DTIC ADA512850: Influence of Experimental Conditions on the Outcome of Laboratory Investigations Using Natural Coastal Seawaters

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The Influence of Experimental Conditions on the Outcome of Laboratory
Investigations Using Natural Coastal Seawaters
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0601153N
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Jason S. Lee, Richard I. Ray, Brenda J. Little
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73-5052-19-5
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Naval Research Laboratory
NRL/JA/7330-09-9297
Oceanography Division
Stennis Space Center, MS 39529-5004
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14. ABSTRACT
Methods for handling and storing natural coastal seawater, particularly methods for deaeration over time, influenced the
chemistry and microflora. Bubbling nitrogen into natural seawater to achieve an anaerobic condition was not conducive
to the growth of sulfate-reducing bacteria (SRB). Despite a higher initial dissolved sulfate concentration and higher total
organic carbon in Persian Gulf seawater, higher dissolved sulfide concentrations and SRB populations were measured in
Key West, Florida, seawater under all exposure conditions.
15. SUBJECT TERMS
carbon steel, marine corrosion, seawater, sulfate-reducing bacteria
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The Influence of Expermental Conditions on the Outcome of Laboratory Investigations Using Natural Coastal Seawaters
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Jason S. Lee, Richard I. Ray, Brenda J. Little
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The influence of Expermental Conditions on the Outcome of Laboratory Investigations Using Natural Coastal Seawatera
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Jason S. Lee, Richard I. Ray, Brenda J. LttUe
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CORROSION SCIENCE SECTION
Technical Note: Influence of Experimental
Conditions on the Outcome of Laboratory
Investigations Using Natural Coastal Seawaters
J.S. Lee," R.I. Ray* and BJ. Little"
a 1986 volume of contributed papers, 10 of 36 papers
ABSTRACT
2
specifically mention SRB in the title. In the 2009
Methods for handling and storing natural coastal seawater,
session on MIC at CORROSION 2009, 3 of 10 papers
particularly methods for deaeration over time, influenced the
dealt with some aspect of SRB-influenced corrosion.
chemistry and microflora. Bubbling nitrogen into natural sea-
Today the list of causative and contributing bacte-
water to achieve an anaerobic condition was not conducive to
3
ria, fungi, and archaea includes numerous groups;
the growth of sulfate-reducing bacteria (SRB). Despite a higher
however, SRB are arguably the microorganisms most
initial dissolved sulfate concentration and higher total organic
closely identified with MIC.
carbon in Persian Gulf seawater, higher dissolved sulfide con-
SRB are a group of ubiquitous, diverse anaer-
centrations and SRB populations were measured in Key West,
2
obes that use sulfate (SO ;) as the terminal electron
Florida, seawater under all exposure conditions.
acceptor, producing sulfide (S"). SRB have been iso-
KEY WORDS: carbon steel marine corrosion, seawater,
4 5
lated from a variety of environments. " Because of
sulfate-reducing bacteria
the consistently high concentration of sulfate in sea-
water (>2 g/L), SRB are extremely important in sea-
INTRODUCTION
water corrosion. For many years there has been a
debate over the best laboratory solution for studying
Sulfate-reducing bacteria (SRB) were the first group of
6
marine corrosion, i.e., natural or artificial seawater.
1
microorganisms identified as causing corrosion. SRB
Dexter carefully compared laboratory solutions and
have been the focus of many investigations involving
natural seawater for studying the corrosion of alumi-
microbiologically influenced corrosion (MIC), and sev-
num alloys and prepared a specific artificial medium
eral corrosion mechanisms have been attributed to
that reproduced the short-term behavior of alumi-
SRB, including cathodic depolarization by the enzyme
6
num alloys in natural seawater. However, none of the
dehydrogenase, anodic depolarization, production of
laboratory solutions were able to simulate the long-
iron sulfides, release of exopolymers capable of bind-
term corrosion behavior of aluminum alloys in natu-
ing metal ions, sulfide-induced stress corrosion crack-
ral seawater. The selection of a laboratory medium for
ing, and hydrogen-induced cracking or blistering. In
MIC experiments is even more complicated. Investiga-
Submitted for publication July 2009; in revised form. September
tors have used natural seawater with naturally occur-
2009. Presented as paper no. 07518 at CORROSION/2007. March
7 9
ring microorganisms, " artificial seawater to which
2007. Nashville. TN.
10
nutrients and microorganisms have been added, ""
Corresponding author. E-mail: [email protected].
Naval Research Laboratory. Code 7332. Stennis Space Center. MS
and inoculated synthetic media, e.g., Postgate B with
39529.
12 13
3.5% sodium chloride (NaCl) and Postgate C in MIC
Naval Research Laboratory. Code 7303, Stennis Space Center. MS
39529.
experiments. The use of the term synthetic seawa-
ISSN 0010-9312 (print), 1938-159X (online)
CORROSION—Vol. 66, No. 1
015001-1
10/000001/$5.00+$0.50/0 ©2010, NACE International
CORROSION SCIENCE SECTION
ter is not always defined. Vargas-Avila and coworkers ered by suction pumps. Seawater was shipped to the
12
defined 0.6 M NaCl as synthetic seawater. In other Naval Research Laboratory (NRLSSC), Stennis Space
cases, the term synthetic seawater connotes commer- Center, Mississippi, in three 5-gal (18.93 L) plastic
cially available carbonate-buffered salt solutions that
containers, each containing 4 gal (15.14 L) of seawa-
mimic the composition of seawater.
ter. Both waters had been collected 48 h previous to
To further complicate interpretation and compari- arrival at NRLSSC and testing began within 24 h of
son of data, many laboratory experiments with SRB arrival.
rely on gas purging to remove oxygen and create an
Exposure Conditions
anaerobic environment in the laboratory solution of
choice. The pH of carbonate-buffered synthetic and Seawater samples from the two locations were
natural seawater is controlled (buffered) by carbon
maintained in the following conditions in the labora-
dioxide (C0 ). Gaseous carbon dioxide (C0 [gl) from tory:
2 2
the atmosphere dissolves into seawater forming aque-
—PG-A/KW-A: 450 mL stagnant seawater in a
ous carbon dioxide (C0 [aq]) (Equation [1]), reacts loosely covered glass container exposed to air.
2
with water, and forms carbonic acid (HjCOJ (Equation —PG-D/KW-D: 1,500 mL stagnant seawater
[2]). Proton dissociation from H C0 proceeds with maintained inside an anaerobic hood in a
2 3
interdependence on pH. The first proton dissociation 2,000-mL glass flask sealed with a rubber
forms bicarbonate (HCOj) (Equation [3]), with carbon- stopper.
ate (CO3") formation from subsequent proton dissocia-
—PG-B/KW-B: 1,500 mL stagnant seawater con-
tion (Equation [4]). tained in a 2,000-mL glass flask, bubbled with
house-supplied nitrogen gas, and sealed with a
rubber stopper.
C0 (g) ^ C0 (aq)
(1)
2 2
1
The atmosphere in the anaerobic chamber " was
maintained using a gas mixture of 10% H , 5% C0 ,
C0 (aq) + H O^H C0
(2)
2 2
2 2 2 3
and 85% N . Hydrogen was added to the anaerobic
2
gas mixture to aid in the removal of 0 . A palladium
+
H C0 H + HCO3
2
(3)
=F^
2 3
catalyst inside the anaerobic chamber served as an
0 and H reaction site resulting in the formation of
2 2
+
HCO3 ^ H + CO3"
(4)
water and increased humidity. Relative humidity was
maintained between 20% and 30% using an alumina
Increasing atmospheric [C0 (g)] increases dissolved
desiccant. Atmospheric 0 concentration was moni-
2
2
[C0 (aq)], which elevates [H C0 ] resulting in decreased
tored continuously at less than 1 part per million
2 2 3
seawater pH. If C0 (aq) is removed from seawater, pH
(ppm). House-supplied N gas specifications called for
2
2
increases. In some cases, researchers using a seawa-
less than 100 ppm atmospheric 0 ; however, during
2
ter medium and a gaseous nitrogen (N ) purge have
the duration of the experiment the average concentra-
2
14
indicated the need for pH control," whereas others
tion was -10 ppm 0 . Bubbling was not vigorous with
2
have not. Experiments described in this paper were
approximately 2 bubbles per second. No nutrients or
designed to evaluate microbial sulfide production in
other additions were made to either of the seawaters.
two natural coastal seawaters collected from Key
West, Florida, and the Persian Gulf deaerated under
Seawater Analysis
two laboratory conditions: purging with N and main-
At the onset of the experiment and continuing
2
tenance in a hood with a mixture of gases, including
at 30-day intervals, 50 mL of seawater was removed
N , hydrogen (HJ, and C0 .
from each exposure condition for analysis. The pH
2 2
was measured using a calibrated pH meter. Dissolved
MATERIALS AND METHODS
sulfide concentrations were determined in triplicate
15
using the methylene blue method 228-C. Four ster-
Seawater Collection
ile 3-mL syringes were used to remove 4 mL (1 mL
Persian Gulf (PG) seawater was collected from the
in each syringe) from the 50-mL seawater sample.
10
United States Navy pier at Mina Sulman, Bahrain.
One mL was used to inoculate serial dilutions (10 )
121
Key West (KW), Florida, seawater was collected at the
in each of the following seawater media: phenol red
Naval Research Laboratory's Marine Corrosion facility.
dextrose broth, Postgate medium B, nutrient broth,
PG seawater was collected within the first meter of the
and thioglycollate medium to determine most prob-
seawater surface by immersing each container under-
able numbers (MPN) of acid-producing bacteria (APB).
water by hand. KW seawater was collected at a depth
SRB, general heterotrophic aerobes, and anaerobes,
of 1.2 m to 1.5 m under water by intake pipes pow-
respectively. Dilutions were incubated for 28 days at
25°C. Salinity, total organic carbon, and sulfate were
Coy Laboratory Products. Grass Lake. Michigan.
also quantified at the onset of the experiment for both
Dixie Testing and Products. Inc.. Houston. Texas.
131
seawaters.
AccuLab Inc.. Marrero. Louisiana.
2010
015001-2
CORROSIONS ANUARY
CORROSION SCIENCE SECTION
C0 vs. pH
TABLE 1
2
141
Seawater Physical Chemistry
Gas mixtures were prepared with C0 % rang-
2
ing from 0.01% to 0.1%, 10% H , and balanced with
Total
2
N . Bottled laboratory-grade N with 0% C0 was also
Organic
2 2 2
Salinity
Carbon
Sulfate
used. KW seawater (500 mL) was added to a sealed
Seawaters (mg/L)
(g/L)
PH (mg/L)
1,000-mL flask with an electronic pH meter. Start-
ing with N and 0% C0 , gas was bubbled into sea-
7.82 38
Key West 1.79 3,864
2 2
44
Persian Gulf 7.98 1.94
4,696
water using a pressure regulator set to 1 psi. The pH
was allowed to stabilize over a 1-h period, pH was
recorded, and the gas mixture with the next highest
The pH values were monitored over the expo-
C0 % value was introduced.
2
sure period (Figure 1). There were no significant dif-
ferences in pH between the two coastal seawaters for
RESULTS
the same exposure condition. However, different expo-
sure conditions produced a range of pH values that
Seawater Analysis
varied by 3 pH units. Stagnant seawaters exposed to
Seawater chemistry analyses for both seawa-
air maintained a pH near 8.0 (PG-A/KW-A). Exposure
ters at the onset of the experiment are listed in Table
to the mixed gas anaerobic environment decreased
1. PG seawater had higher values for all parameters
pH values to below 7.0, with KW-D having the lowest
when compared to KW seawater. Table 2 lists the bac-
recorded value of 6.5. Bubbling N gas had the oppo-
2
terial counts for the four different bacterial groups
site effect, causing an increase in pH to above 9.0.
tested for both seawaters at the onset of the experi-
Dissolved sulfide concentrations were monitored
ment. KW seawater had equal or greater numbers
over time for all exposure conditions (Figure 2). Con-
of each group of bacteria when compared to PG
ditions exposing the sample to air produced dissolved
seawater.
sulfide levels below the resolution (<1 parts per bil-
lion [ppb]) of the detection method and are not shown
141
Nordan-Smith Specialty Gases. Hattiesburg. Mississippi.
TABLE 2
Bacterial Cell Counts at Onset of Experiment
Sulfate-
Acid-
Heterotrophic Producing Reducing
Heterotrophic
Aerobes Bacteria
Anaerobes Bacteria
(10*)
Seawaters (10*)
(10*) (10*)
Key West
Persian Gulf
10,000
Q-
s
c 1,000
o
c
CD
o
100
c
O
o
CD
"a
c/3
o
200
200
50 100 150
50 100 150
Exposure Time (day)
Exposure Time (day)
FIGURE 2. Log sulfide concentration in ppb as a function of exposure
FIGURE 1. pH values as a function of exposure time (days) for
time (days) for Key West seawater (KW-) and Persian Gulf seawater
Key West seawater (KW-) and Persian Gulf seawater (PG-) under
(PG-) under aerobic (A), nitrogen-bubbled (B), and anaerobic (D)
aerobic (A), nitrogen-bubbled (B), and anaerobic (D) conditions.
conditions.
vol. 66, 1
CORROSION— NO.
015001-3
CORROSION SCIENCE SECTION
a Aero
• Aero
PG-A
KW-A
• Anaero
• Anaero
• APB
• APB
• SRB
• SRB
in • i
n i i i i
184
91 125 157 184
35 63 99 134 166
Exposure Time (day) Exposure Time (day)
• Aero
PG-B
• Anaero
nAPB
• SRB
u
I
161 184
61 95 130
60 89 124
Exposure Time (day) Exposure Time (day)
167
61 100 133 184
69 94 129
Exposure Time (day)
Exposure Time (day)
FIGURE 3. Bacterial cell numbers (10*) as a function of exposure time (days) for Key West seawater (KW-) and Persian Gulf
seawater (PG-) under aerobic (A), nitrogen-bubbled (B), and anaerobic (D). Bacterial groups include general heterotrophic
aerobes (AERO) and anaerobes (ANAERO), acid-producing bacteria (APB), and sulfate-reducing bacteria (SRB).
in Figure 2. Anaerobic stagnant seawaters maintained are shown in Figure 3. Generally, KW seawater had
in a hood with mixed gases (KW-D/PG-D) produced higher bacterial numbers in all exposure conditions
1 3
when compared to PG seawater. Bubbling N gas
more than 10 ppb to 10 ppb dissolved sulfide. Sea-
2
waters maintained with bubbled nitrogen produced through the seawaters produced the lowest concen-
10' ppm dissolved sulfide during the course of the tration of bacteria in comparison to seawater left open
experiment, but dissolved sulfide was below detection to air and that tested in the anaerobic chamber. Bub-
limits at the conclusion of the experiment. bling N gas did not promote SRB growth.
2
pH values were recorded in KW seawater bub-
Planktonic bacterial populations were monitored
bled with mixed gases containing C0 ranging from
over time for all exposure conditions and the results
2
2010
CORROSION—JANUARY
015001-4
CORROSION SCIENCE SECTION
0 to 0.1%. Figure 4 shows pH values as a function of
C0 %. Increase in C0 % resulted in decreased pH val-
2 2
ues. The highest pH value (8.74) was recorded with
the 0% C0 , and the lowest (7.73) with the 0.1% C0
2 2
gas mixture.
DISCUSSION
It is generally recognized that seawater surrogates
do not approximate the complexity of natural seawa-
ter. However, many of the classic papers dealing with
0.04 0.06
0.1
SRB corrosion in the marine environment have been
C0 (%)
conducted in artificial media to which nutrients and
2
10 16
microorganisms have been added. One motivation
FIGURE 4. KW seawater pH as a function ofC0 % in bubbled mixed
2
for using synthetic seawater instead of natural sea-
gas containing 10% H and balance N .
2 2
water in MIC experiments is the goal of getting repro-
ducible results. The dilemma is whether to start with
a well-characterized controlled medium that may not
chemistry and microflora. It is the intention of the
replicate any natural environment or with a natural
authors to alert present and future researchers to the
environment that cannot be thoroughly characterized
possibility that deaeration using pure nitrogen may
and that varies with time. Few investigators have con-
influence microbial sulfide production. Currently, the
sidered the differences that can be induced in natural
authors use an anaerobic atmosphere consisting of
seawater and synthetic seawater maintained under
0.1% C0 , 10% H , and balance N , which maintains
2 2 2
different experimental conditions. Dexter concluded
seawater under anaerobic conditions with a pH of 7.8.
that changes in pH in natural and synthetic seawater
6
influenced the corrosivity of both toward aluminum.
CONCLUSIONS
In the experiments described in this paper, sul-
fide production in two natural coastal seawaters was
• Methods for deaerating natural coastal seawaters
evaluated under two laboratory conditions: purging
influenced the chemistry and microflora, particularly
with nitrogen and maintenance in an anaerobic hood
over time.
with an atmosphere of 10% H , 5% C0 , and 85%
• Laboratory experiments that created anaerobic
2 2
N . It was demonstrated that the microbial popula-
conditions by bubbling pure nitrogen gas into natural
2
tion that one is able to detect is determined by the
seawater produced pH changes that were not condu-
way the water is maintained. The microbial popula-
cive to the growth of SRB.
tions were followed using liquid culture techniques
• KW seawater consistently had equal or greater
that are recognized as capable of growing only a small
bacterial numbers in all conditions when compared
percentage of the natural populations. Liquid culture
with PG seawater.
techniques can be used to make comparisons, but not
• Despite the higher initial dissolved sulfate concen-
to make determinations about population diversity.
tration in PG water, higher DS concentrations were
In the experiments described in this paper, bubbling
measured in KW seawater.
N into natural seawater produced a pH shift from 8.0
• An anaerobic atmosphere consisting of 0.1% C0 ,
2
2
to above 9.0. Maintenance of seawater in an anaero-
10% H , and balance N can maintain seawater under
2 2
bic hood with an anaerobic mixture of gases produced
anaerobic conditions at a pH of 7.8.
a pH shift of 8.0 to below 7.0. Using Figure 4, a gas
mixture with 0.05% C0 will produce a seawater pH of
ACKNOWLEDGMENTS
2
8.0, matching the measured pH values in the aerobic
exposures (KW-A/PG-A) (Figure 1).
This work was supported by the Office of Naval
The reasons for higher SRB in KW seawater com-
Research Program element 0601153N (6.1 Research
pared to PG water have not been explored. While eval-
Program). NRL Publication number NRL/JA/7330-09-
uating the corrosion of aluminum, Dexter reported
9297.
results that were internally consistent within a sin-
gle batch of natural coastal seawater from a variety of
REFERENCES
locations, but were inconsistent from batch to batch
C.A.H. von Wolzogen Kuhr. L.S. van der Vlugt. Water 18. 16
6
at the same site and between sites.
(1934): p. 147-165.
The point of this short technical note is not to
S.C. Dexter, ed.. Biologically Induced Corrosion (Houston. TX:
NACE International. 1986).
question the conclusions of past research involving
B.J. Little. J.S. Lee. "Mierobiologieally Influenced Corrosion," in
SRB in natural or synthetic seawater media purged
KirkOthmer Encyclopedia of Chemical Technology (Hoboken. New
with nitrogen without regard to potential changes in
Jersey: John Wiley and Sons, Inc.. 2009). p. 1-38.
vol. 66, 1
015001-5
CORROSION— NO.
CORROSION SCIENCE SECTION
N. Pfennig. F. Widdel. H.G. Truper, The Dissimulatory Sulfate-
11. W.C. Lee. W.G. Characklis, "Corrosion of Mild Steel Under an
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Anaerobic Biofilm," CORROSION/90, paper no. 126 (Houston.
eds. M.P. Starr, M. Stolp, H.G. Truper, A. Balows, H.G. Schlegel TX: NACE, 1990).
12
(New York. NY: Springer-Verlag. 1981), p. 926-940. M.M. Vargas-Avila, R.K. Singh Raman. C. Panter. B.W. Cherry.
P. Farinha. H.A. Videla, "Corrosion Behavior of Duplex Stainless
J.R. Postgate. The Suljate Reducing Bacteria (New York, NY:
Steel in Marine Media Containing Sulfate Reducing Bacteria. A
Cambridge University Press, 1979).
S.C. Dexter, "Laboratory Solutions for Studying Corrosion of Alu- Laboratory Study," CORROSION/2009, paper no. 09388 (Hous-
ton, TX: NACE, 2009).
minum Alloys in Seawater," in The Use of Synthetic Environments
13
for Corrosion Testing, vol. STP 970. eds. P.E. Francis, T.S. Lee H.A. Videla, "Corrosion of Mild Steel Induced by Sulfate Reducing
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