Field Experience With an SRB Rapid Detection Test Kit
- G.L. Horacek (Conoco Inc.)
- Document ID
- Society of Petroleum Engineers
- SPE Drilling Engineering
- Publication Date
- December 1992
- Document Type
- Journal Paper
- 275 - 278
- 1992. Society of Petroleum Engineers
- 4.9.3 Pipeline Pigging, 4.9 Facilities Operations, 4.2 Pipelines, Flowlines and Risers, 6.5.2 Water use, produced water discharge and disposal, 4.2.3 Materials and Corrosion
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This paper reports field experience with a new rapid method to detect and count sulfate-reducing bacteria (SRB) accurately. Test results are available in 15 minutes, compared with the 3 to 4 weeks required with traditional culture methods. This new test method detects SRB that grow poorly or not at all on culture media and gives superior results with such solids samples as biofilms, soils, and sludge.
SRB are indigenous to the oil field and cause severe operational problems (i.e., corrosion, hydrocarbon souring, increased problems (i.e., corrosion, hydrocarbon souring, increased emulsions, increased suspended solids, and reservoir plugging) that increase costs significantly throughout the petroleum industry. 14 In addition, the presence of hydrogen sulfide produced by SRB leads to safety and environmental concerns. These problems can be prevented or alleviated by controlling SRB populations. Quick, prevented or alleviated by controlling SRB populations. Quick, accurate SRB population estimates can reduce operating costs significantly, improve oilfield safety, and decrease sulfide releases into the environment.
Historically, SRB have been detected and enumerated in oilfield waters by use of various culture techniques. While several culture techniques commonly are used, the API RP 386 method remains the most widely accepted procedure. SRB, however, constitute a diverse group of microorganisms often interrelated only by their ability to reduce sulfate to sulfide under anaerobic conditions. Isolating, purifying, and identifying many SRB strains are difficult tasks. Furthermore, practical experience indicates that only a fraction of the SRB in the typical oilfield environment win grow on culture media, and growth often is slow. For example, the culture medium specified by API RP 386 grows only lactate-using SRB and requires 28 days of incubation before results may be tabulated. Most SRB media use lactate as the primary carbon source. It is becoming widely recognized, however, that some common oilfield SRB strains do not grow on lactate. In addition, some SRB strains exhibit elaborate trace organic requirements that must be met before they will grow. As a result, an increasing variety of media and incubation conditions are being used to detect and cultivate SRB.
As media formulations become more complex, however, particularly as to potential sulfur sources, the risk of obtaining false results increases. This occurs because SRB culture media actually detect sulfide production and not SRB per se. Therefore, selecting a medium solely because it recovers higher populations of sulfide-producing bacteria does not mean that SRB are also recovered efficiently. As media formulas become more complex, biological communities or "consortia" can develop in the media. These consortia can produce sulfide from sulfite, thiosulfate, cysteine hydrochloride, and other sulfur-containing compounds that might be added to the medium. In fact, SRB may not be present even when a positive result is obtained in such media. The key point is, unless the sulfide is generated stoichiometry from sulfate (under anaerobic conditions), then bacteria other than SRB are involved. Therefore, a simple defined medium with sulfate as the only sulfur source is preferable to grow SRB. However, this leads to a problem: while simple defined media are preferable for proving that SRB are present, many SRB's will not grow on these media.
Clearly, rapid, accurate detection of SRB is highly desirable. It was discovered recently that all eubacterial SRB share a common enzyme, adenosine 5-phosphosulfate reductase (APS reductase), which catalyzes the reduction of adenosine 5-phosphosulfate to sulfite and adenosine monophosphate. APS reductase is required for respiratory sulfate reduction (i.e., dissimilatory sulfate reduction) to occur in the eubacteria. @ enzyme is present in all SRB studied, including thermophiles. APS reductase is not used in assimilatory sulfate reduction and thus is not present in non-SRB eubacteria. This enzyme is present in some colorless sulfur bacteria and photosynthetic present in some colorless sulfur bacteria and photosynthetic microorganisms but not in Significant quantities. It is not known whether the enzyme would be present in any archaebacterial SRB that might be discovered in the future.
With APS reductase as a marker, a highly selective and sensitive immunoassay method was developed and patented to detect SRB. Non-SRB bacteria, including sulfur and sulfite-oxidizing bacteria, do not cross-react with the immunoassay or otherwise impede accurate SRB determinations.
An SRB field test kit was developed from this immunoassay. Tatnall et al. reported laboratory comparisons of this rapid test kit with common culture methods. Horacek and Gawel also reported field tests of an early version of the field test kit. That testing showed that the presence of other bacteria, oil, chemicals, oxidized metals, and sulfide did not interfere with the performance of the test kit in oilfield waters. This paper presents field data obtained with the now commercially available version of the rapid test method.
Sample Collection and Testing. Water Samples. The SRB rapid was field evaluated within several geographically diverse company operating divisions. Water samples generally were collected from producing wells, battery tanks, and various points within water-injection producing wells, battery tanks, and various points within water-injection systems. Usually these samples contained nominal quantities of oil. Depending on their source, some samples also contain small amounts of such normal production chemicals as emulsion breakers, reverse breakers, and corrosion inhibitors. All fluids were tested in an unaltered state. Except where noted, all testing was done on site and consisted of duplicate, parallel SRB population enumerations by different test methods.
Initially, each sample was analyzed for SRB populations by a most probable number adaptation of the standard API dilution-to-extinction probable number adaptation of the standard API dilution-to-extinction test. In some cases, SRB media other than the API medium were used; these occasions are noted. Then each sample was analyzed by the SRB rapid test kit. Each test kit includes detailed instructions; additional information on the test kit is also available.
High-Solids Samples. Samples containing suspended solids often are difficult to analyze. Examples of such samples include biofilms, soil, sludge, and tank bottoms. When < 10 cm3 of sample was processed (usually the case), distilled water was added to bring the analyzed fluid volume to the normal 10 cm3. The test kit was run as usual on this diluted sample, but the SRB result reported by the test kit was adjusted accordingly. That is, if the test result was 1,000 SRB/g, the result was interpreted as being 10,000 SRB/g in the original sample. Instructions are packaged with the test kits. packaged with the test kits. Test-Kit Sensitivity and Scoring Considerations. Each SRB rapid test kit contains a color card to report SRB populations, calibrated with microscopically counted dilutions of a pure culture of Desulfovibrio desulfuricans. The nominal lower detection limit (i.e., least color visible) for the test kit is 1,000 SRB/cm3 of sample.
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