Detection of Microbial sulfate-reduction associated with buried stainless steel coupons Page: 5 of 12
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fastened securely in place. Coupons were maintained under humid anaerobic (N2
atmosphere) conditions in a sealed plastic container and incubated in the dark at 200 C for 2
Positive and Negative Control Experiments
A separate set of imaging experiments were conducted as positive controls and to
provide baseline data for estimating the rate at which steel coupons may be colonized by
sulfate-reducing microorganisms. In these experiments sets of coupons recently obtained
from a metals supplier were prepared at INL according to the procedures described by NBS for
preparation of the original buried materials. They were then immersed and incubated in an
active sulfate-reducing bacterial culture. Three sets of coupons for three time points of
observation were immersed in the culture. The sets consisted of the same metal types as the
original 4 types used in the main treatment of this study with the exception that no type 316
stainless steel could be found, so type 316L coupons were used instead. Each set consisted
of types 301, 304, 316L, and 316L sensitized coupons. In addition, 3 mild steel coupons were
included to provide a metal that rapidly reacts with products of microbial sulfate-reduction.
Coupons to be used in the control experiments were washed in acetone, ethanol, and
sterilized de-ionized water before immersion in the sulfate-reducing microbial culture. The
bacterial strain used for the positive control sulfate-reducing microorganism was Desulfovibrio
thiooxidans from INL laboratory stock; growth medium consisted of a dilute minimal salts
solution that was developed in this laboratory to be used in studies of microbial interactions
with buried steel waste canisters. Salt concentration in the medium was below drinking water
standards; lactate and sulfate were added as energy source and electron accepter respectively
Incubation of coupons in the sulfate-reducing culture was conducted at room
temperature. Evidence of bacterial sulfate-reduction was indicated by production of black
metal sulfides visible in the culture vessel (Figures 1A and 1 B).
Negative control experiments consisted of steam sterilized foils and coupons that were
coated as described above with sterile lactate and labeled sulfate solutions and incubated for 2
Silver Foil Imaging
Images were created using a phosphorescence imaging system (3). After incubation,
foils were detached from coupons, thoroughly rinsed with sterile de-ionized water to remove
residual radioactive sulfate, and sealed in sterile plastic bags. The foil containing bags were
exposed face down on the phosphor imager plate. Foils were placed on and removed from the
plate surface by use of the sample exposure platform that allows for transfer without light
exposure of the phosphor coated surface. Exposure times were standardized to 24 hours after
comparing image densities from different exposure durations. Digital images of foils from each
coupon were created on the system imaging platform (Figures 4 A-D).
(3) Molecular Imaging System (Bio-Rad, Hercules, CA), including a Sample Exposure Platform and Model GS-525
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Delwiche, Mark E.; Flitton, M. Kay Adler & Olson, Alicia. Detection of Microbial sulfate-reduction associated with buried stainless steel coupons, article, March 1, 2007; [Idaho Falls, Idaho]. (digital.library.unt.edu/ark:/67531/metadc888535/m1/5/: accessed February 20, 2019), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.