BACrERIOLOGICAL REVIEWS, Mar., 1966Copyright © 1966 American Society for Microbiology

Symposium on Bioelectrochemistryof Microorganisms,IV. Biochemical Fuel Cells

KENNETH LEWIS

Department ofAgricultural Microbiology, Rutgers, The State University, New Brunswick, New Jersey

INTRODUCTION................................................................ 101

FUEL CELL CONCEM .................................................. 101CONCEPTS OF BIocHiEmICAL FuEL CELLS AND THEIR APPLICATION.................. 104

Microbial Corrosion .................................................. 105Exploratory Investigations .................................................. 106Urea-Air Bio-Cell .................................................. 106Hydrogen-Air Bio-Cell.................................................. 108

Electroactive Enzyme Systems................................................. 109

Organic Fuels.................................................. 110

CONCLUSIONS .............................................................. 111LrThEATURE Cru ............................................................ 111

INTRODUCTION

On the first page of a recent volume on fuelcells, the following statement appeared, "Perhapsthe most highly refined fuel cell system today isthe human body, a mechanism that catalytically(enzymes) bums (oxidizes) food (fuel) in an elec-trolyte (blood) to produce energy, some of whichis electrical" (28). Attempts to attain some of therefinement of the living fuel cell have now beenpursued in biochemical fuel cell investigationsduring the past 5 years or so. There have been re-views (14, 20, 35) outlining the general approachesto such a cell, and initial results have been re-

ported, but no comprehensive review has cometo my attention. Herein, it is proposed to presenta general summary of biochemical fuel cell investi-gations.For the purpose of orienting those who are un-

familiar with the term, a fuel cell may be broadlydefined as an electrochemical energy conversiondevice. A brief review of fuel cell concepts will begiven to clarify this definition. Further informa-tion on fuel cells can be obtained from two recentbooks (27, 49), and references contained therein.

FUEL CELL CONCEPTS

Historically the fuel cell is not new; the firstsuccessful cell has been attributed to Sir William

This symposium was held at the Annual Meetingof the American Society for Microbiology, AtlanticCity, N.J., 26 April 1965, with R. L. Starkey as con-vener and consultant editor. Paper of the JournalSeries, New Jersey Agricultural Experiment Station,New Brunswick.

Grove in England in 1839 (19), which makes itmore than 100 years old. There were relativelyfew subsequent fuel cell investigations until ap-proximately 10 years ago, when the governmentand industries concerned with fuels and powerproduction became interested in new and moreefficient power sources.One of the common means of producing elec-

trical power today involves a series of steps con-sisting of the combustion of conventional fuels(petroleum, natural gas, and coal) to produceheat to run a steam engine which drives a dynamothat produces electricity. In this conversion proc-ess, one employs a heat engine, the efficiency ofwhich is subject to a theoretical limit stated in thesecond law of thermodynamics. The chemicalenergy of the fuel is converted to thermal energy,but this in turn cannot be converted completelyto electrical energy. Thus, the production of elec-trical power by the process outlined is not themost efficient means of using the chemical energyof the fuel.Ostwald recognized this problem in 1894 (29),

when he recommended development of an elec-trochemical cell with the fuel and the oxidantseparated so as not to combine by direct chemicalreaction. The fuel and the oxidant would eachreact electrochemically by an exchange of elec-trons at the surfaces of the electrodes. Chemicalenergy of the reaction would be converted di-rectly into electrical energy, and would do workwhen the two electrodes were connected, leadingto a flow of current in the external circuit. Thus,the electrochemical "combustion" of a fuel resultsin direct conversion of chemical energy into elec-

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LOAD

ANODE L\ tCATHODEELECTROLYTE

MEMBRANEFIG. 1. Schematic diagram of a fuel cell.

trical energy, and avoids intermediate productionof heat which, as noted above, lowers the effi-ciency of the conversion process. This is the majoradvantage of a fuel cell in energy conversion, andis the primary reason for interest in the device.The operation of a typical cell is illustrated in

Fig. 1. This cell operates with hydrogen gas as thefuel and oxygen gas as the oxidant. The hydrogen-oxygen fuel cell, as it is called, is the most highlydeveloped, since hydrogen is the most reactivefuel known at present. The cell consists of twoelectrodes, an anode at which oxidation occursand a cathode at which reduction occurs. Theyare in contact with and separated by an electrolyte.Often a membrane also separates the anode andcathode compartments. In operation, the fuel,hydrogen gas, passes over the surface of theanode and is electrochemically oxidized to hy-drogen ions, which enter the electrolyte and mi-grate toward the cathode. Oxygen gas passes overthe surface of the cathode and is reduced, com-bining with the hydrogen ions of the electrolyteto form water. The net result of the operation ofthe cell is combination of hydrogen and oxygento form water, and release of electrons that flowthrough the external circuit.From the diagram of the cell, it can be seen

that it is possible to vary the electrolyte, the elec-trodes, the fuel and oxidant, and the conditionsof operation, such as temperature and pressure.In fuel cell investigations, all of these have beenstudied. Thus, one finds cells with solid, aqueous,and molten salt electrolytes, electrode materialsvarying in composition and form, and cells run atlow, medium, and high temperatures and also atdifferent pressures (27, 47, 48, 49). At this point,consideration will be given to the fuel only.A fuel, to be useful, must be favorable both

thermodynamically and kinetically. Thermody-namically, the potential difference between thefuel and oxidant in an electrochemical cell is di-rectly related to the free energy change; therefore,thermodynamic data on the free energy of thereaction indicate whether a material can serve asa fuel. In Table 1 materials are listed which aretypical of those being considered as fuels today.With each substance, the total reaction is thecombination of the fuel with oxygen, leading tothe complete combustion of the fuel. The theo-retical voltages from thermodynamic calculationsare given in the second column of the table. Asidefrom hydrogen, which has been mentioned, thematerials are hydrocarbons or partially oxidizedhydrocarbons. Particular interest in these car-bonaceous fuels is due to their ready availabilityand low cost; they are being produced by thepetrochemical industry and serve now as fuelsfor other power devices. All the materials listedare thermodynamically useful fuels capable ofyielding large quantities of energy.The kinetics are indicated by data in the last

column of the table, where the relative electro-chemical reactivity of the fuels is compared withhydrogen as a standard at a value of 100. A fuelwhich exhibits very poor reactivity, such as meth-ane, does not readily release the large quantitiesof energy which it potentially is capable of yield-ing. These data illustrate the importance of kinet-ics in fuel cells.The kinetics of a reaction are concerned with

the rate of the reaction. Thus, an oxidation reac-tion such as A - B + e would be studied bymeasuring the rate of disappearance of A or therate of appearance of B or electrons. Since elec-

TABLE 1. Relative fuel activities

Fuel Voltage Relative(theoretical) activity

Hydrogen .............. 1.23 100Formaldehyde .......... 1.13 50Methanol............... 1.21 30Propane ................ 1.10 3Methane................ 1.10 2

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trons cannot accumulate, but flow through a cir-cuit, their rate of appearance is measured by de-termining the current. Thus, current is a measureof reaction velocity of an electrode reaction.

In a fuel cell, as the reaction proceeds at afaster rate, i.e., as the current increases, a drop inthe cell voltage is usually observed. Since thepower obtained from the cell depends on theproduct of the current and the voltage, the per-formance of a fuel is measured by determiningwhat is called a current-voltage curve or polariza-tion curve.An example of this type of curve is illustrated

in Fig. 2. The current is plotted on the abscissaagainst the voltage on the ordinate. The twocurves indicate that with increasing current drainthe voltage of cell B falls more rapidly than thatof A. Thus, cell A is capable of producing morepower than B. These are referred to as polariza-tion curves, because when current is drawn thedecline in cell voltage is called polarization. Thereactivity values used in Table 1 were obtained bycomparing polarization curves for each of the fuelmaterials.

It is evident, then, that the polarization be-havior of an electrode is important in fuel cellstudies. There are several forms of polarizationand several factors that determine the degree ofpolarization of an electrode. Only one form willbe considered here, chemical or activation polari-zation. Information on other types of polarizationcan be found in the volume on fuel cells (27).Any electrode reaction consists of a series of

steps: (i) the transport of the reactant species tothe electrodes, (ii) adsorption of the reactant,(iii) electron transfer, (iv) surface reactions, (v)desorption of products, and (vi) transport ofproducts away from the electrode. If any of thesesteps involves an appreciable energy barrier, thenet result is polarization of the electrode, causinga drop in voltage which is revealed by the polari-zation curve.

I-

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20

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As regards polarization, there are large differ-ences between hydrogen and a hydrocarbon, suchas methane. It was indicated previously that wherethe reactivity of hydrogen was 100, the reactivityof methane was 2. The hydrogen molecule issmall and the electrochemical reaction involvesonly one 2-electron exchange. As seen by thefollowing reaction sequence, the complete anodicoxidation of one molecule of methane involvesthe transfer of 8 electrons to the electrode, andconsidering a simple oxidation scheme there arefour 2-electron steps:

CH4 + H20 CH30H + 2H+ + 2eCH30H - HCHO + 2H+ + 2e

HCHO + H2O )- HCOOH + 2H+ + 2eHCOOH > CO2 + 2H+ + 2e

CH4 + 2H20 - CO + 8H+ + 8e

Furthermore, the intermediates can be ad-sorbed to different extents and react at differentrates. Since these all affect the reactivity of meth-ane, it is obvious why there is a difference in re-activity of methane and hydrogen.One of the primary concerns in fuel cell in-

vestigations today is how to obtain greater reac-tivity from carbonaceous fuels, i.e., how to over-come the activation polarization. One means ofaccomplishing this is to change the reaction con-ditions, such as raising the temperature. Mostreactions are sensitive to temperature changes,and electrode reactions are no exception. Thus,by raising the temperature of operation of a cell,the reactivity can often be increased. This is oneof the reasons for using cells with molten electro-lytes or high temperatures. Though the fuel be-comes more reactive, with increased temperaturenew problems arise, such as those resulting fromrapid corrosion.

Another approach to the reactivity problem isto use a catalyst for the reaction. Much effort hasgone into screening programs and mechanismstudies to obtain catalysts that overcome theenergy barriers to reaction under moderate con-ditions. Some of these studies are summarized inthe volume edited by Mitchell (27), and an out-line of a field the authors refer to as electrocataly-sis has been presented by Bockris and Wroblowa(7).Based on the concepts that have been men-

tioned, the following are criteria of an ideal fuelcell. (i) The cell should have high conductivity;though this has not been discussed, it should beevident that large internal resistance in a cell re-sults in loss of power in the external circuit. (ii)The electrodes and electrolyte should be invari-ant; i.e., there should not be any change in thesedue to corrosion reactions or to accumulation of

CURRENT DENSITY

FIG. 2. Typical polarization curves. Current densitywas measured as milliamps per square centimeter.

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products which could lead to a change in the cellreactivity in time. (iii) The cell should exhibithigh reactivity; the fuel and the oxidant shouldreact rapidly, completely and electrochemically.

Aside from these major criteria, it is desirableto have an inexpensive fuel, and to operate underconditions of moderate temperature and pressure.

CONCEPTS OF BIocHEMIcAL FUEL CELLS ANDTHEIR APPLICATION

The concepts of the biochemical fuel cell canbe introduced by reference to the analogy made inthe opening statement between the living organ-ism and the fuel cell. Energy for life processes isderived from the combustion of food materialsjust as energy is obtained from a fuel cell by virtueof the oxidation of the fuel. It is significant thatthe living organism is able to make use of vastlymore complex "fuels" than a fuel cell can handle,and to do this under what are considered to beextremely mild conditions of temperature, pres-sure, and concentration of reactants.The first work in which microbial activity was

followed by the measurement of an electrode po-tential was carried out by Potter in 1911 (30). Hemeasured the potential difference between elec-trodes inserted respectively in a bacterial cultureand the sterile culture medium. He actually madewhat could be considered the first biochemicalfuel cell battery by assembling six cells, each ofwhich consisted of a yeast-glucose half-cell and aglucose half-cell. In 1931, Cohen (12) similarlystudied the potential differences arising betweenvarious cultures and sterile media, and also builta bacterial battery which produced a small cur-rent for a short period of time. From the time ofthese observations until the last few years, therewas no reported interest in the possibility of abiochemical battery. Investigations with the useof electrode potentials to follow changes resultingfrom development of bacteria have continued,and this work was reviewed in a book by Hewittin 1950 (21). Recently, coincident with effortsto develop fuel cells, there has been concern forbiochemical fuel cells. Shaw (35) presented ageneral outline of the subject in 1963, and partof that work will be followed here.

In the overall metabolism of living things, elec-tron-rich substances or foods are converted toelectron-poor substances or wastes.The high-energy, electron-rich substances such

as carbohydrates, lipids, and proteins are notusually electroactive, but the intermediatesformed during biological oxidation may often beactive at an electrode. Also, some final productscan serve as fuels in a fuel cell. Thus, biochem-ically, certain food materials which are not elec-

TYPES OF 810-FUEL CELLS

F OiO D

ORGANISM

BIOLOGICAL ELECTROCHEMICALMETABOLISM METABOLISM

P RODUCTS j

IT~~~~~~~~~~~~~~~~~IBIO-FUEL CELL B10 -FUEL CELL

TYPE A TYPE B

DIRECT INDIRECT

FiG. 3. Bio-fuel cell classification scheme adaptedfrom Shaw (35).

troactive can be transformed to electroactivesubstances.As pointed out by Rohrback (33), any one of

three different biochemical preparations can beused to carry out the initial transformations:living organisms, cell-free extracts, and purifiedenzymes. The latter two preparations becomespent and are more difficult to maintain thanwhole cells.A classification of bio-cells based on that given

by Shaw (35) is shown in Fig. 3. Food enters theorganism and is oxidized, with the microbial cellsfunctioning in fuel cells in one of two ways. Inthe type A cell, the organism converts the foodinto a useful fuel. This may be done either di-rectly with the organisms located at the electrodeand producing the fuel at the electrode surface, orindirectly with the microorganisms in a separatevessel from which the fuel is separated and fed tothe fuel cell. There is an obvious advantage of thedirect fuel cell, but there are disadvantages also,because the best conditions for biological activitymay not be the best ones for electrochemicalactivity, as will be indicated later.The B type of bio-fuel cell, according to Shaw,

is one where the redox reactions of metabolismare performed electrochemically, presumably invivo. The organism derives no energy from theoxidations; instead, the energy is used in the fuelcell, with the microbial cell serving as the catalystfor the reaction. Therefore, it perishes. It is sug-gested that a portion of the population wouldmaintain growth, and the rest of the cells wouldprovide the enzymes that would release the elec-trons.As an example of such an electrochemical

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metabolic process Shaw suggested a step-by-stepoxidation of glucose with hydrogen carriers suchas nicotinamide adenine dinucleotide (NAD) andmediators. Thus, glucose would be oxidized togluconolactone with the concomitant reductionof NAD. This would be reoxidized by an electro-active mediator that transfers electrons to theelectrode.A series of reaction sequences can be conceived

whereby the substrate is oxidized completely. Amajor obstacle to the functioning of such a systemis that the sequence of events involves interplayof materials inside and outside the cell, in a sensedisregarding the cell membrane, since the electrontransfer must finally be made to the electrode.Attempts to obtain something like electrochemi-cal metabolism will be described subsequently.

It is thus likely that a type A cell would bemore practical than a type B cell. The followingare some of the suggested bio-anode reactions andbacteria involved: urea -+ ammonia (Micrococcusureae, Bacillus pasteurii); carbohydrate -÷ ethylalcohol (Saccharomyces sp., Pseudomonas lind-neri); carbohydrate hydrogen (Clostridiumcellobioparus); sulfate - hydrogen sulfide (De-sulfovibrio desulfuricans).The first bio-anode provides for use of the

waste, urine, which contains considerable urea,readily hydrolyzed to ammonia by certain micro-organisms. Ammonia is a potential fuel (44), be-ing oxidized to nitrogen which is a desirable prod-uct because it is easily eliminated. With this fuelthe major difficulty is its low reactivity.For the next two electrodes, a single substrate

becomes converted to two different fuels by differ-ent bacteria. Ethyl alcohol is a possible fuel, butits complete oxidation at fuel cell electrodes hasnot been demonstrated (32). Hydrogen, whichcan be produced from the same substrate as ethylalcohol is an excellent fuel, as has been stated.It is highly reactive, and, on the basis of currentinformation, has the greatest potentiality of allmicrobial products for use in a bioelectrode sys-tem. With the last bio-anode, sulfide produced byD. desulfuricans during growth on a suitable or-ganic substrate would serve as the electroactivematerial. If the sulfide is oxidized to sulfate at theelectrode, the sulfide-sulfate couple would act asa mediator, with the organic substrate being theinitial source of energy. Unfortunately, sulfideapparently is oxidized at electrodes only to sulfurand not to sulfate (1, 34). Both sulfate and theorganic substrate would have to be supplied forsuch an electrode system, and sulfur would ac-cumulate.

Several bio-cathode systems have been sug-gested also. One of these is an oxygen cathode inwhich the oxygen is supplied to the electrode

through photosynthesis of algae. A possible ad-vantage here would be production of the oxygendirectly at the electrode surface. An obvious dis-advantage is dependence of the cell on a source oflight energy.

Other suggested bio-cathodes are based onelectrochemical metabolism, which actually maynot function, as has been mentioned. Thus, as anexample, the reduction of nitrate to N2 by use ofM. denitrificans has been suggested.The results obtained with a few of the bio-fuel

cells will be considered to provide information onlimiting factors and potentialities for workingcells.

Microbial CorrosionPotter and Cohen, as mentioned earlier, assem-

bled the first biochemical fuel cells, but the re-vival of interest in bio-fuel cells apparently de-veloped from interest in microbial corrosionprocesses. Investigations during 1959-1960 in-volved bio-cells consisting of an active metalanode (usually Mg) and an iron cathode, col-onized by a mixed culture of sulfate-reducingbacteria (36, 43).The basic idea behind this cell was outlined by

von Wolzogen Kuhr and van der Vlugt (42) as anexplanation for the anaerobic corrosion of iron inthe presence of sulfate-reducing microorganisms.Briefly, the anodic reaction is the oxidation of theactive metal to metal ions, and the cathodic re-action is the evolution of hydrogen. If the hy-drogen accumulated, the cathodic reaction wouldstop (because of a type of polarization called con-centration polarization which has not been dis-cussed). Since it was known that extensive anaero-bic corrosion occurred, von Wolzogen Kihr andvan der Vlugt suggested that sulfate-reducing bac-teria, which thrive well under these conditions,could utilize the hygrogen to reduce sulfate. Theorganisms would thus remove the accumulatinghydrogen (a process referred to as depolarization)and allow the corrosion reaction to proceed. Nu-merous microbial corrosion studies have beenmade to test the above theory, and the resultshave been summarized in reviews by Starkey (38)and Booth (8).

Initial reports of investigations based on con-sideration of this type of mechanism for a fuelcell were made by Sisler (36, 37). Preliminary re-sults and limiting currents were indicated, butexperimental details were not included.

In 1962 Magna investigators (18) outlinedstudies aimed at determining the nutritional andphysiological parameters of bacterial sulfate re-duction applicable to the microbial cathode re-action. They demonstrated that an enrichmentculture of D. desulfuricans oxidized hydrogen in a

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seawater medium containing a small quantity ofyeast extract and ammonium ion. Reasonablehydrogen oxidation was also obtained whenmarine muds replaced yeast extract. They did notreport any investigations of an actual bio-fuel cell.

Wilson (43) made an extensive study of a mag-nesium-seawater cell with a cathode colonized bysulfate-reducing bacteria, contrasting it with asimilar cell without the bacteria. The bio-cell ex-hibited better performance at low current densi-ties (less than 1 ma/cm2) than did the cell withoutbacteria, but in a 5-month test both cells decayedto zero output owing to calcareous deposits.The performance of cells of this type has not

been any better than that obtained with other bio-cells, and no further reports of investigations ofsuch a cell have come to my attention since 1962.

Exploratory Investigations

Exploratory investigations using enzymes andmicrobial systems were reported by Davis andYarbrough (15) in 1962. Their objective was toreplace part of the biological electron-transportmechanism with an electrode wire which wouldcouple oxygen to the microbial hydrogen trans-fers.The first system they tested was the oxidation of

glucose to gluconic acid by glucose oxidase. Theglucose and enzyme in an anaerobic chamber pro-vided with a platinum electrode was coupled toan oxygen cathode. No reaction occurred underthese conditions; i.e., glucose was not oxidizedat the anaerobic anode. On addition of methyleneblue, the potential changed and current was pro-duced in the external circuit coincident with de-colorization of the dye. The authors concludedthat methylene blue acted as a substitute hydrogenacceptor, and the low currents obtained were dueto slow and inefficient anodic oxidation of the re-duced dye.Davis and Yarbrough also attempted to use a

hydrocarbon in a microbial fuel cell. An activeethane-oxidizing culture of Nocardia was used inthe anode compartment, and ethane gas wasbubbled into the cell. Under anaerobic conditions,the ethane was not oxidized, and the addition ofmethylene blue led to no change. Lack of reactionunder anaerobic conditions may be due to re-quirement for physical incorporation of molecu-lar oxygen in the initial reaction of biological hy-drocarbon oxidation (39).The experiment with Nocardia is an attempt to

obtain electrochemical metabolism, whereby theorganism's enzyme system would oxidize the sub-strate with release of the energy to the externalenvironment by means of the electrode. Thedifficulty of tapping reactions within the cell to ob-

tain energy outside the cell is evident from the re-sults.Methane is an inexpensive hydrocarbon which

is a desirable fuel, but, as previously mentioned,is unreactive under moderate conditions. Usingthe aerobic methane-oxidizing bacterium, Pseu-domonas methanica, van Hees (40) attempted toset up a bio-anode with methane as the bacterialsubstrate. Open circuit potentials and polariza-tion measurements were obtained with culturesin an anaerobic anode compartment both withand without a mediator. Experiments were alsoperformed with the organism separated from theelectrode by a dialysis membrane, in which casethe currents obtained were insignificant. Fromthese results, and a comparison of the observedanodic potentials with known potentials of oxida-tive enzymes, van Hees concluded that "the elec-trochemistry takes place in or on the bacteria, notvia an excreted (direct) fuel" (41). On the basis ofthe potentials obtained, he concluded also thatthis electrochemical metabolism occurs at theflavoprotein or cytochrome b level.The author claims that no deterioration of the

organisms occurs if a moderate current drain (3jia/cm2) is maintained in the cell, whereas pro-longed periods of open circuit or high currentdrain are detrimental. This should be investigatedfurther, because verification of the results wouldseem to indicate that the electrode can act as anelectron acceptor in the reaction.The currents obtained are very small, and

energy calculations indicate that little improve-ment can be expected.

Urea-Air Bio-Cell

The cell based on the conversion of urea to am-monia has been the object of several studies. In-terest in this cell stems from the availability ofurea in human waste, and the fact that the productof the electrochemical oxidation of ammonia isnitrogen, which is a nontoxic gas.

In 1962 a group from Magna Corp. (33) re-ported on preliminary results with a urea-air cell.They obtained an open circuit voltage, i.e., avoltage measured with no current drawn from thecell, of 0.8 v, and a short circuit current of 3.6amp/ft2. It should be noted that under both ofthese conditions no power is obtained from thecell. Power is the product of current and voltage,and at open circuit the current is zero, and atshort circuit the voltage is zero.

In a report by Thompson-Ramo-Wooldridge,Inc. (16), an indirect bio-fuel cell was used inwhich the ammonia was produced by a urea-urease system in a urea-air bio-cell. A 64-cell unitdeveloped 28 v and 0.7 amp when the cell oper-

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ated at 26 C, and 1 atm and when the electrolytewas 3 M KCl in a citrate buffer atpH 6.

It has already been noted that current is re-lated to the rate of an electrochemical reaction.Actually, it is the current density, i.e., the currentper unit area of electrode surface, which is theuseful rate measure. For orientation purposes, acurrent density of 100 amp/ft2 is obtained easilywith a H2/02 cell. In sharp contrast, the currentof most bio-cells is considerably smaller than this.

In a report of a group from Magna Corp. in1963 (9) the biochemical reaction and electro-chemical reaction of the urea bio-cell were studiedseparately, and then coupled under mutually satis-factory operating conditions. For the biochemicalreaction, which is the enzymatic hydrolysis ofurea to ammonia, Bacillus pasteurii and the en-zyme urease were used. With both, the influenceof pH and temperature on urea hydrolysis wasdetermined. The bacterium was active betweenpH 5 and 9 with a maximum atpH 7. The effect oftemperature was determined manometrically bymeasuring CO2 production during hydrolysis.Activity was maximal at 50 C and dropped tozero above 65 C. Activity of the enzyme, urease,was maximal atpH 8 and 47 C.The initial electrochemical studies of ammonia

oxidation were performed under optimal condi-tions for biological ammonia production. Theammonia of the ammonium carbonate was oxi-dized at an exceedingly slow rate at pH 7and 47 C. Further study provided the followinginformation. The concentration of ammoniumcarbonate had little effect on the reaction rate.Increase in the temperature increased reactivityand raised the limiting current density. Increasein the ionic strength of the solution reduced solu-bility of ammonia and thus lowered the effectiveammonia concentration at the electrode, resultingin decrease in the limiting current density. Theoxidation rate increased slightly with increase inpH presumably due to an equilibrium shift to-ward free ammonia.

Results were obtained with a cell operating inthe presence and absence of the bacterium underthe optimal conditions. Current values in the ab-sence of the bacteria were ascribed to limited hy-drolysis and electrochemical activity of impurities.Bacterial activity resulted in better open-circuitvalues, higher limiting currents, and higher over-all activity. It was concluded that the urea-am-monia system was capable of sustaining currentdensities of the order of 3 amp/ft2, but the princi-pal problem appeared to be that of how to obtaina high rate per unit volume.

In another report, the Magna Corp. (11) ob-tained rapid steady-state anaerobic developmentof B. pasteurii on urine in continuous culture,

with production of 0.5 mg of ammonia per ml ofculture per hr.Assuming that there would be 22 g of urea per

man per day, and that the ammonia fuel cellwould operate with a voltage of 1.13 v, it was cal-culated that the reaction would yield 66 watthours. With an efficiency of 70%, there would be47 watt hours available per man day or the poweravailable would be about 3 w per man.Some information was obtained also on the

effect of the organism on the electrode process.Polarization curves for the system consisting ofB. pasteuri and urine showed that the limitingcurrent densities before and after growth do notdiffer significantly. Nevertheless, up to the limit-ing currents, the electrode was more active andthe polarization was lower due to development ofthe bacterium. It was noted in these studies thatthe system changed with time, leading to decay ofactivity which was attributed to poisoning of theelectrode by the ammonia. The general conclusionwas made that the amount of power obtainablefrom these systems is insufficient to be a usefulsource of energy.From studies of the urea-urease system at the

Philco Research Laboratories (3), it was recog-nized that the hydrolytic enzyme reaction wouldnot lead to significant electron redistribution, andit was noted that the product of hydrolysis, NH3,did not exhibit significant electrochemical ac-tivity under conditions favorable for the microbialprocess. Polarization curves of buffer with andwithout ammonium nitrate were essentially identi-cal over a large range of current densities. The re-sults indicate that the course of polarization forthe ammonium nitrate solution was not due toactivity of the ammonia, since the same result wasobtained in its absence.The action of the enzyme urease on urea was

also investigated. When urea was added to com-mercial urease preparations, there was a signifi-cant increase in current at a fixed polarization.However, when a dialyzed enzyme preparationwas used, the electrochemical activity was re-duced. Further studies indicated that the activityof the dialysis solution was greater than that ofthe dialyzed enzyme, indicating that the com-mercial enzyme preparations contained electro-chemically active materials, but the addition ofurea to the active dialysis solution resulted in nochange in the electrochemical activity. On theother hand, addition of urea to the dialyzed en-zyme resulted in significant increase in current.The results also disclosed that pH had an effect

on electrochemical activity of the urease prepara-tions. Activity was significantly greater at pH 8than atpH 6.5.The rate of the hydrolytic reaction was com-

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pared with the rate of change of current uponaddition of urea to the system. The current ob-tained was proportional to the extent of hydroly-sis. Further tests indicated that changes inpH andcurrent followed the same course, indicating thatthe change in pH was intimately related to thecurrent obtained in the urea-urease system. There-fore, it was concluded that the electrochemicalactivity of the urea-urease system is probably dueto the oxidation of impurities in the urease prepa-rations which are actively oxidized at high pH.All of the potentialities of ammonia as a fuel arenot known, but the information that is availableshows that great care should be used in inter-preting the significance of electrochemical datainvolving complex organic systems.

Hydrogen-Air Bio-CellAnother system of interest is a cell based on the

production and utilization of hydrogen. It hasbeen noted already that the hydrogen-oxygencell utilizing the anodic oxidation of hydrogen asthe fuel reaction is at present the best and mosthighly developed fuel cell. There are storage diffi-culties, such as the requirement for a metal cylin-der for high pressure storage that weighs morethan the fuel gas itself. As an alternative to start-ing with the gas, the hydrogen can be generatedfrom a hydride reacting with water, by catalyticdecomposition of ammonia, or by reforming a hy-drocarbon with steam. Also, hydrogen can be pro-duced by microorganisms.

Investigators at Magna Corp. (33) suggestedand experimented with one of the first bio-fuelcell systems aimed at using hydrogen. They re-ported on the production of hydrogen from glu-cose by C. butyricum. When the electrode wasplaced in the glucose medium, no current wasgenerated in the cell, whereas when the bacteriumwas active the electrode performed better, but itwas still strongly polarized at currents of 200 to400 ,ua per cm2.

In additional electrochemical studies (10), itwas noted that the poor performance of the hy-drogen system was due to the low solubility ofhydrogen in the electrolyte. To overcome this,palladium metal, which absorbs hydrogen, wasused as the anode, and the electrode was initiallysaturated with hydrogen before it was used as ananode. This procedure improved the electrodeperformance, as indicated by differences in thelimiting current; with Pt it was approximately0.8 ma/cm2, and with Pd, 4 ma/cm2. However,the performance of the Pd anode decays withtime, apparently due to loss of absorbed hy-drogen.For studies with the bacterium, an electrode

was prepared of a mixture of carbon, Pt black,cells of C. butyricum, and glucose in phosphatebuffer pressed into a disc. The electrode per-formed poorly, presumably due to poor reactivityof He on carbon. The limiting current was only0.1 ma/cm2, which was one-tenth that expected.Berk and Canfield (4) reported on studies which

presumably involve the formation of a hydrogen-oxygen fuel cell. Pt electrodes in the presence ofphotosynthetic microorganisms were found toundergo shifts in potential upon illumination. Aplatinum electrode immersed in water containingsuspended cells of Rhodospirillum rubrum andmalate exhibited a shift to more anodic potentialswhen illuminated, and an electrode coated withmarine algae developed more cathodic potentialswhen lighted.They assembled a total cell from the above two

half cells, which produced an open circuit poten-tial of 0.97v and, on short circuit through a meter,yielded 75 tza/cm2. The cell deteriorated with time.The authors ascribe the results to the photo-

synthetic production of products which reactelectrochemically. Thus, in the presence of lightR. rubrum produces hydrogen (and other prod-ucts) which is anodically oxidized. Similarly, thealgae yield oxygen which is reduced at the cath-ode. With platinized Pt electrodes, the highestcurrent density obtained was 160 Jua/cm2 undershort-circuit conditions. The authors concludedthat it is doubtful that bio-cells could comparewith conventional fuel cells or solar cells.There are differences of a few orders of magni-

tude in the values of the limiting currents ob-tained with the bio-fuel cell systems and with thehydrogen-oxygen fuel cell. Apparently, the differ-ence is due, at least in part, to conditions underwhich the cells operate. In a standard chemicalfuel cell,the conditionsare muchmore severe (hightemperatue, pressure, and fuel concentration)than in the bio-cells.One way to avoid this difficulty is to use the in-

direct approach and carry out the biochemicalreactions at a distance from the electrochemicalones. Initial studies of such a system were re-ported by Melpar Inc. (26). They attempted todevelop systems capable of producing large vol-umes of hydrogen efficiently from plant residues.In this report no mention was made of use of thehydrogen in a direct bio-fuel cell.

In studies of hydrogen production from glu-cose, it was found that, of the bacteria tested,Clostridium welchii and Escherichia coli producedthe greatest amounts. Under optimal conditionsof pH, buffer, substrate, and cell concentrationwith a 10-liter fermentor, 45 liters of hydrogenwas produced in 8 hr with a conversion efficiency

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of 44%. Compared with generation of hydrogenchemically by the catalytic decomposition of am-monia (17), the bacterial system produced only0.1 to 0.6 as much hydrogen, but it is consideredlikely that the bacterial process could be im-proved.

Electroactive Enzyme SystemsBiological oxidations occur under mild condi-

tions by virtue of the enzyme systems involved.Thus, if the reduced form of an enzyme could beoxidized electrochemically, and its oxidized formcould react with a substrate, the enzyme couldcycle back and forth, being oxidized at the elec-trode surface, and reduced by chemical reactionin the solution. The net reaction would be theapparent oxidation of the substrate at the elec-trode. It will be noted that such an electroactiveenzyme system represents a greatly simplifiedtype of electrochemical metabolism which avoidsthe major difficulty involved in that process oftransferring the electrons across the cell mem-brane.

Several enzyme systems have been examinedfor electrochemical activity. Usually there was noactivity, but there have been some suggestive re-sults.Yahiro et al. (46) studied a number of enzyme-

substrate systems. Evidence of electroactivity wasobtained with glucose oxidase, D-amino acid oxi-dase, and peroxidase. It was concluded thatflavoprotein systems are capable of generatingelectrical energy, whereas systems involving pyri-dinoproteins did not do so, and that the resultsprovided physicochemical means of distinguish-ing enzymatic oxidations proceeding by electrontransfer from those due to hydrogen transfer. Itwas later reported (45) that the flavoenzymes(glucose oxidase and D-amino acid oxidase) prob-ably generated electrical energy as a result of elec-tron transfer via the oxidation of the substrates.In contrast to this, the system ethyl alcohol dehy-drogenase-NAD did not produce electrical en-

ergy. Their results were based primarily onvoltages of cells in which the enzyme systems werein the anode compartments. In contrast to theresults of Davis and Yarbrough (15), methyleneblue was not needed to mediate the electron trans-fer from the glucose-glucose oxidase system to theelectrode. It should be pointed out that Davis andYarbrough attempted to obtain electrochemicalreactivity in an anaerobic system, whereas the re-sults of Yahiro et al. were obtained in an aerobicsystem. The presence of oxygen can be a majorfactor in some enzyme reactions.A number of hydrolytic and oxidative enzyme

systems were examined at the Philco Research

Laboratories (3) to determine whether any directelectrochemical reactions of the enzymes oc-curred. The effect of mediators was determined,but direct enzyme-electrode reaction was con-sidered to occur only if reactivity appeared in theabsence of the mediator.Among the hydrolytic enzymes, only urease

showed possible reactivity, and this was due toimpurities in the preparations.The activity of the oxidative enzymes, D-aminO

acid oxidase, glucose oxidase, lactic dehydrogen-ase, and alcohol dehydrogenase, and of mito-chondria and reduced NAD was tested. Only withD-amino acid oxidase was there an indication ofdirect electrochemical reactivity, but some systemswere active in the presence of mediators.The results of Davis and Yarbrough on glucose

oxidase were verified. In an anaerobic system, noelectrochemical activity was evident in the ab-sence of a mediator. Aerobically, a small amountof current was obtained that was ascribed to HP2produced in the enzyme reaction.

Initial studies provided evidence of activity ofthe D-amino acid oxidase-tryptophan system.Further investigation showed that this activitywas apparently due to the presence of oxygen.Whereas there was little or no activity underanaerobic conditions, the system became electro-active when oxygen was introduced. If the enzymewere reacting with the electrode, the activityshould be greatest under anaerobic conditions,and introduction of oxygen should lead to itscompetition with the anode for oxidation of theenzyme, resulting in reduction in the electrochemi-cal activity. Since the opposite occurs, they con-cluded that the product of the reaction of theoxidase on tryptophan must be an electroactivespecies.

Further studies of the system were carried outby an electrochemical technique called chrono-potentiometry, in which a constant current ofknown value is imposed between two electrodesand the potential of the electrode at which the re-action is taking place is recorded against time. Achronopotentiogram for the system D-amino acidoxidase-tryptophan obtained by the Philco groupis shown in Fig. 4 (3). Curve 1 represents thechronopotentiogram obtained by use of trypto-phan alone or tryptophan with D-amino acid oxi-dase under anaerobic conditions. The fact thatthe results were the same with the two systemsindicates that the enzyme did not exhibit anyelectrochemical activity. This could be due eitherto the reduced enzyme being nonelectroactive orto the concentrations of the enzyme being too lowto be detectable. Curve 2 is for the same systemexposed to air; the results were different in that

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there was oxidation at a constant potential otherthan that for the oxidation of tryptophan. Oxygenresulted in formation of an electrochemically ac-tive product. Even with a concentrated solution ofhighly purified enzyme and tryptophan, there wasno evidence of any direct reaction between thereduced enzyme and the electrode.The evidence indicates that direct electrochemi-

cal reaction of enzyme systems either does not oc-cur or is extremely slow. Therefore, there is nobasis for use of a cyclic mechanism employing anenzyme alone. Nevertheless, since the systemsmay be activated by mediators, additional infor-mation is needed on the electrochemistry of vari-ous mediators, the chemistry of mediator-enzymereactions, and the electrochemistry of the inter-acting systems as possible means of obtainingincreased bio-fuel cell reactivity.

Organic Fuels

Simpler organic compounds produced in fairlyhigh yields by microorganisms from more com-plex but readily available substances might serveas fuels in fuel cells. There will be less energy avail-able from the microbial product than from theinitial compound, but there could be a gain in theelectrochemical reactivity of this fuel.

This was the approach of Reynolds andKonikoff (31), who assembled a cell consistingof a yeast-glucose half-cell and an air cathode.The yeast-glucose system was chosen becauseglucose was nonelectroactive, and thus should notaffect the observed potentials. Cell potentialswere obtained only after addition of yeast to glu-cose or in cell-free supernatant fluid from a yeastculture. Further studies with known inhibitors ofglycolytic enzymes, and paper chromatographicanalysis of the culture media led to the conclusionthat yeast metabolism produced electrochemicallyactive fuels, but these were not identified.

Later studies (23) showed that a distillate of theyeast supernatant fluid had the same activity asthe yeast culture. Ethyl alcohol was identified asthe active fuel by gas chromatography and com-parison of polarization curves. Thus, indirectly,these workers studied ethyl alcohol as a fuel.The indirect type of cell with the microbiologi-

cal and electrochemical systems separated hasbeen investigated in our laboratories; the electro-chemistry of compounds produced in high yieldsby microorganisms has been studied directly.

Oxalic acid was chosen for the first tests for thefollowing reasons: (i) large yields of the acid canbe obtained from sugars by the action of fungi(22); (ii) oxalic acid is one of the simpler organicproducts of microorganisms, and thus the chem-istry of its oxidation should be relatively simple;

1.01.

0.81.

C,)-

-J0

w

U)CoCn

n

0.eL

0.4p

0.2L

0.01

-0.2L

TIMEFIG. 4. Chronopotentiogram of the tryptophan

-amino acid oxidase system. (1) Tryptophan ortryptophan plus D-amino acid oxidase (anaerobic). (2)Tryptophan plus D-amino acid oxidase (aerobic). FromBean et al. (3).

(iii) the expected products of oxidation are CO2and water, which should have little or no unde-sirable effect on the electrode process; (iv) thethermodynamic data indicate a favorable elec-trode potential for the oxalic acid-CO2 system(24); and (v) the electrochemical literature indi-cated that oxalic acid could be anodically oxi-dized (2, 25).The anodic behavior of oxalic acid was char-

acterized from current voltage curves obtainedunder varying conditions, such as electrode ma-terial, temperature, and concentration of theacid. The desired anodic reaction is the completeoxidation of oxalic acid to carbon dioxide, hy-drogen ions, and electrons, according to the equa-tion

H2C204 -4 2CO2 + 2H+ + 2e

The coulombic efficiency of the reaction was de-termined from comparison between the amountof carbon dioxide produced at constant currentand the theoretical yield.

Oxalic acid does not exhibit a reversible poten-tial at Pt electrodes in 1 M H2S04 solution. It isseverely polarized from the theoretical open cir-cuit value, and does not determine the potential

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1.0

Uf)

0 0.6

n

0.4

x 0.20

0 0.1M HE Ct 046 0.001M HeCtO4

leCURRENT DENSITY (MA/CM8)

FIG. 5. Dependence of anodic oxidation of oxalicacid on concentration.

in the presence of another electroactive materialsuch as oxygen.

Steady-state current-voltage curves can be ob-tained, but the response of the electrode is slug-gish and, at low current densities (less than 0.1ma/cm2), there is continual decay in performancewith time. Current-voltage curves show that theperformance of oxalic acid is considerably betteron platinized Pt than on bright Pt, undoubtedlydue to the increased surface area of the former.The dependence of reactivity on concentration

is shown in Fig. 5. With 0.001 M oxalic acid, thelimiting current density is approximately 1 ma/cm2, whereas with 0.1 M acid the limiting currentdensity is above 10 ma/cm2. Furthermore, at thelower current densities the 0.001 M solution ismore highly polarized. At 25 C, a 0.1 M oxalicacid solution is approximately saturated. There-fore, low solubility establishes a limit to the use ofconcentration to improve performance.The reaction proceeds with essentially 100%

conversion to carbon dioxide. For this determina-tion, the electrode was maintained at a currentdensity of 30 ma/cm2 for 3 hr, during which timethe electrode potential change was negligible, in-dicating that a true steady state was attained.The principal limitation of oxalic acid as a fuel

is the severe polarization on drawing useful cur-

rent densities. Continued efforts in fuel cell labo-ratories have led to improved catalytic electrodesfor hydrocarbon fuels (5), and presumably thereare electrodes which can catalyze the oxalic acidoxidation and make this system a more usefulsource of energy.

Bockris, Piersma, and Gileadi (6) investigatedthe anodic oxidation of several carbohydrates, in-cluding cellulose and glucose. Current-potentialcurves were obtained at 80 and 100 C in 5 NNaOH and 40% H3PO4. From coulombic effi-ciency determinations with and without currentflow, they suggested that glucose was oxidized inacid solutions by a chemical oxidative decar-

boxylation leading to a five carbon product whichcould be electrochemically oxidized completely toCO2. The possibility was suggested that cellulosecould serve as a fuel under special cell conditions.

CONCLUSIONSThe obvious conclusion from the available in-

formation is that no successful biochemical fuelcell has been demonstrated. In other words, nodevice has been developed that produces electricalenergy in large amounts continuously and at auniform rate. This is not unexpected. Results withconventional fuel cells before initiation of pro-grams on bio-fuel cells indicated that conditionsnecessary for fuel cell reactivity were extremecompared with those favorable for microbial de-velopment. This was recognized by Cohn (13),who remarked at one of the first symposia on bio-chemical fuel cells (1962) that the development ofsuch cells would require full understanding ofbioelectrochemistry, which would take years ifnot decades of study. He indicated also that bio-cells would probably be low power devices usefulonly under special circumstances.The available results indicate that a direct bio-

fuel cell in which the microbial cells are locatedat the electrode has little value. Indirect cells inwhich the microorganisms generate the fuel at adistance from the electrode have more promise,but even these will be useful only under specialconditions. The systems are not competitive withconventional power sources.

Studies of biochemical fuel cells have had thedesirable result of focusing attention on bioelec-trochemistry. Examination of enzyme systemshas indicated that none of the enzymes studiedwas electroactive, even though some were in-volved in electron transport. In the presence ofmediator dyes, some enzyme systems were elec-troactive, but others were not. Thus, electro-chemical activity represents another techniquefor studying enzyme systems and learning moreabout their mechanisms. Studies in this new areahave promoted development of new electrochemi-cal and biochemical techniques and new conceptswhich will increase understanding of both elec-trochemistry and biochemistry.

AcKNowLEimGEmsI wish to express my gratitude to R. L. Starkey

and D. Pramer for their interest, encouragement, andcriticism of this work.

This investigation was supported by Office of NavalResearch Contract NONr 404(17).

LimERATruR Crrm1. ALLEN, P. L., AND A. HICKLING. 1957. Electro-

chemistry of sulphur. 1. Overpotential in the

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discharge of the sulphide ion. Trans. FaradaySoc. 53:1626-1635.

2. ANSON, F. C., AND F. A. SCHULTZ. 1963. Effect ofadsorption and electrode oxidation on theoxidation of oxalic acid at platinum electrodes.Anal. Chem. 35:1114-1116.

3. BEAN, R. C., Y. H. INAMI, P. R. BASFORD, M. H.BOYER, W. C. SHEPHERD, E. R. WALWICK, ANDR. E. KAY. 1964. Study of the fundamentalprinciples of bio-electrochemistry. Final Tech-nical Report Philco Corp., Contract NASw-655,March 1963-March 1964.

4. BERK, R. S., AND J. H. CANFIELD. 1964. Bioelec-trochemical energy conversion. Appl. Micro-biol. 12:10-12.

5. BINDER, H., A. K6HLING, H. KRUPP, K. RICHTER,AND G. SANDSTEDE. 1965. Electrochemicaloxidation of certain hydrocarbons and carbonmonoxide in dilute sulfuric acid. J. Electro-chem. Soc. 112:355-359.

6. BOCKRIS, J. O., B. J. PIERSMA, AND E. GILEADI.1964. Anodic oxidation of cellulose and lowercarbohydrates. Electrochim. Acta 9:1329-1332.

7. BOCKRIS, J. O., AND H. WROBLOWA. 1964. Electro-catalysis. J. Electroanal. Chem. 7:428-451.

8. BOOTH, G. H. 1964. Sulphur bacteria in relationto corrosion. J. Appl. Bacteriol. 27:174-181.

9. BRAKE, J., W. MOMYER, J. CAVALLO, AND H.SILVERMAN. 1963. Biochemical fuel cells. Part2. Proc. 17th Annual Power Sources Conf., p.56-59.

10. BRAKE, J., W. MOMYER, H. SILVERMAN, ANDW. R. SCOTT. 1963. Biochemical fuel cells.Third Quarterly Report, Magna Corp., Con-tract No. DA 36-039-SC 90866 USAERDL.

11. CANFIELD, J. H., AND B. H. GOLDNER. 1964. Re-search on applied bioelectrochemistry. FinalReport, Magna Corp. Contract No. NASw-623.

12. COHEN, B. 1931. The bacterial culture as an elec-trical half-cell. J. Bacteriol. 21:18.

13. COHN, E. M. 1963. Perspectives on biochemicalelectricity. Develop. Ind. Microbiol. 4:53-58.

14. DAVIS, J. B. 1963. Generation of electricity bymicrobial action. Advan. Appl. Microbiol.5:51-64.

15. DAVIS, J. B., AND H. F. YARBROUGH, JR. 1962.Preliminary experiments on a microbial fuelcell. Science 137:615-616.

16. DEL DUCA, M. G., J. M. FUSCOE, AND R. W.ZURILLA. 1963. Direct and indirect bioelectro-chemical energy conversion systems. Develop.Ind. Microbiol. 4:81-91.

17. GEISSLER, H. H. 1963. Compact H2 generators forfuel cells. Proc. 17th Annual Power SourcesConf., p. 75-77.

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19. GROVE, W. R. 1839. On voltaic series and thecombination of gases by platinum. Phil. Mag.14:127.

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