The electrochemical diminution of surface bacterial concentration

HARI P. DHAR Chemistry Department, Texas A&M University, College Station, TX, U.S.A. 77843

DONALD H. LEWIS Department of Veterinary Microbiology, Texas A&M University, College Station, T X , U.S.A. 77843

AND

JOHN O'M. BOCKRIS' Chemistry Department, Texas A&M University, College Station, TX, U.S.A. 77843

Accepted June 16, 1981

DHAR, H. P., D. H. LEWIS, and J . O'M. BOCKRIS. 1981. The electrochemical diminution of surface bacterial concentration. Can. J. Microbiol. 27: 998-1010.

An investigation of the electrodic effects on bactericidal activity is presented while applying electrode potentials. Dark-field and phase-contrast microscopy were used to examine regions adjacent to surfaces of Sn02 glass electrodes for bactericidal activity, while scanning electron microscopy (SEM) was used to visualize microbicidal effects on the surfaces of S n 0 2 electrodes.

As the cathode potential approached -0.3 V (normal hydrogen electrode) the bacterial concentration near the cathode was reduced by 99%. The SEM results showed bacterial concentration on cathode to be reduced to 0.1- 1 % of the original value. Anodes showed reduction of 0.01-1%. The application of the same potential in pulses resulted in enhanced cleaning of the surfaces. The off periods using the pulses could be more than 10 times the on ones.

Models considered to explain the observed results involved effects due to change of surface energy with potential, the depletion of O2 in the solution, the production of bactericides (e.g., Hz02), and the change of pH near the surface. Results are inconsistent with the first two models. Many of them are consistent with both the latter two. Diagnostic experiments are suggested.

DHAR, H. P., D. H. LEWIS et J. O'M. BOCKRIS. 1981. The electrochemical diminution of surface bacterial concentration. Can. J. Microbiol. 27: 998- 1010.

Nous prCsentons une Ctude sur l'effet bactericide du potentiel Clectrique d'ilectrodes. Nous avons utilist la microscopie 2 champ obscurci et h contraste de phase pour observer llactivitC bactCricide au voisinage de la surface d'Clectrodes de verre contenant du SnOz; nous avons utilisC la microscopie Clectronique 2 balayage (SEM) pour visualiser les effets microbicides sur la surface de ces Clectrodes.

Lorsque le potentiel de la cathode approchait -0.3 V ("normal hydrogen electrode") la concentration bactkrienne au voisinage de cette dernikre Ctait rCduite de 99%. Les rCsultats obtenus par SEM ont dtmontrC que la concentration bacterienne sur la cathode Ctait rCduite 2 des valeurs de 0 , l 2 1% du compte original. Les anodes montraient des reductions de 0,01 2 1 %. L'application du m&me potentiel sous forme de pulsations avait un meilleur effet nettoyant sur les surfaces. Les ptriodes mortes au cours des pulsations pouvaient avoir plus de 10 fois la longueur des ptriodes vives.

Les modkles que nous avons considCrCs pour expliquer ces rCsultats impliquent des effets rCsultant du changementd'knergie de surface en fonction du potentiel, de l'kpuisement de 1'02 dissout, de la production de bactericides (e.g., H202) et duchangement de pH au voisinage de la surface. Les rCsultats ne peuvent &tre expliquts par les deux premiers modeles; plusieurs le sont B la fois par les deux derniers. Nous suggCrons des essais diagnostiques.

[Traduit par le journal]

Introduction Microbial fouling is a well-recognized problem in

technological situations in which natural water comes in contact with metals. Such films (Characklis 1979; Picologlou et al. 1980) reduce water flow in boiler pipelines (Corpe 1978) and may enhance corrosion of a metallic substratum (Gerchakov 1978).

Attempts to destroy microbial films have primarily centered around the use of C12 (Fava and Thomas 1978; Norman et al. 1977; Characklis 1978) dissolved in the

'Author to whom correspondence should be addressed.

surrounding liquid. Concern over the toxic effect of this material (Jolley et al. 1978) in the effluent water makes it environmentally unacceptable for use in large quantities and for prolonged time. As alternatives to chlorination, a number of chemical and mechanical techniques have been mentioned with regard to their use in the Ocean Thermal Energy Conversion (OTEC) power plant, and these techniques have had limited success (Conn et al. 1977).

In the past, attempted electrochemical approaches (Castle 1951; Littauer and Jennings 1968) involved cathodic evolution of Hz and achieved partial success,

0008-41661811100998-13$01 .OO/O 0 198 1 National Research Council of CanadaIConseil national de recherches du Canada

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

DHAR ET AL. 999

but the high current densities (or rates of evolution) used caused the interface to become highly alkaline and favored the development of calcareous deposits.

In a recent study A. S. Gordon, S. M. Gerchakov, and L. R. Udey (submitted for publication to Can. J . Micro- biol.) examined the effect of electrical polarization on the attachment of bacteria on Cu and Pt surfaces and found that anodic polarization retarded bacterial attachment.

In the present approach (Dhar et al. 198 I ) , an electro- chemical potential in the region of H202 generation is applied across a surface. H202 is formed cathodically on the surface of the electrode (i.e., metal surface) from the dissolved O2 present (ca. 3 X lop4 mo1.L- ') in the saline electrolyte. The lethal effect of H202 on bacteria (Meynell and Meynell 1970) in the interphasial layer is expected to reduce the number of bacteria for attachment on the surface. A much lesser current density would be expected to be effective in keeping bacteria away in this approach than in that used to produce Hz. The electro- chemical reaction for the generation of H202 can be represented by

(Eo = 0.68 V) in the acid medium, and in the alkaline medium by

(Eo = -0.146 V). With a~~~~ = m o l . ~ - ' (see footnote 2) and PO2 = 0.2 atm (1 atm = 101.325 kPa), the reversible electrochemical potential, Ere,, for the generation of H202 is 0.43 V versus normal hydrogen electrode (NHE13 for an electrolyte of pH 7. In fact, the saline solution used had a pH of 6.5, the corresponding thermodynamic potential is 0.45 V.

It is possible to construct current-potential curves by making some speculative assumptions concerning the exchange current densities appropriate to reactions 1 and 2 on the substratum used in the present work (Sn02). Thus, the (cathodic) potential at any current at least 10 times below the limiting current is given by

where qi is the overpotentia14 at current density i. For a cathodic reaction the overpotential is given by

[4] qi = RTIaF In i d i

where io is the exchange current density, i is the actual

his value is used (see Results) because the bacteria began to become inactivated at this concentration.

3 ~ 1 1 potentials are expressed on the NHE scale. 40verpotential is the excess potential required over and

above the reversible potential.

current density at which the reaction is to function, and a has been assumed to be 1 12 (Bockris and Reddy 1973).

The value of io is dependent upon the electrode material used. The relevant values for Sn02 are not available. The following values of io have been used in the calculations of the current-potential curves: for 0, to H202 reduction, the value for Pt, 1 x 10- ' ' ~ . c m - ~ (Damjanovic et al. 1967a), for O2 to H,O reduction, the value for Pt, 1 X lo-'' Aecm-' (Damjanovic et al. 1967a), and for H' to Hz reduction, the value for Sn, 1 X A-cm-'.

Using these values, one obtains Fig. 1. Thus, accord- ing to the calculated situation with the parameters stated, the reaction for H202 never functions at above 10% efficiency, the competing reaction being the four elec- tron reduction of O2 to H 2 0 or to OH-. It is noteworthy that in these potential regions, the evolution of Hz does not yet occur. In the disc-ring electrode work (Damjan- ovic et al. 1967a, 1967b) using Au as the electrode material, the current efficiency for reduction of O2 to H202 was found to be 20-30%. The optimal situation (maximum production of H202) according to Fig. 1 will be approximately -0.4 V. However, the uncertainty of io values and the shape of current-potential curves in the limiting current region make it better to operate between approximately -0.2 and -0.6 V.

The above concerns the reaction carried out on the cathode. It is also relevant to consider the reactions occurring on the anodic counter electrode. The follow- ing reactions are possible for the Sn02 counter electrode in a solution containing NaCl:

[5] 2H20 -, O2 + 4H' + 4e

[6] 40H- -, 2 H 2 0 + 4e

[7] 2C1- -, C12 + 2e

[8] Sn02 + 4C1- -+ SnC14 + O2 + 4e

[9] Organics (impurity) -, organicsn+ + ne Generation of C1, according to reaction 7 will provide

a toxic substance at the anode. To distinguish the action of H202 at the cathode and C12 at the anode, a cationic membrane was used in the experiments (see below). Reaction 8 implies attack on the anode. However, with current densities of a few microamperes used for approximately 3 h, the anode would lose only a fraction of a micrometre in thickness. Reaction 9 represents oxidation of any organic impurities adventitiously pres- ent in the electrolyte.

The present work has been carried out on the assumption that C12 could not be used because of the danger of electrochemical corrosion at the anode, induced by the anodic potential necessary in C12 evolu- tion. In cathodic reactions, the metal would be cornpara- tively protected.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

1000 CAN. I . MICROBIOL. VOL. 27, 1981

L I I I I I I

-12 -10 -8 -6 -4 -2

Log i (A.crn")

FIG. 1. A hypothetical construction of the potential versus log i relationship for the cathodic processes O2 + OH-, O2 + H202, and Hf + H2 for an electrolyte of pH 7.

Materials and methods Microorganisms and culture conditions

A marine bacterium, Vibrio anguillarum, was used in the studies. The bacteria were cultured batchwise (Pirt 1975) in marine broth (Difco) at 21°C for 24 h. A portion of the culture medium with the bacteria (109.mL- I) was diluted appropri- ately in 3% NaCl solutions before subjecting the bacteria to experimental studies. Because the age of a culture can be important in the studies of bacterial attachment (Fletcher 1977), fresh (24 h) culture was used to initiate each experi- ment.

Electrolyte and electrode materials The bacteria were suspended in 3% NaCl solutions, pH 6.5. Transparent and conducting materials which would facili-

tate observations of bacteria under a light microscope were used as electrodes. Tin oxide glass (Nesa) and indium oxide glass (Nesatron), both obtained from PPG Industries, have the desired properties. Surface resistivity of these glasses is approximately 80 ohm per square area.' Either variety of conducting glass could be used with no change in the results.

Electrical connections to tin oxide were established with conducting silver epoxy (Acme Chemicals) over which a coating of epoxy resin was applied.

Surface preparation Tin oxide glass was cut in pieces of ca. 5 cm2 in area. The

pieces were degreased in acetone, followed by 10 min of immersion in absolute ethanol, and then dried in an oven.

Application of electrochemical potentials A potentiostat (ECO Inc., model 549) was used to apply an

'Surface resistivity is given by p, = plt = Rwll where p is specific volume resistivity, t is thickness, w is width, 1 is length, and R is bulk resistance of any rectangular conducting material. When w = I, p, = R.

appropriate potential in the range 0.2 to -0.56 V (NHE) to the working electrode. A reference electrode (saturated calomel) was used in the working electrode compartment. In pulsing experiment pulses were applied from a pulse generator (Princeton Applied Research, model 175) through the poten- tiostat to the working electrode. Pulse length was generally varied from 0.1 to 100 s, and pulse height from 0.24 to -1.ov.

Fixing procedure for adsorbed bacteria Bacteria adsorbed on the electrode surface were fixed in 3%

NaCl solution containing 4% (vlv) glutaraldehyde for 2 h. Afterwards, the preparations were placed for 10 min in saline solutions of decreasing salinity 3-0.2% NaCl, distilled water, aqueous acetone solution containing 25-100% acetone, and finally in acetone-xylene solution containing 50 and 100% xylene. The samples were then air-dried and coated with Au-Pd in a vacuum evaporator (Hammer I by Technics Inc.) and examined in a scanning electron microscope (Japanese Electron Optical Labs, model JSM-35) operated at 25 kV accelerating voltage.

Optical arrangements An Olympus microscope (model BHA) at magnification

400-800x equipped for phase-contrast and dark-field illumi- nation was used to observe bacterial movements in very thin electrochemical cells made of tin oxide glass (Fig. 2). The dimensions of each compartment were 3cm X 2cm X 0.02 cm. A Nafion (Du Pont) cationic membrane No. 425 was used to separate the cathode and anode compartments. The membrane was kept immersed in water for 2-3 days to allow leaching out of any acidic organic groups before its use in the electrochemical cell.

Assessment of number of bacteria Solution from the electrode compartment was withdrawn by

means of a disposable plastic syringe, and the bacterial concentration was determined by the serial dilution technique of counting (Frobisher et al. 1974).

Numbers of attached bacteria on surfaces were determined by microscopic counts as well as by counting adsorbed bacteria from scanning electron micrographs. Caution was taken to compare results with the same optical method with and without application of potential.

Tests for H202 and C12 Chemical spot tests (Feigl 1958) for H202 and C12, in the

anode and cathode compartments, respectively, were carried out. A bigger cell of ca. 10-mL volume and a cathode surface area of 30cm2 was prepared for this purpose. Hz02 reduces potassium ferricyanide to potassium ferrocyanide which with acidic femc chloride solution produces a Prussian blue coloration. A test for H202 was positive in the electrolyte from the cathode compartment at the end of 15 min of an experiment at a constant potential of -0.3 V. A light blue coloration (as opposed to a yellow coloration for a blank test) was obtained. The identification limit of H202 in the above test is 1.6 Fg.mL-l and the interfering chemicals are SnC12, Na2S03, Na2S203 which were assumed to be absent from the electrolyte. A spot test for C12 in the anodic solution was carried out by using fluorescein - KBr solution mixture. To a 2-mL solution of the anode chamber, one drop of fluorescein -

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

DHAR ET AL. 100 1

FIG. 2. Experimental cell. (A) Top view; (B) horizontal sectional view; a , anode; b , cathode; c, reference electrode position; d, membrane; e and f, electrical connections; g, glass spacer.

0 C

Results Bactericidal activity of H202

The number of bacteria versus molarity of H202 in 3% saline solutions is shown in Fig. 3. It is seen that substantial reduction of the bacterial concentration begins at approximately I O - ~ mo1.L- ' H202.

e b COMPARTMENT

KBr solution mixture was added. A faint red coloration indicated the presence of C12. C12 was also recognized by the smell. Spot tests for H202 in the anode chamber and C12 in the cathode chamber were negative. Thus, there was no migration

i of H202 from catholyte to anolyte and of C12 from anolyte to catholyte, due to the membrane which separated them. 5

Cells made of both tin oxide glass and indium oxide glass II: W

were used separately in the above tests; no significant + 10' 0 a

difference was noticed in the results. -E m 'I: lo0

Bacterial viability in H202 solutions Bacteria of an approximate concentration of 1O4.mL- ' in '

Direct observations of bacteria under an electricaljeld To examine the effectiveness of the present electro-

chemical concept efforts were made to observe bacterial

fl

motility directly during the application of electrical potential to the transparent Sn02 electrodes in contact with solution. After introduction of the marine bacteria

3% saline solutions were added to test tubes containing H202 1 0 I lo-= I - lo-9

of different concentrations varying from lo- ' to [ti202] (mol L - I )

mo1.L-' in saline solutions. After approximately 15 min, samples from these test tubes were counted by the serial FIG. 3. A plot showing the effect of Hz02 concentration on dilution technique (Frobisher et al. 1974). bacteria, Vibrio anguillarum.

at a concentration of 107.mL-' into the ex~erimental

/ d

--9

I -\ J $.

cell (Fig. 2), the bacteria were observed to cease movement in approximately 5 min when a potential of -0.3 V (NHE) was applied through the potentiostat.

CI A

f a COATI N G \GLASS

SUBSTRATE 0

Dependence of bacterial concentration as a function of electrode potential (steady state)

Assessments of bacterial viability in thin (0.02 cm) layers adjacent to the electrode surface were made by collecting samples from each compartment of the cell. For each experiment, a fresh sample of bacterial solution containing approximately lo4 cells.mL-' was intro- duced in the cell, and at the end of an experiment (5-30 mins) 0.1 mL of solution was collected for counting. The data presented in Fig. 4 represent concentrations of bacteria during the production of Hz02 and C12 at steady currents in the cathode and anode compartments, re- spectively. computation of the half-lives (ti,$ from

,

; '-----< lo! I I I I

6~alf- l i fe is defined as the time required for concentration to become half the original concentration.

I I

ArF

I 1- - - - - - - I- L - - - - - - - - - ,.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

1002 CAN. J. MICROBIOL. VOL. 27, 1981

I I I I

TlME ( m i n )

F I G . 4 . Bacteria concentration versus time of experiment. 0, blank, no potential being applied; A and 0, cathode and anode compartment results, respectively. Thickness of each compartment, 0.02 cm; cathode potential, -0.36 V; cell potential, 1.95 V; current density, 5 x lop2 ~ . m - * .

results such as those in Fig. 4 shows values of approximately 1 and 2 min for the anode and cathode compartments. Such values do not refer to the electrode surface but to the solution adjacent to the electrode. On the electrode the t , , 2 should be less (see below). The t l12 values and Fig. 4 suggest that H 2 0 2 is less bactericidal than Clz

Four cells similar to that in Fig. 2 were fabricated so that distances between the plates were varied (0.02- 0.15 cm). Data are presented in Fig. 5 reflecting bacterial concentration as a function of time after applying the potential appropriate for the formation of H 2 0 2 on the cathode in cells of varying distance between the membrane and the electrode. At a constant time, the number of bacteria in the cell decreases with decreasing cell thickness. From Fig. 5, bacterial t I l2 corresponding to a concentration of 5 x 103.mL- ' can be read for each of the curves. Such values of tl12 are plotted versus cell thickness in Fig. 6. The extrapolated t I12 value of 0.3 * 0.3 min (least squares) should pertain to the electrode surface. It is assumed that linear extrapolation is possible.

Electrode su$ace after application of a steady-state potential

Degreased tin oxide glass samples were kept im- mersed (without potential control) in 3% NaCl contain- ing lo7 bacteria.mLP1 at room temperature. The time of immersion was varied from approximately 10 to 1000 min. The electrodes were taken out and adsorbed bacteria fixed according to procedures described above. A scanning electron micrograph of adsorbed bacteria after 6 h is shown in Fig. 7.

The effect of potential on the concentration of adsorbed bacteria was examined in a beaker-type cell

I I I I I I I J 0 5 10 15 20 25 X)

TlME ( m i d

F I G . 5 . Bacteria concentration versus time of experiment in cells of various thickness. 0, 0.15 cm; 0, 0.09 cm; A, 0.06 cm; 0, 0.02 cm. Cathode potential, -0.36 V ; current density, 5 X ~ . m - ~ .

FIG. 6. Cell thickness versus r l l z of bacteria

divided in the middle by a Nafion membrane No. 425. Potential in the range of 0.2 to -0.56 V was applied to the surface concerned within 1 s of the immersion of the electrodes in the cell containing lo7 bacteriaeml- '. Tin oxide glass was used both as cathode and anode material.

After 3 h, the electrodes were removed, the bacteria fixed, and photographs taken in the scanning electron microscope.

Results are shown in Fig. 8. Potentials negative to 0.0 V are effective in reducing the number of bacteria on the surface. This region of potentials is consistent with the theoretical values speculatively calculated above for generating H 2 0 2 Up to 500 times reduction in the number of adsorbed bacteria was achieved in these experiments with continuous application of potential in the appropriate region. Initially, the anode surfaces

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

DHAR ET AL. 1003

FIG. 7. Scanning electron micrograph showing adsorbed Vibrio anguillarum on tin oxide glass substratum. Immersion time, 6 h; number of adsorbed bacteria, 7.5 X 10'O.m-*. Bar scale = 10 pm.

ANODIC POTENTIAL ,V (NHE) , 0 0 8 0 2 4 I , . .,8 ,

CATHODIC P0TENTIAL.V (NHE)

FIG. 8. A plot showing bacterial adsorption versus steady electrode potentials. Time of each experiment, 3 h.

seem to adsorb more bacteria than the cathode surface. currents at a potential of -0.36 V for periods of up to At higher anodic potentials, e.g., 1.7 V, bacterial 24 h. Adsorbed bacterial concentration on the cathode concentration is less at the anode than at the cathode. and anode surfaces is shown in Fig. 9 as a function of

An electrochemical potential of -0.36 V was chosen time of experiments. Results are also shown in Fig. 9 for (cf. Fig. 8) for the following set of experiments. In the surfaces which were put into bacterial solutions but did beaker-type cell, electrodes were subjected to cathodic not receive any potential applications. From Fig. 9 it can

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

CAN. J. MICROBIOL. VOL. 27. 1981

T

BLANK

01 6 1 T

CL W CATHODIC

i, T T T

ANODIC

TIME ( h )

FIG. 9. Attached bacteria versus time of experiments. 0, no potential; 0, cathode results; A , anode results.

be seen that during experimentation of up to 24 h, the anode surface remained cleaner than the cathode surface by ca. 10 times, and the cathode by ca. 100 times, than a surface not polarized.

The power consumption in the electrochemical exper- iments is approximately 10 p,~.cm-'. A smaller power density would be required upon intermittent application of the appropriate electrochemical potentials.

Electrode surface after application of potential pulses The effects of potential pulses, their heights and

lengths, were examined in the studies of adsorbed bacteria onto surfaces. The effect of variation of the pulse height at constant pulse length and the variation of pulse length at constant pulse height was studied.

In Fig. 10, the effect of pulse height for an off and an on period of 10 s is shown. During the off period the electrode rests at a potential of 0.24 V, and during the on period the electrode reverts to the chosen cathode potential. It can be seen from Fig. 10 that the pulses are as effective in reducing adsorbed bacterial layers as are those applied at constant potential (Fig. 8).

In Figs. 1 1, 12, and 13, the effect of various pulsing conditions is shown for a pulse height potential of -0.36 V. During the off period the electrodes rested at 0.24 V. Each experiment continued for 6 h.

In Fig. 13 the situation is shown for 0.1 s on and a series of off times up to 1 s. The noteworthy result is the absence of change in bacterial concentration in the range of off times indicated. The reduction in the bacterial

concentration is about the same as for the steady state. Corresponding results are shown for 1 s on and up to

10s off(Fig. 12). InFig. 11, theuseofonpulsesof 10s followed by off pulses of 5 s seems to result in 1 O4 times reduction in bacterial concentration, better than any other result obtained. In the absence of any applied potential, attached bacterial number is nearly 10'' (see Fig. 9).

Effect of pH on bacterial adsorption on Sn02 Since the reduction of 0' to H202 or to H20 is

accompanied by a change of pH near the vicinity of the electrode, it was necessary to examine the adsorption of bacteria from electrolytes of known pH, without apply- ing any potential. Tin oxide glass samples were im- mersed in saline solutions of pH ranging from 1 1 to 3 for 6 h. Adsorbed bacteria versus pH of electrolytes is shown in Fig. 14.7 The plot has a flat maximum covering a pH range of 9 to 5, and the pH effect seems to be symmetrical about pH 7.

Discussion Factual summary

The facts reported in this paper can be divided into two categories: (1) facts about the viability of Vibrio anguillarum in solution near (10-2cm) a tin oxide surface in contact with the solution containing bacteria

7~ssistance of Mr. David Howell in obtaining this diagram is acknowledged.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

DHAR ET AL.

ANODIC PULSE, V (NHE) , 0,.8 l;O l.,2 l.,4 1.; li8 2;0,

0.0 -0.2 -0.4 - 0.6 - 0.8 - 1.0 CATHODIC PULSE, V(NHE)

FIG. 10. Bacterial adsorption versus pulse height potentials. Pulse length: 10 s on, 10 s off; rest potential for offpulse, 0.24 V; time of each experiment, 6 h.

and (2) facts about the surface of tin oxide glass and the bacterial content on it.

From the first category are the following: (i) The efficacy of H202 in killing bacteria has been shown (Fig. 3) to begin at concentrations of approximately lop6 and lo-' mo1.L- ' and reduces the number to zero at mo1.L- '. (ii) At ca. 0.02 cm from the electrode surface the bacterial concentration fell exponentially with time after attaining a given potential. A reduction of lo4 times came in the first 15 min. (iii) As the cathode-anode distance was reduced from 3 to 0.4 mm, the rate of reduction of bacteria increased. A tlI2 of bacteria "at" the surface of approximately 0.3 -+ 0.3 min was estimated.

From the second category are the following: (i) A parabolic relationship between concentrations of bacte- ria on the surface and pH exists (Fig. 14). It has a flat maximum. (ii) A reduction of bacterial coverage begins when the potential is approximately 0.00 V (NHE). A corresponding reduction on the anode begins at 1.0 V. The anodic behavior is significantly different from the cathodic behavior in that the reduction of bacteria near the anode takes place over a longer potential span than near the cathode (Fig. 8). (iii) A remarkable improve- ment in the ability to take bacteria off the surface was

observed if short pulses were used (0.1 s) (Fig. 13). The off time could be increased to at least 10 times the on time.

Models Four models of the electrical effects upon the attach-

ment of bacteria to metals may be proposed. Model 1. Changes in surface energy or conjguration As the potential of an electrode changes from the

potential at which the electrode has zero charge (pzc) the surface energy decreases. As bacteria can be regarded (Fletcher 1977) as large molecules, and as molecules adsorb increasingly on surfaces of high energy, one expects a decrease of bacterial presence on a surface as the potential departs from the pzc.

Correspondingly, as the surface free energy changes, there must be a small change in interatomic distance. Could this change be felt by microorganisms having a radius as small as 1000 atomic diameters?

The surface tension of a metal is ca. lo3 dynes-cm- ' (1 dyne = 10 pN) and the diminution of surface tension during the course of an electrocapillary curve can be taken as, e. 250 dynes-cm- ' or approximately 250 X

dyne.k" (I A = 0.1 nm). For a typical interbond

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

CAN. J. MICROBIOL. VOL. 27, 1981

ON PULSE 10 s

TlME OF OFF PULSE ( s )

FIG. 11. Adsorbed bacteria versus time of offpulse. On pulse, 10 s; pulse height, -0.36 V;restpotential for offpulse, 0.24 V.

FIG. 12. Adsorbed bacteria versus time of off pulse. On pulse, 1 s; pulse height, -0.36 V; rest potential for off pulse, 0.24V.

10"

lon

N l o 9

'E

5 [r. loa W I- 0

3 lo7

lo6

lo5

- CATHODIC

T T

-

\ANODIC -

ON PULSE I s -

-

1 I I I I I I I I 2 3 4 5 6 7 8 9 10

TlME OF OFF PULSE ( s

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

DHAR ET AL. 1007

TIN OXIDE GLASS

10"

10"-

10'- 'E 4 cc

lo8-

. . Qm lo7

lo6-

FIG. 14. Adsorbed bacteria on tin oxide glass samples versus pH of solutions.

a CATHODIC

1 T I

T T, I -

ON PULSE 0.1 s

I I I I I I I I I

0. I 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 .O

distance of 3 x cm, the force would be (250 X 3) X lo-' dynes.

A typical force constant is 5 X lo5 dynesecm-' and hence (5 X 105)Ax = 7.5 x lop6, i.e., Ax would be approximately 1.5 X 10- " cm. Such a movement would not affect a bacterium which has a length of ca.

cm. It seems, therefore, unlikely that surface tensional changes affect the situation.

TIME OF OFF PULSE ( s )

FIG. 13. Adsorbed bacteria versus time of off pulse. On pulse, 0.1 s; pulse height, -0.36 V; rest potential for offpulse, 0.24 V.

Model 2. Depletion of O2 Vibrio anguillarurn is a facultative anaerobe; thus,

removal of O2 from the medium would cause metabolic changes in the bacteria. The following is a general model presented for the depletion of O2 from the electrolyte and its effects on bacteria in the present studies.

The flow of O2 into the elecrolyte within the electro- lytic cells being examined is only from the sides of the cell. Thus the area of the solution exposed to air (see Fig. 2) would be ca. 0.2cm2. As O2 is being removed by reduction at the electrode surface, the exhaustion of 0 2

in the vessel could be the cause of bacterial inactivation. An order of magnitude calculation of the flow of 0 2 into the cell can be made by assuming that the central section of the cell is exhausted of O2 and the edges of the cell are saturated with O2 from the atmosphere (Fig. 2). The rate of diffusion of O2 into the solution from the sides of the cell would be given by

[lo] Diffusion rate = DA(dcldx)

TakingD= 1.3X 1 0 - 5 c m 2 ~ s - 1 , A = 0 . 2 c m 2 , c = 3 ~ lo-' m ~ l . c m - ~ , andx -- 1 cm, the diffusion rateof O2 is approximately lo-'' mo1.s-', while at a current density of 5 x lop6 ~ecm-', in an electrode area of 6cm2, the 0 2 consumption rate is

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

1008 CAN. J . MICROBIOL. VOL. 27, 1981

[ I l l itlnF = (5 X lop6 x 6 X 1)/(4 X 10') TABLE 1. Calculated pH changes near the electrodes at various potentials

= 7.5 x lo-' ' rn0l.s-'

Thus, the consumption of O2 seems to be about an order Cathode Anode of magnitude greater than the diffusion of 0 2 from the potential, potential, Current, PH PH surroundings. This conservative calculation suggests V V ~ . c m - ~ cathode anode that there Gay be a danger of 0 2 exhaustion in thethin cells, and the O2 depletion model must therefore be +0.04 considered. On the other hand? the calculations show -0.06

-0.16 that such a model would only be applicable to experi- ments in the small cells. Behavior in the bigger cells -0,36 does not seem qualitatively different from that in the -0.56 small cells.

Model 3. Change of pH The mechanism of a pH effect might arise from the

effect of pH on the double layer surrounding the bacteria, and thus upon their interaction with the electric field provided by the electrode.

The cathodic reduction of O2 to H202 or to OH- is accompanied by change in pH in the vicinity of the electrode surface (cOH-, electrode) causing an increase in pH. Correspondingly, at the anode, the likely reactions are the evolution of O2 which would consume OH-, or the evolution of C12 which would react with water to give HCI and HOCI. Hence, the cathode compartment will tend to become alkaline and the anode compartment acidic.

Thus,

[ 121 io, = (nFDIZ)(cOH-, electrode - COH-, soln) The concentration of OH- in solution (cOH-, corresponding to bulk pH 6.5 for 3% NaCl solution is 10- 10.5 m~ l . cm-~ . The calculated pH values in the cathode and anode compartments are listed in Table 1.

Thus, on the anodic side, the pH in the solution near the electrode is reduced. Correspondingly, the negative charge which exists on bacteria (Daniels 1980) in solution will be reduced and attraction to the positive electrode correspondingly lessened. On the cathodic side,8 the pH is increased, and it may be inferred that increasing OH- adsorption will increase the bacterial charges, thus reducing adsorption of charged bacteria on the cathode.

Comparison of Table 1 with Fig. 14 shows that pH conditions near the electrode do change significantly. It is a moot point as to whether for the potentials concerned (e.g., -0.1 V for the cathode, 1.2 V the anode) the pH change (Table 1 and Fig. 14) brings about significant removal to interpret the change observed.

Model 4. Production of H202 and C12 With respect to H202, the reversible potential for

'1, this statement it is assumed that the charge on the anode is positive. Although this seems likely, it cannot be maintained with clear evidence until data on the potential of zero charge of the substrata (i.e., the electrodes) have been measured.

H202 generation at 6.5 pH (3% NaCl solution) is0.45 V. The theoretically calculated potential range for H202 production at the range of current densities is 0.45 to -0.5 V (Fig. l) , the range being due to the difference in overpotential. Bacterial life on a tin oxide surface seems to begin to be affected ca. 0.00 V. Thus, if the H202 view is applicable, a cathodic overpotential of approxi- mately 0.5 V is required.

Correspondingly, from a consideration of the limiting current obtained (ca. 5 x A.cmP2) in the electro- chemical experiments, at 10% current efficiency for H202 production, the concentration of HZ02 is approxi- mately 5 x mo1.L- ' at ca. 0.02 cm from the inter- face, and this concentration (see Fig. 3) is in the lethal range. Thus, reasonable assumptions are consistent with t he i202 model.

With respect to the anodic evolution of Cl,, the situation is less clear because of the lesser definition of the current efficiency of C12 evolution. It is possible arbitrarily to assume that this is 10% of the total anodic current, the rest being the evolution of 02 . The reversi- ble potential for C12 generation in 3% NaCl can be calculated on the assumption of 10% current efficiency. Thus, with a typical anodic current density for C12 of 5 x

~ - c m - ~ ,

[13] 5 X = (DzFIZ)cc12

with D = 1 X lop5 cm2.s-', F = 10' C-mol- ', 6 - 0.02 cm, and cclz = 8 x m ~ l . c m - ~ . The solubility of C12 in water is approximately 0.1 mo1.L-' and hence the chlorine concentration is equivalent to approxi- mately (8 x 10-9)/0.1 = 8 X atms. Utilizing the Nernst equation, = 1.22 V.

This potential is ca. 0.2 V more positive than that at which bacterial adsorption on the anode begins to be affected.

Facts compared with models Change in internuclear distance upon change of

potential is too small to dislodge bacteria. Correspon- dingly, the dependence of the degree of surface cover- age, 0 , for organic molecules upon change of potential (Gileadi et al. 1975) is generally up to 10 times.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

However, in our observations, the bacterial concentra- tion on the electrode surface was reduced by more than 100 (and up to lo4) times. Surface free energy changes would not give rise to such high values of the change of occupancy.

The depletion of O2 concept is inconsistent with the fact that experiments showing the removal of bacteria are also carried out in beakers in contact with air.

With respect to model 3, the pH change, it has already been shown that the current density reached would bring the pH in the vicinity of lethal range in the interface. The same conclusion applies to the applicability of the theory concerned with the production of H202.

The fact that (Fig. 5) the bacteria are killed more readily in thinner vessels fits all theories except the surface tension theory. The tl12 of 20 s near the interface is also not diagnostic among models 2, 3, and 4, although inconsistent with model 1.

The greater stability in the anode compartment (Fig. 8) may support model 3. It may reflect subtle differences in the stability of a charged bacterium in the acid and alkaline medium.

From Fig. 6, the validity of the diffusion equation

DHAR ET AL. 1009

can be checked. Taking A, the thickness of the cell as the diffusion length covered by the toxic material in time t, the tlI2 of bacteria, the diffusion coefficient D can be calculated provided the tl12 at zero distance, 0.3 rnin, is subtracted from the tl12 values for each of the four cells. The results of such calculations are listed in Table 2.

It is of interest to note that the value for the diffusion coefficient of OH- is ca. 5 x lop5 cm2.s- ' and that for H202 is 1.3 X ~ m - ~ . s - ' .

Pulse length and height Results of Fig. 11 seem to show that there is an

optimal pulse situation (10" bacteria becomes lo7) when

Pulse onlpulse off = 2

and the pulse on is 10 s. It is possible that the resting period is used by the electrode (which relaxes to a potential of ca. 0.2 V) to adsorb C1- from the solution which diminishes the host condition for bacterial adsorption.

It is possible to calculate the minimum pulse on and off. The minimum on period would be that which gives a concentration of H202 around the bacteria of at least lop6 mo1.L-'. At an apparent current density of 5 X

A . c ~ - ~ , and assuming 10% current efficiency, the coulombs in t are 0.5 x t and the moles per square centimetre are (0.5 X lop6 t)l(2 x lo5). The volume occupied by these molecules, assuming diffusion is the mode of their dispersal, is cm-3 (D in square centimetres per second, t in seconds). Hence, the

TABLE 2. Values of diffusion coefficients of the toxic material produced in each cell

A , tli?, t112 - 0.3, D,* crn min min cm2.s-

concentration after time t will be

(0.5 x ty(2 x 105)v.2Dt

= (5 x l ~ - ~ ~ ) f i m o l . c r n - ~ = (5 x 1 0 - ~ ) f i m o l . ~ - '

Therefore, (5 X fi> and t > 2 s. This seems to be discrepant with the efficacy of pulses

at 0.1 s. However, solution to the discrepancy may be found in the fact that the observations of the minimum lethal concentration of H202 were bulk determinations: a t the surface the H202 may be more effective.

To get a rough idea of how long this concentration may be effective, i.e., what could be the maximum off period of the pulse length, one calculates the time for a volume of H202 within of the surface to pass through the diffusion layer at the electrode. Assuming this to be the usually quoted value for unstirred solutions (0.05 cm), one gets:

This discussion is at best a very crude one. Diffusion tends to give over to natural convection at about 1-10 s and the use of A2 = 2Dt becomes affected by natural convection near the surface. One concludes only that an on time of seconds, perhaps even 0.1 s, and an off time of hundreds of seconds are likely indications of what either model 3 or model 4 would demand.

Most likely model At present, both the models in which H202 and Cl2

cause bacteria to remain off the surface, and pH changes near the electrode, explain the facts available.

Dynamic and static experiments The present experiments have been carried out under

static conditions. It is desirable to cany them out under dynamic conditions, i .e., with flow up to several feet per second. The Prandtl layer, a layer stationary to the solid in a flowing electrolyte, is approximately 1110th the thickness of the diffusion layer. This is 0.05 cm in stationary solutions but is reduced to cm in high flow rates. The Prandtl (or stationary) layer is, therefore, approximately a micrometre in thickness which is the

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.

1010 CAN. J. MICROBIOL. VOL. 27. 1981

same order as that of a bacterium. It is therefore possible that the bacteria themselves will not be disturbed at flow rates of feet per second. 0 2 diffusion to the surface would be increased about 10 times.

A critical experiment Critical experiments are needed to distinguish be-

tween the H202 and pH models. Among these may be the following.

The deductions which have been made above with respect to the presence of H202 in the vicinity of the electrode arise from estimates of the exchange current density on tin oxide glass for H202 formation. The rotating disc electrode with ring (Damjanovic et al. 1967a, 1967b) could be used to examine the bacterial viability on rotating electrodes which could test whether H202 was being evolved or not. By utilizing various disc materials, all of which will change the pH to the same extent but not produce H202 at the same rate, it would be possible to obtain an indication of whether H202 or pH is the vital feature. Something similar could be done for the anode because one can obtain materials with a rotat- ing disc electrode and ring which will indicate how much chlorine is being evolved and how much 0 2 .

Future possibilities Whether the present experiments have practical pos-

sibilities in antifouling has yet to be assessed. The calcareous deposits found in previous work may be possible to avoid if the current density (and hence the pH change) are relatively low. Pulse methods should be used in a way which would avoid the region in which calcareous deposits are formed.

Acknowledgments This work was supported in part by the Texas A&M

University Sea Grant College Program of the National Oceanic and Atmospheric Administration Office of Sea Grant, under grant No. NA79AA-D-00127. Support in part by the Office of Water Research and Technology during the summer months of 1980 is also acknowl- edged. The authors particularly thank Mr. F. D. Jennings, Director of the Texas A&M University Sea Grant Program, for his interest during the progress of the work.

BOCKRIS, J. O'M., and A. K. N. REDDY. 1973. Modem electrochemistry. Chapters 8 and 9. Plenum Press, New York.

CASTLE, E. S. 1951. Electrical control of marine fouling. Ind. Eng. Chem. 43: 901-904.

CHARACKLIS, W. G. 1978. Biofilm development and destmc- tion: experimental systems. Proc. Symp. Microbiol. Power Plant Ther. Effluents, Iowa City, IA. pp. 73-83.

1979. Biofilm development and distribution in turbu- lent flow. Ozone Sci. Eng. 1: 167-181.

CONN, A. F., M. S. RICE, and D. HAGEL. 1977. Ultra clean heat exchangers-a critical OTEC requirement. Proc. Annu. Conf. Ocean Therm. Energy Convers., 4th, Univer- sity of New Orleans. pp. VII 11 - VII 14.

CORPE, W. A. 1978. Ecology of microbial attachment and growth on solid surfaces. Proc. Symp. Microbiol. Power Plant Therm. Effluents, Iowa City, IA. pp. 57-65.

DAMJANOVIC, A., M. A. GENSHAW, and J. O'M. BOCKRIS. 1967a. The mechanism of O2 reduction at Pt in alkaline solutions with special reference to H202. J. Electrochem. SOC. 114: 1107-1 112.

1967b. Hydrogen peroxide formation in O2 reduction at Au electrodes. J. Electroanal. Chem. 15: 173- 180.

DANIELS, S. C. 1980. Mechanisms involved in sorption to solid surfaces. In Adsorption of microorganisms to sur- faces. Edited by G. Bitton and K. C. Marshall. John Wiley and Sons, New York. pp. 7-58.

DHAR, H. P., J. O'M. BOCKRIS, and D. H. LEWIS. 1981. Electrochemical inactivation of marine bacteria. J. Elec- trochem. Soc. 128: 229-23 1.

FAVA, J. A., and D. L. THOMAS. 1978. Use of C12 to control OTEC biofouling. Ocean Eng. 5: 269-288.

FEIGL, F. 1958.Spot tests in inorganic analysis. Elsevier Publishing Co. pp. 353-37 1.

FLETCHER, M. 1977. The effects of culture, concentration and age, time, and temperature on microbial attachment to polystyrene. Can. J. Microbiol. 23: 1-6.

FROBISHER, M., R. HINSDILL, K. CRABTREE, and C. GOOD- HEART. 1974. Fundamentals of microbiology. W. B. Saunders Co., Philadelphia. p. 136.

GERCHAKOV, S. M. 1978. Biofouling and effects of organic compounds and microorganisms on corrosion processes. Proc. Symp. Microbiol. Power Plant Therm. Effluents, Iowa City, IA. pp. 67-72.

GILEADI, E., E. KIROWA-EISNER, and J. PENCINER. 1975. Interfacial electrochemistry. Addison-Wesley Publishing Co., Inc., Reading, MA. pp. 462-469.

JOLLEY, R. L., R. B. CUMMING, W. W. PITT, F. G. TAYLOR, G. E. THOMPSON, and S. J. HARTMAN. 1978. Ecological impact of chloro-organics produced by chlorination of cooling tower waters. Roc. Sym. Microbiol. Power Plant Therm. Effluents, Iowa City, IA. pp. 85-91.

LITTAUER, E., and D. M JENNINGS. 1968. Prevention of marine fouling by electricalcurrents. Proc. Int. Congr. Mar. Corros. Fouling, 2nd, Athens. pp. 527-536.

MEYNELL, G. G., and E. MEYNELL. 1970. The theory and practice of experimental bacteriology. Cambridge Univer- sity Press. pp. 88-90.

NORRMAN, G., W. G. CHARACKLIS, and J. D. BRYERS. 1977. Control of microbial fouling in circular tubes with C12. Dev. Ind. Microbiol. 18: 58 1-590.

PICOLOGLOU, B. F., N. ZELVER, and W. G. CHARACKLIS. 1980. Biofilm growth and hydraulic performance. J. Hy- draul. Div. Am. Soc. Civ. Eng. 106: 733-746.

RRT, S. J. 1975. Principles of microbe and cell cultivation, John Wiley and Sons, New York. pp. 22-28.

Can

. J. M

icro

biol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

YO

RK

UN

IV o

n 11

/22/

14Fo

r pe

rson

al u

se o

nly.