Detection of Microbial sulfate-reduction associated with buried stainless steel coupons Page: 6 of 12
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Soil Activity Serum Vial Experiments
Soil samples for measurement of microbial activity that were collected at the same time
as coupon recovery, were handled in the same manner as described above for steel coupons.
Samples were taken from areas central to the flat surface of and within 2 cm distant from
Sulfate-reduction activity in four soil samples was quantified by measuring production of
radioactive sulfide from 35SO4. One soil sample was tested from locations adjacent to the
types 301, 304, and 316 coupons used in the imaging experiments. A fourth sample was
tested that came from adjacent to a type 301 sensitized coupon which at the time of unearthing
was observed to be coated with "corrosion products" consisting of friable and discolored
material. For each of those 4 soil samples, 6 crimp-top serum vials were inoculated each with
2 g of soil plus 4 ml liquid medium formulated for culture of sulfate-reducing organisms (4).
Experiments were started by addition of 35S stock solution to give a final sulfate concentration
of 0.0125 mM and activity of 2 pCi. Microbial activity in vials was sequentially stopped, by
addition of concentrated base, after elapsed times of 0, 1, 2, 4, 8 and 18 hours. Sulfide
present from microbial sulfate-reduction was driven from solution by acidification, flushed from
headspace by a stream of ultra high purity (99.9995%) nitrogen, and captured by bubbling into
1 M NaOH. The radioactive sulfide containing NaOH was placed in scintillation cocktail (5) (2
ml per 10 ml) and quantified by liquid scintillation counting (6). Vials were counted for 30
minutes each. Quench standards used were low energy 14C, and sample disintegrations per
minute (DPM) were converted to 35S concentration by comparison to standards prepared from
the original isotope stock.
Coupon images and corresponding foil phosphor images are shown in Figures 2A-D.
Phosphor images of the foils from 3 of the 4 metal coupons showed localized very low rates of
sulfate-reduction. It appeared that only a tiny fraction of the 2 pCi of 35SO4 spread on these
plates was reduced to sulfide. Based on correlation of the phosphor images with the sites of
their incubation, activity on the coupons appears to have been associated with dark stained
streaks residual from organic material in the ground, and did not correspond to any signs of
incipient corrosion such as pitting. Faint darkening visible in the foil image from the fourth
coupon, the un-treated type 316 SS (Figure 2C), may have been an artifact of residual un-
Serum vial experiments also indicated very low sulfate-reduction activity (Table 3). Of
28 enrichments from 4 soils in the 35S rate experiment, 6 showed measurable activity. Of the
6, based on standards and correcting for decay (half life = 88 days), sulfate-reduction rate
ranged from 0.5 to 55 pmol/g sediment/day. Another 8 enrichments had activity around
experimental detection limit; these enrichments had sulfate-reduction activity less than 0.5
pmol/g/day. Enrichments had a seemingly random distribution of activity, with some series
having a positive response in earlier time points and none in later time points, the reverse of
what might be expected. There was no evidence of a time series with increasing response
over time. No correlation between soil activity and coupon metal type was noticeable. Positive
(4) C & S Laboratories Inc., Tulsa, OK
(5) Ultima Gold AF
(6) Perkin Elmer Tri-Carb 3170 TR/SL, Downers Grove, IL
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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/m1/6/: accessed November 16, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.