol, only a small amount of ethanol has diffused across the RO membrane to the fuel cell. As shown in Figure S1, 0.1 M ethanol is significantly below the optimal ethanol concentration for the fuel cell (~1-1.5 M) and this accounts for the low currents; control runs at 0.2 M ethanol produced current densities of only 0.4-1.3 mA/cm2 (2-8 mA total). However, over time more ethanol is able to diffuse into the fuel cell chamber; after 24 hours the ethanol concentration is 0.2 M and it reaches 0.5 M by the end of the experiment. Despite the increase in the ethanol concentration, the fuel cell currents drop steadily over several days of operation, ultimately going negative. (The negative current is an artifact of using a potentiostat to measure the current; the potentiostat will supply power if necessary to keep the


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potential at 200 mV and this results in a negative current.) These results illustrate the problems of the incomplete rejection of the fermentation media by the RO membrane. This problem can be partially ameliorated by using a 3-chamber setup as in Figure 4, where average currents greater than 1 mA are maintained throughout the 1 week experiment. However, even though the ethanol concentration is increasing slightly after day 3, the currents in the three chamber fuel cell continue to decline, suggesting that poisoning remains an issue. While the RO membrane provides some protection against the rich media that would otherwise rapidly poison the fuel cells,13 during extended operation fuel cell performance still degrades. Another long term concern with RO membrane systems is fouling.

In commercial RO

systems, fouling is a major issue that has limited its use due to the need to frequently clean the membrane. Even without net flow, biofilms can readily form on the polyamide membranes commonly used in RO systems.27 To examine the issue of fouling when coupling a fuel cell with a fermentation, water flux data was collected for never-used RO membranes and compared with the RO membranes of two 3-chamber bio-hybrid fuel cells with partially deuterated water used as a tracer. For the 3-chamber bio-hybrid fuel cells, flux across the fermentation-amelioration chamber RO membrane was examined, since the buildup of biological components on the fermentation side of the RO membrane is the most likely source of fouling. The fuel cells had both been extensively used (over 1 month of total operation time each) and the measurements were conducted following a 1 week experiment without any cleaning of the RO membranes. A summary of the flux data is shown in Table 1. The never-used membranes have slightly higher water fluxes but the decline in the self-diffusion is very modest. The decline is likely modest in part because the backing of the RO membrane was placed towards the fermentation, rather than the polyamide layer, which readily supports biofilms.27 However, if extended operation of the


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bio-hybrid fuel cells becomes feasible, it is likely the flux would continue to decline and eventually necessitate periodic cleaning of the system. Table 1. Water flux (in mmol d-1 cm-2 M-1) through never-used RO membranes and through RO membranes after use in the fermentation. The measurements done in duplicate; the ± values represent 1 standard deviation.

Never-used RO membrane

RO membrane after use with fermentation

0.45 ± 0.04

0.38 ± 0.02

CONCLUSIONS Here we have examined the use of RO membranes to link a fermentation with a DEFC to create a bio-hybrid fuel cell. This creates several challenges, as both the fuel cell and the fermentation have components that poison the other. Ethanol electro-oxidation by the fuel cell primarily produces acetic acid, which readily crosses RO membranes, due to its small size and weak acidity. This has, until now, limited operation of fuel cells with ongoing fermentations as acetic acid is toxic to yeast at low concentrations. Its diffusion across the RO membranes can be stopped if it is converted to acetate ions. We have shown that this property can be used to develop a three chamber bio-hybrid fuel cell where the middle chamber contained sodium bicarbonate which reacts with acetic acid to form sodium acetate. Long-term operation of a biohybrid fuel cell is also limited by diffusion of fermentation media components across the RO membrane to the fuel cell. In particular, DEFCs are highly sensitive to amino acids commonly present in rich growth media.

Therefore, when rich growth media are used, the currents

produced by the fuel cell rapidly decline to near zero. It should be possible to extend the fuel cell lifetimes by using more minimal growth media, or by regenerating the fuel cell using strong


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acids (e.g., sulfuric acid). Alternatively, RO membranes could be combined with a second method to remediate the small quantities of amino acids that bleed through. Overall, these results demonstrate the utility and limitations of RO membranes for bio-separations and for linking biological and artificial systems. Corresponding Author * Email: [email protected] Tel: +1-301-394-2522 Fax: +1-301-394-0310 ACKNOWLEDGMENT This contribution was identified by Prof. Taejin Kim (SUNY Stony Brook) as the Best Presentation in the session “ENFL: Fuel Cells” of the 2016 ACS Spring National Meeting in San Diego, CA. Jahnke was supported from the U.S. Army Research Laboratory Postdoctoral Fellowship Program administered by the Oak Ridge Associated Universities. Benyamin was supported from the College Qualified Leaders (CQL) program of the U.S. Army Educational Outreach Program (AEOP). ABBREVIATIONS RO: Reverse Osmosis; DEFC: Direct Ethanol Fuel Cell, FTIR: Fourier Transform Infrared REFERENCES (1) Badwal, S. P. S.; Giddey, S.; Kulkarni, A.; Goel, J.; Basu, S. "Direct ethanol fuel cells for transport and stationary applications – A comprehensive review." Applied Energy 2015, 145, 80. (2) Soloveichik, G. L. "Liquid fuel cells." Beilstein Journal of Nanotechnology 2014, 5, 1399. (3) Zhang, C.; Su, H.; Baeyens, J.; Tan, T. "Reviewing the anaerobic digestion of food waste for biogas production." Renewable and Sustainable Energy Reviews 2014, 38, 383. (4) Fukuda, H.; Kondo, A.; Tamalampudi, S. "Bioenergy: Sustainable fuels from biomass by yeast and fungal whole-cell biocatalysts." Biochem. Eng. J. 2009, 44 (1), 2. (5) Yasin, N. H. M.; Mumtaz, T.; Hassan, M. A.; Abd Rahman, N. A. "Food waste and food processing waste for biohydrogen production: A review." Journal of Environmental Management 2013, 130, 375.


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Using Reverse Osmosis Membranes to Couple Direct Ethanol Fuel

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