Bioelectrochemistry

BIOCHIMICA ET BIOPHYSICA ACTA

375

BBA 45057

BIOELECTROCHEMISTRY I. ENZYME U T I L I Z I N G B I O - F U E L CELL S T U D I E S A. T. Y A H I R O , S. M. L E E AND D. O. K I M B L E

Space-General Corporation, E1 Monte, Calif. (U.S.A.) (Received D e c e m b e r 3ist, 1963)

SUMMARY

Electron transfer as opposed to hydrogen transfer was demonstrated to be involved in the oxidation-reduction of the flavoprotein enzyme system. A bioelectrochemical investigation of glucose oxidase (EC 1.1.3.4), D-amino acid oxidase (EC 1.4.3.3), and yeast alcohol dehydrogenase (EC i . i . i . I ) systems was conducted in an a t t e m p t to utilize the electron-transferring process as a potential anodic reaction in a biochemical fuel cell. Utilizing a bio-fuel cell constructed of plexiglass, platinum-foil electrodes, and an ion-exchange membrane for conduction between the anolyte and catholyte, the flavoprotein enzymes, both glucose oxidase and D-amino acid oxidase systems in conjunction with an 02 cathode, generated 175-35o mV. In contrast, alcohol dehydrogenase (yeast), a pyridinoprotein enzyme which requires coenzyme I (NAD+), did not produce any electrical voltage. Elemental iron was found to potentiate the flavoprotein enzyme reaction yielding voltages ranging from 625 to 75o mV. The potentiating effect was probably due to a faster turnover rate of F A D H to FAD ÷ coupled with the additional net oxidation potential of iron.

INTRODUCTION

The present space age has initiated a great deal of activity in studies on the generation of electrical energy from biological processes 1, 2. Such studies have involved cells whose construction consisted of (I) electrodes made of platinized nickel wire mesh, platinum foil, carbon, or mild steel and (2) ion-exchange membrane, cellulose membrane or a salt bridge for conduction between the anolyte and catholyte compartments. The biological systems employed at the electrodes included bacteria, fungi, algae, or enzymes. At the present time, due to the paucity of lucid information, half-cell reactions in bio-fuel cells are not clearly understood. To date cathodic half-cells have incorporated an 02 or air cathode while anodic half-cells have employed live microorganisms or enzyme systems. In the case of live microorganisms, the electrical potential m a y arise from a number of different phenomena, i.e., diffusion potential, liquid-junction potential (with or without transference), or an oxidation-reduction potential. Our research as discussed herein is concerned with bio-fuel cells utilizing an anodic oxidation-reduction enzymic system in conjunction with an 02 cathode. To Biochim. Biophys. Acta, 88 (i964) 375-383

376

A. T. YAHIRO, S. M. LEE, D. O. KIMBLE

insure that the electrical output was generated solely as a result of the enzymic reaction only inert platinum-foil electrodes were employed. The results of our study indicate (I) that the oxidation-reduction mechanism of flavoprotein enzymes truly involves electron transfer as contrasted to the pyridinoprotein enzymes which proceed via hydrogen transfer, and (2) that bio-fuel cells utilizing enzymes at the anode and 02 at the cathode have relatively low power output. MATERIALSAND METHODS Enzymes and substrates The enzymes employed in this study were purchased from the Sigma Chemical Company and were not further purified. Listed below are the enzymes, substrate, and buffer solutions : (I) Glucose oxidase (/~-D-glucose: 02 oxidoreduetase, EC 1.1.3.4) (Sigma Type II) 15oo0 units/g. I unit is defined as that amount of enzyme which causes oxidation of I m of glucose to gluconolactone per rain at pH 5.1 at 35 °. The Sigma Type-II grade of glucose oxidase contains approx. 350-370 units of catalase impurity per rag. The catalase activity upon H20 2 which is formed as an end product of glucose oxidase system does not generate electrical energy by the method described herein. (2) D-Amino acid oxidase (D-amino acid: 02 oxidoreductase (deaminating), EC 1.4.3.3), crude, from hog kidney. The acetone powder of the latter was demonstrated to have an activity of 38 units/rag. I unit is defined as that amount of enzyme which will liberate I #g of NH 3 (5.9" Io 2#m) in 30 rain at 37 ° under assay conditions described by the assay data sheet. (3) Alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC I . I . I . I ) (yeast) 400o00 units/rag protein. I unit of alcohol dehydrogenase will form 4.83. lO -a m of DPNH. (4)/%Diphosphopyridine nucleotide (Sigma grade 98-IOO°,~,) or coenzvme I formerly DPN+ is referred to as NAD + in this report. (5) D-Alanine, Sigma grade (Sigma Chemical Co.). (6) D-Glucose, anhydrous, Merck. (7) Phosphate buffer (pH 6-7) was prepared from 17. 4 g K2HPO ~ and 13.6 g KH2PO 4 in 1 1 of distilled water. Reaction vessel A two-compartment membrane cell constructed of plexiglass was used in this study. The compartments had dimensions of 55 × 7o ~ 13 mm and 55 × 7 ° × 20 mm for the cathode and anode compartments, respectively. The anode and cathode were placed approx. 2 mm from the anionic membrane (Ionac No. 3148) which formed the partition between the compartments. The cell was constructed in parts that could be dismantled for cleaning (Fig. 1). The electrodes consisted of smooth-surfaced platinum foils with dimensions of 50 X 64 × o.o127 ram. During the course of a run, the anolyte was stirred by means of a glass propeller regulated to 19oo rev./min with a Heller controller. This high degree of agitation was necessary to achieve maximum voltage output. A glass propeller covered with iron powder (Baker, reagent) on epoxy resin was used to demonstrate the potentiating effects of iron upon the glucose oxidase and D-amino acid oxidase systems. Biochim. Biophys..4cta, 88 (1964) 375 383

BID-FUEL

CELL STUDIES

377

By this means it was possible to eliminate any possible galvanic effects should iron come in contact with the anode.

Electrical measurements The voltage output was recorded on a Varian GI IA recorder and current measured on a Yokogawa Electric Works ammeter. Beckman helipots (Laboratory Model T-Io-A) were used to introduce a resistance into the circuit. Electrode polarization curves were determined by inserting a standard saturated calomel electrode (Radiometer Type K4312 ) against the electrode being tested.

TN I UM ELECTRODE

~

NEOPREN G EASKET-/ ';~''.":';| / ~ STR I RER E ~TAL~ P ~ ~ E !~ N D - END PLATE

SPACER GA SKET SPACERNG EA OS PKR A. EjE TNI CAT;ODE ANOO'% COMPARTMENT COMPARTMENT F i g . ] . B i o e l e c t r o c h e m i c a l cell. E x p l o d e d v i e w s h o w i n g d i s m a n t l e d p a r t s . A n o d e c o m p a r t m e n t w a s 55 × 7 ° × 20 m m a n d c a t h o d e 55 × 7 ° x 13 m m i n d i m e n s i o n . P l a t i n u m e l e c t r o d e s h a d d i m e n sions of 50 x 64 × o.o127 ram.

Analytical methods Iron determination3: Iron, in the reduced ferrous form, was complexed with 2,2'-bipyridine. The intensity of the pink complex formed was used to measure the amount of iron in solution. Glucose oxidase activity: Glucose oxidase activity was determined by the formation of gluconic acid. Enzymic action was stopped by heating a i-ml aliquot for several minutes at 9 o°. The sample was then cooled and titrated against standard o.oi N NaOH to a pH 9.0 using a Radiometer pH meter. D-Amino acid oxidase activity: D-Amino acid oxidase activity was determined by the formation of NH~ as follows : A I-ml sample from a D-amino acid oxidase reaction mixture was transferred to a test tube containing I ml of 20 % trichloroacetic acid. The mixture was then centrifuged and I ml of the supernatant transferred to a test tube containing I ml of Nesslers reagent and I ml of distilled water. The intensityof the colored solution was measured at a wavelength of 505 mff on a Bausch and Lomb Spectronic 20 spectrofotometer. Alcohol dehydrogenase activity: This was determined by the method described by Sigma Chemical Company. I unit of alcohol dehydrogenase activity is defined as that amount of enzyme which causes/JA340 m f f of O.OOI pertain at 25 ° and/or will form 4.83 • lO -4 ffM of D P N H per rain. Absorbancy was measured on a Bausch and Lomb Spectronic 20 spectrofotometer at a wavelength of 340 raft. Biochim. Biophys. Acta, 88 (1964) 3 7 5 3 8 3

378

A . T . YAHIRO, S. M. LEE, D. O. KIMBLE

Experimental methods Flavoprotein enzymes: The phosphate buffer and reaction vessel were warmed to 37 ° in a Sargent Thermoniter controlled constant-temperature bath. Phosphate buffer (35 ml) was added to the cathode compartment. Bubbling 02 through the catholyte was observed to have had no effect upon the voltage output and therefore, was omitted from all experiments. The anode compartment received 30 ml of phosphate buffer followed by 7 ml of a buffer solution containing either glucose or D-alanine. While stirring the anolyte at 19oo rev./min, the enzyme, dissolved in 8 ml of buffer solution, was introduced and reaction allowed to proceed. The following combinations of enzymes and substrates were demonstrated to generate m a x i m u m voltage : Combination I : o.34 g glucose oxidase plus 0.92 g glucose and combination 2 : 0.31 g D-amino acid oxidase plus 0.60 g D-alanine. Pyridinoprotein enzyme: The procedure was essentially the same as above with the exception that the addition sequence to the anolyte consisted of 1-3 ml of 95 % ethanol, 4-1o6-1-1o 7 units of yeast alcohol dehydrogenase and NAD + in varying amounts of IO, 50, and IOO mg. RESULTS

Typical cell voltage curves of glucose/glucose oxidase (Fig. 2, Curve B) and D-alanine/ D-amino acid oxidase (Fig. 2, Curve C) indicate that the glucose oxidase system is a stronger biopotential source than D-amino acid oxidase. In this connection we have observed that the glucose oxidase system produced an average potential of 300-350 mV and the D-amino acid system 175-225 mV. Glucose oxidase and D-amino acid oxidase are flavoprotein enzymes having two and one flavin adenine dinucleotide prosthetic groups per apoenzyme, respectively. Enzymic activities of all enzymes studied were assayed in accordance with the Sigma Chemical Company assay data 6o¢

I

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!

70C

60C

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,F 40

(minute=)

Fig. 2. Cell v o l t a g e o u t p u t d u r i n g 02 c a t h o d e a n d anodic r e a c t i o n i n v o l v i n g t h e following: (A) p h o s p h a t e buffer plus iron, (B) o.91 g glucose plus 0.23 g glucose oxidase; (C) 0.60 g D-alanine plus o.31 g D - a m i n o acid oxidase; (D) o.91 g glucose plus o.34 g glucose o x i d a s e p l u s iron; (E) o.61 g D-alanine p l u s 0.31 g D-amino acid o x i d a s e plus iron; a n d (F) 3 ml 95 % alcohol plus I.lO 7 u n i t s alcohol d e h y d r o g e n a s e p l u s i oo m g N A D +. T h e a n o l y t e a n d c a t h o l y t e r e a c t i o n s were p e r f o r m e d in p h o s p h a t e buffer (pH 6.7).

Biochim. Biophys. Acta, 88 (i964) 375-383

379

BIO-FUEL CELL STUDIES

sheet and were found to have the assayed potency. Enzymic acivity proceeded very rapidly when reacted at optimum concentrations for voltage output. In contrast to the flavoenzymes discussed above, a pyridine nucleotide enzyme, viz., yeast alcohol dehydrogenase, did not demonstrate any signs of voltage output even when the anolyte contained 3 ml of 95 % ethanol, 200 mg of NAD + and I- lO 7units of alcohol dehydrogenase (Fig. 2, Curve F). During the reaction the mixture was assayed for N A D H and the results indicated that a large portion of NAD + wasreduced to NADH. This latter observation demonstrated enzymic reaction without an apparent electron transfer. Iron in the form of iron powder bonded onto a glass propeller demonstrated a dramatic potentiating effect upon the glucose oxidase and D-amino acid oxidase as evidenced in both Fig. 2, Curves D and E, respectively. Voltages as high as 750 mV and 630 mV were generated from glucose oxidase and D-amino acid systems, respectively. When iron powder was deposited onto the anode in a phosphate buffer, a voltage TABLE I A M O U N T S OF I R O N R E L E A S E D

#g of Fe 2+ per ral anolyte

Glucose oxidase* Glucose oxidase* D-Amino acid oxidase** D-Amino acid oxidase** P h o s p h a t e buffer* * *

o rain

15 rain

45 rain

1.05 1.35 -1.4 o

2.22 5.07 1.9i 1.7 o

lO. 7 I 1.6 4-38 4.5 o

* Reaction m i x t u r e contained 0.34 g glucose oxidase and 0.92 g glucose in 45 ml of p h o s p h a t e buffer. ** Reaction m i x t u r e contained o.31 g D-amino acid oxidase and 0.6o g D-alanine in 45 ml of p h o s p h a t e buffer. * * * P h o s p h a t e buffer (45 ml) alone.

400

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~ 300'

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0.03 0.06 .0,09 0.12 0.15 0.18 0.21 0.24 0.27 030 CURRENTDENSITY(microompsper sq.cm )

Fig. 3. C u r r e n t density m e a s u r e m e n t s of the glucose oxidase a n d D-amino acid oxidase s y s t e m s w i t h o u t the influence of iron. M e a s u r e m e n t s of c u r r e n t and voltage were p e r f o r m e d b y i n t r o d u c i n g a resistance into the circuit during the period of m a x i m u m voltage o u t p u t .

Biochim. Biophys. Acta, 88 (1964) 375-383

380

A . T . Y A H I R O , S. M. L E E , 700

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100

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CURRENT DENSITY

18

(microomps per sq.cm)

Fig. 4. C u r r e n t d e n s i t y m e a s u r e m e n t s of t h e g l u c o s e o x i d a s e a n d D - a m i n o a c M o x i d a s e s y s t e m s w i t h t h e i n f l u e n c e of iron. M e a s u r e m e n t s w e r e d e t e r m i n e d a s d e s c r i b e d i n Fig. 3-

output in the neighborhood of IOOO mV was produced through localized galvanic action. This effect was eliminated by bonding the iron powder onto the glass propeller with an epoxy resin which resulted in a negligible amount of electricity being generated (Fig. 2, Curve A). The amounts of ionic iron formed in the anolyte (Table I) during the course of the 3OO

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DENSITY ( m i c r o o m p s per sq. c m ]

F i g . 5. E l e c t r o d e p o l a r i z a t i o n c u r v e s of t h e c a t h o d e a n d a n o d e d u r i n g t h e g l u c o s e - g l u c o s e o x i d a s e r e a c t i o n w i t h a n d w i t h o u t t h e i n f l u e n c e of i r o n i n t h e a n o l y t e . A s a t u r a t e d c a l o m e l e l e c t r o d e (SCE) c o u p l e d w i t h t h e e l e c t r o d e b e i n g t e s t e d v i a a n a m m e t e r w a s p l a c e d n e x t t o t h e e l e c t r o d e . A v a r i a b l e r e s i s t a n c e w a s i n t r o d u c e d i n t o t h e c i r c u i t of t h e cell i n p a r a l l e l w i t h a r e c o r d i n g v o l t meter.

Biochim. Biophys..4cla, 88 (1964) 3 7 5 - 3 8 3

381

BIO-FUEL CELL STUDIES

runs (Fig. 2, Curves A, D, and E) indicated that enzymic action was necessary for elemental iron to actively participate in this observed potentiating effect. This was substantiated by the appearance of increasing amounts of Fe 2+ in the anolyte with respect to time (Table I). The nominal amount of iron present at zero time was probably that iron normally present in the enzyme preparations. Glucose oxidase was found to contain 23,8/~g iron per ioo mg and D-amino acid oxidase 37-63 #g iron per IOO mg. The iron contents of the enzymes were found to vary from batch to batch. I

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CURRENT DENSITY (rnicroomps per sqcm)

Fig. 6. E l e c t r o d e polarization c u r v e s of t h e c a t h o d e a n d a n o d e d u r i n g t h e D - a l a n i n e - D - a m i n o acid o x i d a s e reaction w i t h a n d w i t h o u t t h e influence of iron in t h e anolyte. T h e m e t h o d for m e a s u r i n g t h e c u r r e n t d e n s i t y is described in Fig. 5-

Cell and electrode current density measurements of the two flavoenzyme systems illustrate biopotential differences between these two systems and the potentiating effect of elemental iron (Figs. 3-6). In addition, the presence of a cathodic reaction was demonstrated by obtaining current density measurements in the cathode compartment with phosphate buffer only. No attempts were made to further identify the reaction. DISCUSSION

Two flavoenzymes, glucose oxidase and D-amino acid oxidase, have been demonstrated to generate electrical energy, which appears to be the result of electron transfer via the oxidation of the substrates. The chemical reactions of each enzyme system are as follows : A nodic reactions :

1. Glucose oxidase s y s t e m /5-D-glucopyranose + F A D +-+ 6 - n - g l u c o n o l a c t o n e + F A D H + H + (5-D-gluconolactone + H 2 0 - + I)-gluconic acid F A D H + H + + 02=~ F A D + + H202

(t) (2) (3)

2. D - A m i n o acid oxidase s y s t e m D-alanine + F A D + ~ ~*imino propionic acid + F A D H + H + ~-imino propionic acid + H20--~ p y r u v i c acid + N H a F A D H + H + + O 2-+ F A D + + H202

(4) (5) (6)

Biochim. Biophys. Acta, 88 (1964) 375 383

382

A. T. Y A H I R O , S. M. L E E ,

D. O. K I M B L E

Cathodic reac:ion :

H20 + ~2 02 "+ H20~ (possible reaction, catholyte was not assayed for I-L,O2)

(7)

The initial rates of voltage g e n e r a t i o n w i t h or w i t h o u t iron (Fig. 2) can be p a r t i a l l y e x p l a i n e d b y t h e n u m b e r of F A D + molecules t h e y possess: glucose oxidase c o n t a i n s 2 F A D + whereas D-amino acid oxidase c o n t a i n s o n l y I F A D +. F u r t h e r m o r e , F A D + is n o t held t i g h t l y b y D-amino acid oxidase a n d often requires t h e a d d i t i o n of FAD~u p o n purification. Studies b y DAVIS AND Y A R B R O U G H 4, a n d SCOTT5 i n d i c a t e d t h a t m e t h y l e n e blue was required to m e d i a t e the transfer of electrons from the glucose oxidase o x i d a t i o n r e d u c t i o n r e a c t i o n to the electrode. However, our studies of this s y s t e m d i d not require t h e m e t h y l e n e blue. The p o t e n t i a t i n g effect of elemental iron a p p e a r e d to be caused b v a faster turnover rate of the r e d u c e d F A D H to the oxidized F A D + according to tile following equation

I:ADH -~- II + + Fe = FAD* + H 2 + Fe 2+ + 20

(8)

If m i n u t e a m o u n t s of H 2 were released its d e t e c t i o n would be difficult, p a r t i c u l a r l y since t h e iron was b o n d e d onto the propeller stirrer. E v i d e n c e in s u p p o r t of the a b o v e t h e o r y is shown in Table I which indicates t h a t an active e n z y m i c reaction involving either of t h e flavoenzymes was necessary for iron to become ionic. No ionic iron was d e t e c t a b l e in t h e a n o l y t e when an iron p o w d e r clad stirrer was allowed to stir in a p h o s p h a t e buffer solution with or w i t h o u t s u b s t r a t e a n d in the absence of the enzyme. The r a t e of iron a p p e a r i n g in solution was found to be i n d e p e n d e n t of w h e t h e r the cell was o p e r a t i n g w i t h an open or closed circuit. Other forms of iron such as heine, Fe ~ a n d F e a~- ions h a d no effect. A l t h o u g h the concept of electron transfer b y t t a v o p r o t e i n enzymes v i a an interm e d i a t e semiquinone is generally accepted, its e x a c t m e c h a n i s m as a half-cell r e a c t i o n r e m a i n s in question. The f o r m a t i o n of a free-radical semiquinone in the flavine molecule is d e t e c t a b l e b y electron-spin-resonance m e a s u r e m e n t s . F r e e - r a d i c a l semiquinones have been d e m o n s t r a t e d in t h e D-amino acid oxidase, c - a m i n o acid oxidase a n d x a n t h i n e oxidase 6-s. However, a b s o r p t i o n a n d electron-spin spectral studies of the glucose oxidase s y s t e m d e m o n s t r a t e d t h e absence of a free radicaP, t°. Therefore, it a p p e a r s t h a t glucose oxidase is an exception. The absence of a free r a d i c a l in the glucose oxidase s y s t e m suggests t h a t a n o t h e r system, i.e., one i n v o l v i n g a m e t a l ion, m a y exist. I n this connection. BEINERT ~' r e p o r t e d electron-spin-resonance spectral evidence i n d i c a t i n g t h a t m e t a l s associated with f l a v o p r o t e i n enzymes m a y u n d e r g o o x i d a t i o n d u r i n g e n z y m i c catalysis. The glucose oxidase used in our studies was rep o r t e d b y t h e m a n u f a c t u r e r to contain catalase as an i m p u r i t y which was present in a p p r o x . 35o units/mg. I r o n was found in glucose oxidase exceeding t h a t a c c o u n t a b l e for catalase alone. Thus, it is possible t h a t the iron p r e s e n t in glucose oxidase m a y be responsible for t h e transfer of electrons. Our studies i n d i c a t e t h a t no electron transfer occurred d u r i n g an a l c o h o l - a l c o h o l d e h y d r o g e n a s e - N A D + reaction. This finding is in a g r e e m e n t with the evidence for the o x i d a t i o n of alcohol b y D A D ÷ v i a h y d r o g e n t r a n s f e r as d e m o n s t r a t e d b y d e u t e r i u m t r a c e r studies ~2 a n d with t h e absence of electron-spin-resonance signals la. A c a t h o d i c r e a c t i o n d e m o n s t r a t e d b y t h e p o l a r i z a t i o n curves (Figs. 5 a n d 6) was r e l a t i v e l y weak. A l t h o u g h this r e a c t i o n was n o t characterized, it m a y well be the result of an inefficient o p e r a t i o n of an O 2 cathode. Biochim. Biophys. Acta, $8 096¢) 375 383

B I 0 - F U E L CELL STUDIES

383

REFERENCES 1 C. F. KODA (Ed.), Prec. Soc. Ind. ~}licrobiol., Corvallis, z962, Vol. 4, G a r a m o n d / P r i d e m a r k Press, B a l t i m o r e , 1963, p. 51. 2 E. M. COliN, Prec. Biochem. Fuel Cell Session, Sauta Moniea, z962, P I C - B A T 2o9/5, P o w e r I n f o r m a t i o n Center, P h i l a d e l p h i a , 1962. a W. HORWlTZ, (Ed.), Assoc. OG. Ogri. Chemists. Washington, 9 t h Ed., I96O, p. 159. 4 j . B. DAVIS AND H. F. YARBROUGH, JR., Science, I37 (1962) 6I 5. V~T. 1(. SCOTT, in E. M. COHN, Prec. Biochem. Fuel Cell Sessiou, Santa ~}Ionica, 1962, P I C - B A T 2o9/5, P o w e r I n f o r m a t i o n Center, P h i l a d e l p h i a , 1962, p. 9. G K. YAGI AND T. OZARVA, Biochim. Biophys. Acla, 67 (1963) 685. 7 V. MASSEV, G. PALMER AND R. BENNET, Biochim. Biophys. Acta, 48 (t961) i . s R. C. BRAY, Biochem. J., 81 (196t) 196. 9 T. NAKAURMA AND Y. OGURA, J. Biochem., 52 (i962) 214. 10 H. BRINERT AND 1~. H. SANDS, in M. S. BLOIS JR., H. \V. BROWN, l~. M. LEMMON, R. O. IANDBLOM AND \\'. \~'EISSBLUTH, @~mp. Free Radicals in Biological @'stems, Sla~zford x96o, P a r t 2, A c a d e m i c Press, N e w York, I961, p. 17. 11 H. BEINERT, 2"he E~zT,vmes, Vol. 2. A c a d e m i c Press, N e w Y o r k , 2nd Ed., 196o, p. 415 . 12 F. H. WESTHEIMER, H. F. FISHER, E. E. CONN AND B. VENNESLAND, J. Am. Chem. Soc., 73 (195 l ) 2403. la bl. R. ~I.A-HLER AND L. BRAND, in M. S BLOlS JR., H. \V. BROWN, R. M. LEMMON, R. O. LINDBLOM AND ~V. \VEISSBLUTH, @,rap. Free Radicals in Biological @'stems, Statzford, ±96o, P a r t , 2, A c a d e m i c Press, N e w Y o r k , 1961, p. 163.

Biochim. Bioph3,s. Ac/a, 88 (1964) 375 383