NATIONAL BUREAU OF STANDARDS REPORT

9846

Technical Summary Report Number 1

For the Period

May 1, 1967 to April 30, 1968

Contract N AON R 14-67

ONR Contract Authority Identification Number NR 036-072

Submitted to the Office of Naval Research

DISTRIBUTION OF THIS DOCUMENT IS UNLIMITED

Subject

MICROBIAL CORROSION

U.S. DEPARTMENT OF COMMERCE

NATIONAL BUREAU OF STANDARDS
THE NATIONAL BUREAU OF STANDARDS

The National Bureau of Standards1 provides measurement and technical information services

essential to the efficiency and effectiveness of the work of the Nation’s scientists and engineers. The

Bureau serves also as a focal point in the Federal Government for assuring maximum application of

the physical and engineering sciences to the advancement of technology in industry and commerce. To

accomplish this mission, the Bureau is organized into three institutes covering broad program areas of research and services: THE INSTITUTE FOR BASIC STANDARDS . .

. provides the central basis within the United

States for a complete and consistent system of physical measurements, coordinates that system with the measurement systems of other nations, and furnishes essential services leading to accurate and uniform

physical measurements throughout the Nation’s scientific community, industry, and commerce. This

Institute comprises a series of divisions, each serving a classical subject matter area: —Applied Mathematics—Electricity—Metrology—Mechanics—Heat—Atomic Physics—Physical

Chemistry—Radiation Physics— Laboratory Astrophysics2—Radio Standards Laboratory, 2 which

includes Radio Standards Physics and Radio Standards Engineering—Office of Standard Refer- ence Data. THE INSTITUTE FOR MATERIALS RESEARCH . . . conducts materials research and provides

associated materials services including mainly reference materials and data on the properties of materials. Beyond its direct interest to the Nation’s scientists and engineers, this Institute yields services which are essential to the advancement of technology in industry and commerce. This Institute is or- ganized primarily by technical fields: —Analytical Chemistry—Metallurgy—Reactor Radiations—Polymers—Inorganic Materials—Cryogenics2 —Office of Standard Reference Materials.

THE INSTITUTE FOR APPLIED TECHNOLOGY . .

. provides technical services to promote the use of available technology and to facilitate technological innovation in industry and government. The

principal elements of this Institute are: —Building Research—Electronic Instrumentation—Technical Analysis—Center for Computer Sci- ences and Technology—Textile and Apparel Technology Center—Office of Weights and Measures —Office of Engineering Standards Services—Office of Invention and Innovation—Office of Vehicle

Systems Research—Clearinghouse for Federal Scientific and Technical Information3—Materials

Evaluation Laboratory—NBS/GSA Testing Laboratory.

1 Headquarters and Laboratories at Gaithersburg, Maryland, unless otherwise noted; mailing address Washington, D. C., 20234.

2 Located at Boulder, Colorado, 80302.

3 Located at 5285 Port Royal Road, Springfield, Virginia 22151.
NATIONAL BUREAU OF STANDARDS REPORT

NBS PROJECT

3120442

NBS REPORT

9846

Technical Summary Report Number 1

For the Period

May 1, 1967 to April 30, 1968

Contract NAONR 14-67

ONR Contract Authority Identification Number NR 036 -072

Subject

MICROBIAL CORROSION

Submitted to the Office of Naval Research

by

Warren P. Iverson Corrosion Section

National Bureau of Standards

Washington, D.C. 20234

DISTRIBUTION OF THIS DOCUMENT IS UNLIMITED

IMPORTANT NOTICE NATIONAL BUREAU OF ! for use within the Governmen

and review. For this reason, whole or in part, is not autl Bureau of Standards, Washin

the Report has been specitica

Approved for public release by the

Director of the National Institute of

Standards and Technology (NIST) on October 9, 2015

;ress amounting documents intended

is subjected to additional evaluation

re listing of this Report, either in the Office of the Director, National

, by the Government agency for which

copies for its own use.

U.S. DEPARTMENT OF COMMERCE

NATIONAL BUREAU OF STANDARDS

ABSTRACT

Studies of the effect of microorganisms, particularly sulfate

reducers ( Desul fovi brio ) , on marine corrosion were initiated. The

inability of sulfate reducers to grow on the agar surface of media

appears to be due to the presence of the ferrous salt used as an

indicator for hydrogen sulfide. Evidence indicates that phosphate

may be an electron acceptor in the cathodic depolarization of steel.

Iron phosphide (Fe^P) and vivianite (Fe^PO^^) • 8H

2 O) were produced

by Desul fovi brio growing in contact with mild steel. The organism

reduces phosphate in the presence of hydrogen to form a volatile

phosphorous containing compound which is not phosphine. Light increases

the corrosion rate of steel as indicated by polarization measurements.

The cathodic protection currents required to maintain a potential of

0.8 volts on a steel specimen in indirect sunlight was found to be 1.5

times that required in the dark.

TABLE OF CONTENTS

Page

INTRODUCTION

EXPERIMENTAL WORK 2

1. Growth and Isolation Studies..... 2

(a) Inhibitory Effect of Ferrous Salts on Agar Growth of Desul fovibrio 4

(b) Isolation and Growth of Desul fovibri

o

from Marine Fouling., 6

2. Mechanism of Cathodic Depolarization 7

(a) Growth on Yeast Extract Containing Benzyl

Viologen and Phosphate as Electron

Acceptors.. 8

(b) Formation of Iron Phosphide by

Desul fovibrio ... 11

(c) Formation of Volatile Phosphorous Compound 12

(d) Other Possible Electron Acceptors in +he

Cathodic Depolarization of Iron 17

3. Effect of Microorganisms on the Corrosion

Process in a Marine Environment. 18

(a) Effect of Light on Cathodic Protection of

Steel in Salt Water.... 19

(b) Effect of Light on Corrosion of Steel in

Sal t Water. 21

SUMMARY 23

ACKNOWLEDGMENT 25

REFERENCES 26

APPENDIX 28

TABLE 1.

Page

The Effect of Light on the Corrosion Rate

of Steel 31

FIGURES:

Fig. 1. Crystals of vivianite on 1010 steel coupons

XI. 25 32

Fig. 2. Crystals of vivianite on 1010 steel coupons

X10 32

Fig, 3. Diagram of 1st circuit used for cathodic

protection 33

Fig. 4. Diagram of 2nd circuit used for cathodic

protection 34

Fig. 5. Recorder tracings of cathodic protection

current at night and during the day 35

Fig. 6. Recorder tracing of cathodic protection

protection current after admittance of sun light to the electrode 36

Fig. 7. Effect of high intensity light from halogen

lamp on cathodic protection current 37

Fig. 8. Diagram of potentiostatic circuit for

cathodic protection 38

Fig. 9. Diagram of polarization circuit 39

Fig. 10. Effect of light on polarization curves 40

INTRODUCTION

Corrosion in the marine environment continues to be a problem of

major importance. The marine environment, because of the high concentration of dissolved salts is a most favorable environment for corrosive action. Obviously the great abundance of flora and fauna in the

ocean is a contributing factor to much of this corrosion, particularly

the marine microorgani sms , because of their wide variety of chemical

activities. Just how important the contributions of living forms is

to marine corrosion remains to be determined.

There is quite good evidence, however, that bacteria can cause

corrosion by bringing about local changes in oxygen or ion concentration or by producing corrosive substances such as acids as a result

of their metabolic activities. Although most of this evidence has

been obtained using non-marine forms, there is no doubt that marine

forms would initiate some reactions since they have been shown to

carry out identical chemical reactions.

Certain bacteria, particularly the anaerobic sulfate reducing

bacteria ( Desulfovibrio , sp.) have been associated with corrosion,

probably more than any other group, for a number of years. They have

been postulated to cause corrosion, particularly of iron and steel by

a cathodic depolarization process in which they remove hydrogen from

the metal surface and use the electrons to reduce sulfate to form

1

hydrogen sulfide. As a result of the hydrogen or electron removal,

iron goes into solution (1). It has been demonstrated that this mechanism

does take place if the dye, benzyl viologen, is substituted for sulfate

as an electron acceptor (2).

One of the objectives of this contract was to study this reaction

in greater detail since some previous studies had indicated that an

electron acceptor other than benzyl viologen might be involved. As

marine sulfate reducers ( Desulfovibrio sp.) have been associated with

marine corrosion (3-7), a second objective was to obtain them in pure

culture and determine if they would cause corrosion by the same mechanism

as the non-marine strains.

A third objective was to compare the corrosion process in sea water

with and without microorganisms.

EXPERIMENTAL WORK

1 . Growth and Isolation Studies

It is highly desirable for studies in microbiology or microbial

corrosion that pure cultures be used and maintained. This is much more

easily accomplished if the cultures can be grown on the surface of agar

media rather than in liquid media.

Cultures of the sulfate reducing organism, Desulfovibrio desul furicans ,

have been very difficult to grow on an agar surface and are usually

grown in liquid media. As a result many, if not all the cultures available,

2

are or have become contaminated. Most of these liquid media contain

sodium lactate as a source of hydrogen or electrons, one or more salts

containing sulfate as an electron acceptor, a source of nitrogen such

as ammonium chloride, one or more agents to lower the redix potential

such as ascorbic acid or thioglycol 1 ate , a phosphate buffer, and an

iron salt, usually ferrous ammonium sulfate, to act as an indicator

of hydrogen sulfide (blackening being due to FeS production). Yeast

extract is usually added also, since it affords better browth. Sodium

chloride is usually added for cultivation of marine strains. The ferrous

indicator salt and the redox lowering agents are usually sterilized

separately by filtering and added to the rest of the autoclaved

ingredients before inoculation.

Recently, two commercially available media (trypticase soy agar

and trypticase soy broth--to which agar is added) were reported to be

satisfactory for the surface growth of the organism (Appendix). A

different medium (for distinguishing the sulfate reducers from the

other microorganisms) was developed using the trypticase soy agar as

a base and adding lactate, sulfate and an indicator salt (Appendix).

Investigations carried out in this study appear to indicate that the

failure of the surface growth on agar using media devised for the growth

of the organism (liquid media) is due to the inhibition of growth by the

ferrous indicator (F^S formation) salt.

3

(a. ) Inhibitory Effect of Ferrous Salt on Agar Growth o f

Desulfovi brio

In some previous unpublished studies, it was found that

only yeast extract plus a hydrogen or electron donor and an electron

acceptor would provide excellent growth on the surface of agar.

Luxuriant growth of Desulfovi brio was found on agar plates (2%) of yeast

extract plus magnesium sulfate (0.2%) in a hydrogen atmosphere. A

distinct green color was noted in the agar, probably due to the green

pigment produced by Desulfovibrio strains, namely desul foviridi n . If

the indicator salt, ferrous ammonium sulfate (sterilized by Seitz

filtration) was added, the typical poor growth as described above was

noted.

Further evidence of the presence of the inhibitory action of the

ferrous salt was obtained. In a private communication from Dr. Postgate,

University of Sussex, England, he stated that trypticase soy agar

(Baltimore Biological Laboratories) (Appendix), as well as the corresponding Difco product and a medium reported by Miss Pankhurst (London

Research Station, Gas Council) at the International Microbiology Congress

in Moscow had shown greater promise than any previous techniques using

plates. The formulation of the medium obtained from Miss Pankhurst is

essentially Baar's medium with a little yeast extract (0.1%) and

thioglycol late (0.01%) set with agar (Appendix).

It appears from the formulation that the ferrous ions from ferrous

4

ammonium sulfate would have been oxidized to ferric ions after

steaming for three successive days. Since there were no ferrous ions

present, nc inhibition would be expected.

A total of 12 petri plates of Miss Pankhurst's medium (2% agar)

were prepared. Six of the plates contained ferrous ammonium sulfate

(sterilized by Seitz filtration and immediately added to the medium

before solidification). The ferrous salt was not added to the remaining

six plates. The six plates with the indicator salt were streaked with

k

3 strains of Desul fovi brio (2 plates per strain) and placed in a Brewer

jar containing a dish of saturated sodium carbonate and pyrogallic acid

to remove traces of oxygen. The jar was evacuated and replaced with

hydrogen three times and incubated at 28° t 1°C. The same procedure

was carried out with the six plates minus the indicator salt.

After incubation for 19 days the plates were removed and examined

for growth. Good growth (over the entire area which was streaked) was

present on all the plates where the indicator salt was absent. All of

the six plates containing ferrous ammonium sulfate with the exception

of one were devoid of growth. The agar plate where growth was present

had smaller volume of space between the cover and the surface of the

agar due to the smaller depth of this particular plastic dish. Possibly

enough of the gas, S

2O, produced by the original inoculum, which has a

*The American Petroleum Institute Strain (Mid-Continent A); a strain

isolated from a contaminated jet fuel water bottom; and a strain

isolated from a corrosion pit in an aluminum tank. Pure cultures of

these strains are maintained on trypticase soy broth plus agar plates

under a hydrogen atmosphere at 28°C.

5

stimulatory effect upon the growth of Desulfovibrio on media containing

the ferrous salt accumulated sufficiently in this smaller volume to

initiate growth (8).

It appears from these observations that the only essential

ingredients for a medium which would provide good surface growth might

be: (1) an electron donor, (2) yeast extract and (3) an electron

acceptor. Sodium chloride may be necessary for marine strains. Since

the ferrous salt appears to inhibit growth a differential medium might

be devised which contains the three essential ingredients plus a ferric

salt as an indicator of hydrogen sulfide production.

( b .) Isolation and Growth of Desulfovibrio from Marine Fouling

One of the objectives in this investigation was to isolate

and study the corrosion mechanism or mechanisms of some marine strains

of Desul fovi brio . These organisms have previously been mentioned as

being associated with marine corrosion.

Ten 1 ml samples of supernatant fluid from the fouling detritus

scraped from five pilings off the Virginia Beach Coast were inoculated

into 5 ml vials of API medium (Appendix) within a few hours of sampling.

After 3 days incubation at ambient temperature, blackening of the medium

was found to occur in all the vials. Bottles of Baar's medium (Appendix)

which contained sodium chloride (2.5%) were also inoculated with the

samples. Blackening also took place in these bottles within a few days.

6

As the trypticase soy agar plus salts medium was found to be quite

successful for growth of non-marine forms of Desulfovibrio (Appendix),

it was used for isolation. Agar (2%) plates of this medium containing

3.0% sodium chloride were streaked from the API medium and incubated

under a hydrogen atmosphere. Black areas developed on the plates

within one weeks' incubation. Microscopic examination of growth

typical of Desulfovibrio revealed an abundance of spiral forms

characteristic of the organism. Repeated efforts involving 9 or 10

transfers (with the possibility of adaptation in consideration) to

isolate colonies of Desulfovibrio on this medium using the streak

dilution technique were not too successful. Although growth of

Desulfovibrio occurred on each transfer, no good separation could be

achieved. At present, work is in progress to develop a better isolation

medium. The effect of salt concentration will be investigated as well

as the use of sea water instead of distilled water for preparation of

the medium. Various media used for the growth of Desulfovibrio in

liquid cultures containing ferric salts as an indicator of H^S will

also be employed.

When the marine strains are isolated in pure culture it is planned

to investigate their influence on the rate of corrosion of ferrous metals

in sea water and their mechanism or mechanisms of corrosion.

2 . Mechanism of Cathodic Depolarization

As indicated in the introduction, Desulfovibrio has been postulated

7

to cause corrosion by a process of cathodic depolarization (removal

of hydrogen or electrons at the cathode by the enzyme hydrogenase)

,

if a suitable electron acceptor were present. This acceptor was

postulated to be sulfate. Benzyl viologen has been substituted for

sulfate and found to be a satisfactory electron acceptor in the presence

of iron. It seemed of interest to determine whether this compound

could replace sulfate as an electron acceptor for growth. The utilization of other electron acceptors (for growth and for cathodic

depolarization) was also investigated.

( a ) Growth on Yeast Extract Containing Benzyl Viologen and

Phosphate as Electron Acceptors

It was previously stated that yeast extract provided an

excellent growth medium for Desulfovibrio if an electron donor (such

as hydrogen) and an acceptor such as sulfate were present. Little, if

any, growth could be found on yeast extract (2%) agar (2%) plates if

one or both were absent for incubation periods up to two weeks. Such

a medium provides an excellent screening medium for electron donors

and acceptors. It was of interest to determine whether yeast extract

without any added electron acceptors would support growth of

Desulfovi brio if the incubation time was greatly prolonged. It was also

of interest to determine whether benzyl viologen was a hydrogen acceptor

would promote growth in the presence of hydrogen. Cells of Desulfovibrio

have been previously demonstrated to reduce benzyl viologen, but benzyl

8

viologen (50 mg/ml) has also been reported to inhibit growth of the

organisms (9). Two brands of commercially available yeast extract

were compared for their inability to support growth in the presence

of electron donors and acceptors.

Three plates of Difco yeast extract (2 %) plus agar (2%) and three

plates of BBL yeast extract (2%) plus agar (2%) were prepared. Three

plates of Difco yeast extract (2%) agar (2%) containing benzyl viologen

(100 mg/ml) were also prepared. The pH of the agar was adjusted to

7.0 ±1 in all cases before autoclaving. After streaking all plates

with the API strain, the plates were placed in dessicator jars. The

three plates containing the benzyl viologen were placed in a separate

jar. Petri dish bottoms containing alkaline pyrogallol were placed

on top of the stacks of petri dishes in the jars. The jars were

evacuated and replaced three times with hydrogen.

After 5 months incubation at 27-28°C, no growth of Desul fovi bri

o

was observed on the plates of BBL yeast extract agar. Good growth,

however, was observed on the three plates of Difco yeast extract agar.

Microscopic examination of this growth showed many cells to be motile.

Very poor growth was found on the three plates of yeast extract agar

plus benzyl viologen. Microscopic observation revealed few, if any,

intact cells. The benzyl viologen was reduced (violet areas), however,

under nearly all of the agar area streaked with the cells.

9

Benzyl viologen does not appear, therefore, to be a satisfactory

electron acceptor for growth of the organism, at least in the concentration used. Preliminary observations would indicate that it might

be based upon the considerable reduction of the dye even by very small

numbers of cells. It appears that the Difco yeast extract does

contain both electron donors and acceptors which are very slowly

utilized whereas the BBL product does not. Further experiments with

the BBL product are planned since it would appear to be an even better

screening agent than the Difco yeast extract.

As it was reported that iron phosphide is produced by Desul fovi bri

o

( vide infra, 10), it appeared from these observations that phosphate

might be an electron acceptor. This was verified by ovserving good

growth on three plates of Difco yeast extract (2%) agar (2%) with

I^HPO^ (0.5 gm/100 ml agar) and the absence of growth on three plates

of the same medium without the phosphate, after one week observation

under a hydrogen atmosphere at 28°C. (The pH of the yeast extract

agar without the phosphate was brought to 7.0 ± 1 by the addition of

KOH. The agar containing KgHPO^ (pH 7.0 ± .1) needed no pH adjustment.)

The growth was not nearly as luxuriant as on the yeast extract agar with

sulfate as the electron acceptor. Microscopic examination of the growth

on the phosphate agar revealed long loosely coiled spirals to predominate instead of the short curved rods, normally found.

10

( b ) Formation of Iron Phosphide by Desul fovi brio

The black materials produced (at Fort Detrick) by

Desul fovi brio growing in yeast extract (2%) containing 1010 steel

was non-magnetic and no X-ray diffraction pattern was produced from

it. Upon heating to 2,250°F for 15 minutes in a vacuum and allowing

to cool to room temperature for 17 hours, a diffraction pattern for

Fe^P was obtained. The material had also become magnetic. Comparison

of this material with a commercially available sample (Rocky Mountain

Research, Inc.) by Mossbauer spectroscopy revealed the materials to

be similar.

Comparison of the colloidal non-magnetic material before heating

with the commercial preparation by Mossbauer spectroscopy seemed

essential. Six 50 ml centrifuge tubes containing 40 ml of 1% Difco

yeast extract broth (pH 7.0 ± 0.1) and coupons of 1010 steel were

inoculated with Desul fovi brio (API strain). After 11 days incubation

under a hydrogen atmosphere, the liquid cultures (black) were combined

in a centrifuge bottle with a rubber cap. The atmosphere above the

culture was replaced with hydrogen. Centrifugation was unsuccessful

until the liquid culture was rapidly frozen using dry ice, and thawed.

After washing twice in boiled distilled water (to remove most of the

oxygen) and drying in a vacuum, 11.6 mg of black powder was obtained.

II This sample was compared with the Mossbauer spectrum of the heated

11

sample of both samples appeared identical. It was of interest that

microscopic examination of the culture before centrifugation revealed

extremely long spiral forms of Desul fovi brio to be present. Some

forms extended across the entire field.

Of interest also was the finding of black crystals on the 1010

steel specimens in (2%) yeast extract broth inoculated with the API

strain when allowed to incubate at 28°C in a hydrogen atmosphere for

a long period of time (1 month). They appeared to predominate on

areas not covered with a dark film (presumably iron phosphide). No

X-ray diffraction pattern could be obtained from the film (Figures 1

and 2), however.

X-ray diffraction analysis of the crystals revealed them to be

vivianite (Fe^PO^ • 8^0). No pits were found under the crystals.

At present no definite explanation can be given for their formation although it probably is related to the process of iron phosphide

formation or degradation. No similar crystals were noted on steel in

uninoculated yeast extract broth inoculated for longer periods of time,

(c) Formation of Volatile Phosphorous Compound

The evidence obtained (namely, growth on yeast extract

agar containing phosphate and the production of iron phosphide) indicated that the organisms were probably reducing phosphate. Some early

investigations had already reported the reduction of phosphate to

12

phosphine by some microorganisms (11, 12).

Several attempts to determine the presence of phosphine (PH^) by

infrared spectroscopy were negative although no one occasion band

suggestive of P - H bonds appeared. There was some doubt as to the

reliability of the instrument at this particular time, however, In

these investigations the hydrogen atmosphere in a Brewer jar containing

9 inoculated (API strain) plates of Difco yeast extract (2%) agar (2%)

plus a potassium phosphate (0.5 gm/200 ml agar) was flushed with helium

(4-5 hours) through a methanol dry ice trap to remove the moisture and

a liquid nitrogen trap to collect the suspected phosphine. The

cultures were 4 to 5 days old when their atmosphere was flushed out.

A more sensitive chemical method reported for the determination of

phosphine in air was then employed (13). The atmosphere from the

Brewer jar containing 9 inoculated plates of the API strain was flushed

with hydrogen through a trap containing glass beads and acidified (5%

sulfuric acid) N/10 potassium permangenate solution to oxidize the

phosphine to phosphate. After titration with N/10 oxalic acid to

remove the excess permanganate, ammonium molybdate (2% solution) and

stannous chloride solution (0.5%) were added to the acidified solution.

An intense dark molybdenum blue color (indicating the presence of

phosphate) was found to develop. Analysis of the blank solution

(permanganate solution flushed with hydrogen) indicated the presence

of only traces of phosphate.

13

In addition to flushing the gas through a trap containing an

acidified permanganate solution, the solution in a petri dish was

placed on top of a stack of inoculated plates in a Brewer jar.

Analysis of this solution also revealed a much greater concentration of phosphate than a control solution in an atmosphere of hydrogen.

A volatile phosphorous compound was also detected from growth on

plates of trypticase soy broth plus salts (Appendix) of one of the

marine isolates of Desul fovi brio using acidified permanganate solution as the trapping agent.

Phosphine may be detected by its reaction with mercuric chloride

to form a yellow precipitate according to the following reaction:

PH

3 + 3HgCl

2 = P (HgCl

)

3 + 3HC1 (14-17).

A tan-colored precipitate (6.9 mg, dry wt.) was formed when a

petri dish bottom containing 1% HgC

1

2 was placed on top of a stack of

9 inoculated petri plates containing Difco yeast extract (2%) agar

(2%) and dipotassium phosphate (0.5 gm/200 m agar) after 11 days

incubation. Comparison of this material with the precipitate formed

by phosphine (generated by the action of an aqueous 50% sulfuric acid

solution V/V on zinc phosphide) on HgCl

2

» by X-ray diffraction has

revealed them to be dissimilar. The X-ray pattern for the yellow

product produced by the action of phosphine on HgCl

2 appeared identical

to that of HgCl

2

* The pattern for the tan powder precipitated by the

14

bac ter i ally produced gas in HgCl

2 showed certain similarity to

Hg^PO^. It is now strongly suspected, however, that the reactive

produce, whereby iron phosphide may be formed, if indeed this is the

mechanism, is not phosphine but another intermediate, volatile,

phosphorous containing product formed in the reduction of phosphate.

Phosphine does not blacken the agar media containing ferrous salts

as does the gas produced by the organisms. If this blackening of

the agar is due to iron phosphide (and this is being investigated),

positive evidence of the mechanism of its formation would definitely

be indicated. It was also of interest that no distinct odors of

hydrogen sulfide or phosphine could be detected upon opening the

culture jars.

The tan-colored compound appears also to be produced in HgCl

2 by the API strain growing in Difco yeast extract (2 %) agar containing

hypophosphi te (Nal-^PC^). This might indicate that the suspected

intermediate is produced after the reduction of hypophosphi te.

Assuming changes of two electrons in each stage of reduction, since

all the known hydrogen carried in the bacterial cell such as pyridene

nucleotides can apparently accommodate only two hydrogen atoms or

electrons at a time, the steps in the reduction of phosphate would be

as follows

(+ 5 ) — (+ 3 ) — (+ 1 ) — (- 1 ) — (- 3 ) -+ P°4 + P0

3

P0

2 + p?

~v ph

3

15

Postulated intermediates in which phosphorous would have a valence of

-1 would include PH and H^PO. The suggestive evidence from

the X-ray pattern indicates that oxygen may be present and that the

latter compound would be a greater possibility. Many of these intermediates, however, are very short lived. Phosphite (PO^""") and

hypophosphi te (PO^

--

) have been reported, however, as intermediates in

the reduction of soil bacteria (12), using a paper chromatographic

technique. The formation of these compounds by Desul fovi bri

o

, marine

and non-marine strains, will also be investigated.

Phosphine, as produced by the action of dilute acid on zinc

phosphide was found to produce blackening of lead acetate paper,

primarily around the edges. Blackening of lead acetate paper was also

noted when dilute acid was added to the commercial sample of iron

phosphide (Fe

2 P), probably due to other phosphides of iron or other

metals as most of the iron phosphide did not react. This observation

appears interesting since one of the tests used in the field for

detecting iron sulfide (indicating bacterial corrosion) is to add a

few drops of concentrated acid to black corrosion products and test

the resulting gas for the presence of hydrogen sulfide using lead

acetate paper. Usually the odor of hydrogen sulfide can also be

detected. It has been noted by one of the investigators in soil

corrosion at the National Bureau of Standards that occasionally

16

blackening of lead acetate paper occurs without any detectable odors

of hydrogen sulfide. This is suggestive evidence that phosphides

might be formed as corrosion products of bacterial action in the

field. Further investigations in this area will be carried out as

the opportunity arises.

(d) Other Possible Electron Acceptors in the Cathodic

Depolarization of Iron

A preliminary study of nitrate and nitrite as electron

acceptors (in addition to the dye benzyl viologen and phosphate), which

might be utilized by Desul fovi brio in the cathodic depolarization of

steel, was initiated. Nitrite has been reported by Senez et a]_ as an

excellent electron acceptor for Desulfovibrio (18). They did not,

however, find nitrate to be reduced. Baumann and Denk did find

nitrate reduction to be rapid and quantitative when lactate was the

hydrogen (electron) donor (19). The technique previously described (2)

was employed in which a steel coupon is placed on the surface of agar

(containing an organic buffer plus an electron acceptor to be studied)

with one portion resting on cells of the organism to be studied.

Assuming no complicating secondary reactions and that a satisfactory

electron acceptor is present, the organisms have been known to remove

hydrogen or electrons from the surface of the steel and reduce the

electron, benzyl viologen acceptors, at the cathode thus causing ferrous

ions to go into solution at the anode (where they can be demonstrated

17

by adding ferricyanide to the surface of the agar).

Nitrite (NaNC^) and nitrate (KNO^) were employed as potential

hydrogen acceptors in concentrations of 10 mg per 20 ml of washed agar

(BBL) containing 0.01 M tris buffer adjusted to pH 7.0 ± 0.1. Control

plates with no electron acceptors were also prepared. Six day old

cells of the API strain were used and the plates with the coupons

placed in a Brewer jar with a helium atmosphere at 28°C. After 18

hours the plates and coupons were removed. Ferrous ions were detected

in the agar at both cathodic areas (bacterial cells) and anodic areas

with nitrate as a potential hydrogen acceptor. As there seemed to be

no difference between the concentration of ferrous ions at both anodic

and cathodic areas in the control plates (no electron acceptors), as

evidenced by the amount of colored ferrous ferricyanide complex, it

appeared that nitrate was not utilized as an electron acceptor. No

ferrous ions were detected in the agar containing nitrite ions. This

might have been expected as nitrite is a known corrosion inhibitor for

iron. Since phosphate is a corrosion inhibitor for iron and it appears

to be reduced in yeast extract in contact with iron, it is possible that

yeast extract was interfering with the corrosion inhibition action of

phosphate. Further investigation is contemplated in this area.

3. Effect of Microorganisms on the Corrosion Process in a Marine

Envi ronment

In a study of microbial corrosion in the marine environment, it

18

is theoretically desirable to compare the corrosion rate in the

presence and in the absence of microorganisms , all other factors being

equal. Practically, however, this may be difficult to accomplish.

As many structures in the marine environment are cathodically

protected, it was first decided, however, to study the effects of

microorgani sms on such a system. Various circuits which might be

used were first investigated. For these investigations a 3% sodium

chloride solution in tap water was used instead of sea water. It

soon became evident that light had a predominent effect on the protection current. A preliminary study of this factor was initiated.

(a) Effect of Light on Cathodic Protection of Steel in

Salt Water

All of these investigations were carried out in succession

on a single mild steel rod electrode (7,7 cm long and 1.27 cm in diameter)

totally immersed in a 4 1/2 gallon jar (12" high and 12" in outer

diameter) containing 17 liter of tap water plus sodium chloride (3%).

The jar was placed on a table so that the electrode was 4' 9" from a

window facing the north. The auxiliary electrode consisted of a piece

of completely immersed platinum foil (1x5 cm) attached to a platinum

wire. The electrodes were suspended by a plexiglass cover which also

prevented evaporation. Holes in the cover permitted introduction of a

thermometer and a commercially available calomel half cell. The first

circuit employed is shown in Figure 3. The resistance of 1500 ohms

19

was selected to maintain an initial potential on the steel electrode

of 0.8 volts. Variations in the potential and current were soon detected and it appeared that these variations were related to the light

intensity. During the night and early in the morning the potential

remained about 0.84 volts and the current about 0.48 mA. When the

room was fully illuminated during the day the potential dropped to

about 0.74 volts and the current increased to about 0.60 mA. No

relation to the temperature could be detected.

A second circuit was then employed (Figure 4). The changes in

current with illumination were even more pronounced as indicated in

Figure 5. During the daylight the changes in current appeared to be

too sensitive to changes in light intensity caused by the passage of

clouds. In several experiments the Venetian blind was closed and the

changes in current measured after they were opened. This effect is

shown in Figure 6. The effect of short bursts (2 min.) of high

intensity artificial light (commercial ly available halogen bulb used

for photography) on the current is indicated in Figure 7. The light

source was placed about 3 feet from the electrode. Again this effect

could not be related to temperature changes.

In order to maintain a fixed potential on the electrode, the circuit (potentiostatic) indicated in Figure 8 was then employed. The

potential on the electrode was set at 0.8V by means of the variable

1000 ohm resistance and was maintained to within ± 0.02V during the

20

experiments. Over a consecutive period of seven days the current

required to maintain the potential at 0.8V generally remained at 0.29

2 mA (0.90 mA/dm ) and rose during the day to a maximum of about 0.44

2 mA (1.37 mA/dm ) during the period of greatest light intensity in

the day. On the basis of these results about 52% more current or

1.5 times the amount of current required in the dark was needed to

maintain the same potential (0.8V) in the light.

( b ) Effect of Light on Corrosion of Steel in Salt Water

The influence of light upon the corrosion rate was then

determined by a polarization technique using the circuit in Figure 9.

Three polarization curves (anodic and cathodic) were obtained from

data recorded on three days (not successive) with the room lights off

and the blind closed. Three other curves were obtained on bright days

with the blind open and the room lights off. The data for the cathodic

curves were obtained in the morning and the data for the cathodic curves

in the afternoon with an interval of at least two hours between. The

current was applied in increments of 0.5 mA for a duration of two

minutes. The potential was recorded at the end of this duration.

Corrosion rates were calculated from the polarization curves

plotted on coordinate paper by the so-called "polarization break"

method (20). The "break" in the cathodic curve is the point Ic, the

intersection of tangents to the two slopes in the curve. The point la

is the intersection of the tangents to the two slopes in the anodic

21

curve. The corrosion current is calculated from the Pearson equation

( 21 ):

The corrosion currents and rates for the three dark periods and

the three light periods are presented in Table 1. The data for a

bright and a dark period are presented in Figure 10. Using the

average values, the corrosion rate in the light is about 1.3 times

the corrosion rate in the dark or about 28% greater in the light than

in the dark. The corrosion ratio is about 100 to 77. Corrosion ratios

of 100 to 83.3 and 84.6 have been reported for light and darkness

based upon weight losses (22). The temperature varied from 22.5 to

23°C during these measurements and there did not appear to be any

correlation between temperatures and corrosion rate.

The potential of the corroding steel was usually found to be displaced 10-15 mV in a more positive direction during the light period.

This is also in agreement with some earlier results (22).

In addition to the effect of light on the corrosion process, the

velocity of the water appears to be another factor to be considered

in such an investigation (23). Future investigations will attempt

to investigate the effect of stagnant sea water (harbor water);

however, cathodic protection and the corrosion rate of steel as

attacked by microorganisms may be more severe in this environment.

22

SUMMARY

1. The inability to grow non-marine sulfate reducers ( Desulfovibrio )

successfully on the agar surface of media (Baar's and yeast extract)

used for their cultivation has been found to be due to the inhibitory

effect of the indicator salt (ferrous ammonium sulfate) for hydrogen

sulfide in the media.

2. Trypticase soy agar plus salts medium (used successfully for the

isolation of non-marine Desul fovi brio strains) was not found to be

suitable for the isolation of pure cultures of marine strains

( Desulfovi brio ) . Development of a suitable medium for direct

counting and isolation of marine strains of Desulfovibrio will be

continued.

3. An agar (2%) medium consisting only of yeast extract (2%) was

found to be useful as a screening agent for hydrogen (electron) donors

and acceptors for the growth of Desul fovi brio .

4. Phosphate was found to be a suitable electron acceptor for growth

in yeast extract agar in the presence of hydrogen as an electron donor.

5. A volatile phosphorous product was detected during the growth of

Desul fovi brio on yeast extract agar containing phosphate. The compound

is not phosphine, but appears to contain oxygen. Hydrogen may also

be present. It appears to be a product of phosphate reduction and may

be involved in the reaction with ferrous ions to produce iron phosphide.

23

Preliminary evidence indicates that marine strains of Desul fovi brio

may also produce this volatile material. The exact composition of

this compound will be determined.

6. The black colloidal material produced in yeast extract cultures

of Desulfovibrio containing mild steel was found to be iron phosphide.

7. Crystals of vivianite (Fe^PO^^-SH^O) were found to develop on mild

steel specimens in inoculated ( Desulfovibrio ) yeast extract broth

after 1 month incubation in a hydrogen atmosphere.

8. In preliminary studies to determine the effect of mi croorgani sms

on cathodically protected steel it was found that light has an effect

upon the current required for protection. On the steel specimen

employed, it was found that 1.5 times the amount of current required

in the dark was needed to maintain the same potential (0.8V) in the

1 i ght.

9. The corrosion rate in indirect sunlight was found to be about 1.3

times the corrosion rate in the dark.

24

Acknowl edgment

The X-ray diffraction studies by C. Bechtoldt and the Mossbauer

studies by Dr. J. Spijkerman are most gratefully appreciated.

Publications and Talks Resulting from Research

Part of the work will be included in a paper to be presented at

the 1st International Biodeterioration Symposium to be held in

Southampton University, England, in September 1968.

25

REFERENCES

1. C.A.H. von Wolzogen Kuhr and L.S. van der Vlugt, Water ]8_, 147

(1934).

2. W.P. Iverson, Science 151_, 986 (1966).

3. R.L. Starkey and J.D. Schenone, J. Bacteriol . 54, 46 (1947).

4. R.L. Starkey, Bacteriol. Proc. 51 (1948).

5. R.L. Starkey, Intern. Congr. Microbiol. Rept. Proc., 6th Congr.

3, 326 (1953).

6. E.E. Corcoran and J.S. Kittredge, Marine Corros. & Fouling Conf . ,

Scripps Inst, of Oceanography, La Jolla, Calif., April 18-20,

1956.

7. W.G. Blaston and C.H. Oppenheimer, Bacteriol. Proc. 36 (1960).

8. W.P. Iverson, Science 156 , 1112 (1967).

9. A.M. Saleh, R. Macpherson and J.D. A. Miller, J. Appl . Microbiol.

27, 281 (1964).

10. W.P. Iverson, Nature 217 , 1265 (1968).

11. K. I. Rudakov, Zentr. Bakteriol. Parasitenk. Abt. II, 7!9, 229 (1 929).

12. G. Tsubota, Soil Plant Food (Tokyo) 5_, 10 (1959).

13. J. Borrowdale and C.E.A. Shanahan, Ann. Occup. Hyg. 4_, 36 (1961 ).

14. L. Moser and A. Brukl , Z. anorg. allgem. chem. 1 21 , 73 (1922).

15. P. Lemhoult, Compt. rend., 145 , 1175 (1907).

16. M. Wilmet, Compt. rend. 185 , 206 (1927).

17. K. Beyer, Z. anorg. allgem. chem. 250 , 312 (1943).

26

18. J.C. Senez, F. Pichinoty and M. Konavol tchi koff-Mozoger , Compt.

rend., 242, 570 (1956).

19. A. Baumann and V. Denk. Arch. Mikrobiol. 1_5, 283 (1951 ).

20. W.J. Schwerdtfeger, J. Research, NBS 6J5, Oct. -Dec. (1961).

21. J.W. Pearson, Trans. Electrochem. Soc. 81_, 485 (1942).

22. C.O. Bannister and R. Rigby, J. Iron & Steel Inst. 133 , 293

(1936).

23. C.A. Smith, Meeting, National Association of Corrosion Engineers,

March 18-21 (1968).

27

USCOMM—DC — NBS

APPENDIX

Pankhurst's medium*

k

2 HP04

nh

4

ci

CaS0

4

0.5 gm.

1.0

1.0 "

MgS0

4

-7H

2 0 2.0 "

Sodium lactate 5.0

Tap water 930 ml

pH adjusted to 8.1 before sterilizing.

Sterilized by autoclaving 10 Ibs/sq in. for 20 min.

plus 50 ml of a 1% (w/v) solution of FeS0

4 (NH

4

) 2 S0

4

• 6H

2 0

(sterilized by steaming 1 hr. on 3 successive days)

plus 10 ml of a 10% (w/v) solution of Difco yeast extract

(sterilized by autoclaving)

plus 10 ml of a 1% (w/v) solution of sodium thiogylcol late

(sterilized by membrane filtration)

pH of medium after prep. = 7.2 - 7.6

*Baars, J.K. (1930) Over Sulfaatreductie door Baterien Dissertation.

Delft: W.D. Meinema

28

Trypticase Soy Agar Plus Salts Medium*

+Trypticase Soy Agar (BBL) 40.0 gm

Sodium lactate (6% sol.) 4.0 ml

Magnesium sulfate 2.0 gm

Agar 5.0 "

Sodium Chloride 25.0 "

Ferrous ammonium sulfate 0.5 "

Distilled water 1000.0 ml

Ferrous ammonium sulfate solution (10 ml dist. b^O)

by Seitz filtration. pH adjusted to 7.0 - 1.

+Trypticase Soy Agar (BBL)*

Trypticase 15.0 gm

Phytone 5.0 "

Sodium Chloride 5.0 "

Agar 15.0 "

Distil led water 1 liter

*W.P. Iverson, J. Appl. Microbiol. 21 (4) 529-534 (1965)

steri 1 i zed

29

API Medium*

Sodium lactate (60%) 4.0 ml

Yeast extract 1 .0 gm

Ascorbic Acid 0.1 "

MgS0

4

-7H

2 0 0.2 "

K

2

HP0

4 (Anhyd) 0.01 gm

Fe(S0

4

) 2 (NH)

2

-6H

2

0 0.1 "

NaCl 10.0 "

Disti 1 led water 1000 ml

pH adjusted to 7.2 - 7.5 with NaOH

*R.C. Allred, Prod. Month. 22, 32 (1958)

30

TABLE 1

The Effect of Light on the Corrosion Rate of Steel

Dark Light

Day

Corrosion

Current (io)

ImA)

Corrosion

Rate

4- (mA/dm^

)

Corrosion

Current (io)

(mA)

Corrosion

Rate

(mA/dm^)

22 Jan — — 0.34 1.06

23 Jan 0.24 0.75 — —

25 Jan 0.23* 0.72 — —

26 Jan — — 0.26 0.81

5 Feb — — 0.27* 0.84

6 Feb 0.20 0.62 — —

Average 0.22 0.70 0.29 0.90

2 +Area of electrode = 32 cm

i o o

p 32cm^ X lOOcnr = mA/dm

*Data plotted in Figure 10

31
: •

S?

:c t , a
1

Fig. 1. Crystals of vivianite on 1010 steel coupons X 1.25.

Coupons were in a vertical position while in the

yeast extract broth (right -*1 eft = top bottom).

Fig. 2. Crystals of vivianite on 1010 steel coupon. X10

32

3 V

Fig. 3. Diagram of 1st circuit used for cathodic protection.

R = recorder, A = ammeter, P = electrometric potentiometer,

AUX = platinum auxiliary electrode, SPEC = steel rod.

33

3 V

Fig. 4. Diagram of 2nd circuit used for cathodic protection.

R = recorder, A = ammeter, P = electrometric potentiometer,

AUX = auxiliary platinum electrode, SPEC = steel rod.

34

Fig. 5. Recorder tracings of cathodic protection current at night

and during the day (during a 1 hour interval).

35

z

rO

OCXOX

r^

CD r—o o

-ao

-E E

cn qj

•r- Q_

03 E

=3 E

CO

O<+- 4-o -a

CD

<d co o o E ,

03

O

4->

4-> X3

•r- E E *r- -o

03 CQ E

ai CO 4- CD 03 +-> 3

4-> E > E *r— _Q

cd E E -O E LT> O) 3 OO -M

C_) 03 * O E E -r- O 3 -a

•r—

O E

03

O

O) <—

-MO 4— E O +-> a. e

r— a>

C_> 03 4-> •I- > o

-a e a. o cd

+-> e • 03 *r- CO O E

4- 03 E o cd cn Q. cn e o E -I—

•r- E O U 3 +-> 03 XJ E E

+-> •>

O

CD -i- e -a e

CD

O Qa e E +J CO O U E

CJ CD 3

CD i—

O

CtC CD .E

36

Fig. 7, Effect of high intensity light from halogen lamp

on cathodic protection current. Potential remained

at 0.74V. L = 2 minute period of light exposure.

37

3 V 3 V

Fig. 8. Diagram of potentiostatic circuit for cathodie protection.

R = recorder, A = ammeter, P = electrometric potentiometer,

B.U. = balancing unit, AUX = auxiliary platinum electrode,

SPEC = steel electrode, REF = calomel reference electrode.

38

3 V 3 V

Fig. 9. Diagram of polarization circuit. A = ammeter, P = electrometric potentiometer, AUX = platinum auxiliary

electrode, REF = calomel reference electrode, SPEC = steel rod specimen.

39

Fig. 10, Effect of light on polarization curves. Several

values for anodic curves not indicated.

40

Unci ass i fi ed

Security Classification DOCUMENT CONTROL DATA - R & D

(Securi ty etessi flcation of tide, body of abstract and indexing annotation must be entered when the overall report la c laaalfled)

1. ORIGINATING activity (Corporate author) National Bureau of Standards

2m. REPORT SECURITY CLASSIFICATION Unci assif i ed

Washington, D.C. 20234 26. GROUP

3. REPORT TITLE MICROBIAL CORROSION.

DESCRIPTIVE NOTES (Type of report and inclusive dateo) TECHNICAL SUMMARY REPORT NO. 1

8- AUTHOR(S) (First siamo, middie initial, last name) Warren P. Iverson

0. REPOR T D A TE

1 May 1968

7 a. TOTAL NO. OF PAGES 40

7b. NO. OF REFS 23

8a. CONTRACT OR GRANT NO. NAONR 14-67, NR 036-072

6. PROJEC T NO. 3120442

9a. ORIGINATOR'S REPORT NUMBER(S)

3120442 - Technical Summary

Report No. 1

96. OTHER REPORT NO(S) (Any other numbers that may be aaaigped

this report)

!0. DISTRIBUTION STATEMENT

Distribution of this document is unlimited

II- SUPPLEMENTARY NOTES 12 SPONSORING Ml LI T ARY ACTIVITY Office of Naval Research

13. ABSTRACT Studies of the effect of microorganisms, particularly sulfate reducers

( Desul fovi brio ) , on marine corrosion were initiated. The inability of

sulfate reducers to grow on the agar surface of media appears to be due to

the presence of the ferrous salt used as an indicator for hydrogen sulfide.

Evidence indicates that phosphate may be an electron acceptor in the

cathodic depolarization of steel. Iron phosphide (Fe 0 P) and vivianite

(Fe o (P0^)p) -8H 90) were produced by Desul fovibrio growfnq in contact with

mild steer. The organism reduces phosphate in the presence of hydrogen to

form a volatile phosphorous containing compound which is not phosphine.

Light increases the corrosion rate of steel as indicated by polarization

measurements. The cathodic protection currents required to maintain a potential of 0.8 volts on a steel specimen in indirect sunlight was found

to be 1.5 times that required in the dark.

DD FORM1 NOV 88 1473 UNCLASSIFIED

Security Classification

Unci assi fi ed

Security Classification

1 4. K EY WO RDS

LINK A LINK B LINK C ROLE W T ROLE W T ROLE W T

Cathodic depolarization

j Microbial corrosion

| Sul fate reducers

Desulfovibrio

;

Mild steel

Iron phosphide

Viviani te

Phosphate reduction

iLight

ICathodic protection

Unclassified

2

Security Classification


1J