Srb & Tcb Tests Evaluation ,

7/31/2019 Srb & Tcb Tests Evaluation ,

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EVALUATION OF SULFATEREDUCING BACTERIA(SRB) TESTS

PROVIDED BY: EMAD BEHDAD

LECTURER: AHMAD SAATCHI,Ph.D

BRANCH OF CORROSION & PROTECTION OF MATERIALS


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INTRODUCTION OF SRB

Sulfate reducing bacteria(SRB) is an anaerobic and sensitiveorganism.

SRB are among the most destructive environmental organisms, and

their industrial impact is widespread. They cause corrosion and stress corrosion cracking of metals and

alloys used in petroleum production and refining , cooling watersystems, waste treatment systems, pulp and paper production ,andin short all aqueous processes

SRB produce hydrogen sulfide and effect on oxygen depletion


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INDICATIONS OF SRB

A reddish or yellowish nodule on metal surface that when brokenexhibits black corrosion by-products

when the complete nodule is removed , a bright silver pit is found.

Corrosion by-products are non-magnetic .

Severe localized pitting is evident.

If hydrochloride acid is added to the black deposit, hydrogen sulfidewill be released giving off a rotten egg odor


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SRB-influenced corrosion of carbon

steel

Fig 15.2


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Metal

surface

Anaerobic cathodic reaction2H+ + 2e- 2H H2

Aerobic cathodic reaction

O2 + 2H2O + 4e-

4OH-

Anodic reactionFe0 Fe2+ + 2e-

Sulfate-reducing bacteria4H2 + SO42- 4H2O + S

2-Fe2+ + S2- FeS

Iron-oxidizing bacteriaFe2+ + O2 + 5H2O 2Fe(OH)3 + 4H

+

Iron corrosion


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SRB Causes Biofilms in SewersResulting in Clogging and CorrosionMIC Corrosion on Heating Oil Tank

Microbiological Induced Corrosion (MIC)

resulted in penetration of this Steel Heating Oil

Storage Tank within 4 years. Most commonly

the microbes are found to be Sulfate ReducingBacteria (SRB) which generate a highly acidic

environment within and under the colony.


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GROWTH OF SRB

SRB can thrive under deposits where no oxygen is present eventhough aerobic conditions exist in the main body of water.

Excessive amounts of dirt , algae , oils and other bacteria will

provide conditions for growth.

Small amounts of oils and grease will provide nutrients for growth.

Stagnant water and low flow conditions will increase the chanceof SRB growth.


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Sulfate reducing bacteria (SRB) operate

best at pH 6.6 to 6.8.


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TESTING METHODS

BROTHBOTTLES

AGARDEEPS

MELT

AGARTUBES

CULTURINGMETHOD

ATP ASSAY

CELLSURFACEANTIBODY

ASPREDUCTASEANTIBODY

DIRECTDETECTION


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CULTURING METHODS


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Drawing 1 mL of sample into a syringe and injecting it into a bottlecontaining 9 mL of liquid growth medium.

The bottle is then shaken thoroughly and a new syringe is used to draw

out 1mL of sample and inject it into a new medium bottle.

This procedure is repeated to produce as many serial dilutions asrequired.

Assumed only a single growing bacterium is required to blacken a bottle

If one bottle is blackened ,the sample contained at least one organism;

If two bottles are blackened the sample contained at least 10 organisms

This method requires the use if sensitive equipment ,namely

hypodermic needles and media bottles, which are difficult to transport

on airplanes and across state lines because of their association withdru abuse.


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AGAR DEEPS

Developed by Biosan Laboratories. The medium is a slightmodification of the RP-3m medium. However , sodium sulfide isincluded as a reducing agent/oxygen scavenger.

The medium is inoculated by dipping a pipe cleaner into the

undiluted sample and inserting it into a single vial of semisolidagar. Mineral oil and a CO2-generating tablet are then added toexclude air, and the vial is capped. It is incubated for up to fivedays and is checked daily for blackening.


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ADVANTAGES OF AGAR DEEP

The agar deep is the easiest of these methods to use.

No special or sensitive equipment is needed

It is easy to wipe surfaces with pipe cleaner and thus samplebiofilms for testing .

Dirty or high-solids samples can be handled easily with thismethod because samples do not have to be forced through a filteror a needle. On the other hand, darkening of the pipe cleaner byvery dirty samples can mask early blackening due to SRB sulfideproduction.

Results are determined in a maximum of five days .this allows theuser to act faster if SRB numbers start to increase.


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DISADVANTAGES OF AGAR DEEP

Five days is often insufficient time for SRB to become acclimatedto the medium and grow.

It may be inconvenient to read the test everyday over the five-day

incubation period because this period always includes at leastpart of a weekend.

The test may yield false positives because the medium containssulfite,which certain common non-SRB can reduce to sulfide.


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MELT AGAR TUBES

The test procedure involves placing the tubes in boiling water toliquify the medium and then cooling the tubes until the mediumreaches a temperature of 40 to 45 C before adding the sample.

These dilutions are pipetted into the bottoms of the tubes.

The tubes are then capped tightly and incubated for three days.

If a tube has general blackening and no visible colonies ,SRBwere considered too numerous to count.


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ADVANTAGES & DISADVANTAGES

+ No special or sensitive equipment is needed.

The procedure involves placing the tubes in cooling water tomelt the medium and then cooling them to 45 C, a process difficult

to control in the field. Results are difficult to interpret .the directions specify that

spherical colonies must be observed ,yet, In many cases, theentire tube turns black so that individual colonies cannot bediscerned.

This medium, as with the agar deeps, contains sulfite sogives a false positive.


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DIRECT METHODSATP ASSAY

The procedure involves filtering a sample to remove dissolvedsolids and salts that might interface with the test.

This filtered sample is then added to a reagent that releases thecell`s ATP.

A special enzyme that reacts with ATP to trigger a photochemicalreaction is added.

The resulting emitted light is measured by a photometer.

The number of bacteria cells is estimated from the total lightcounted.


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+ Results can be obtained in less than one hour. Because thismethod monitors the amount of ATP in the sample.

- the method is suitable for use only on relatively clean samples,.Sample preparation involves filtration to remove substances that nayinterfere with the test , and dirty, high solids samples are not suitablefor filtration.

- the equipment used in this method is fairly expensive . A typicalphotometer costs $5,000 to $10,000.


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EPIFLUORESCENCE CELL SURFACEANTIBODY (ECSA)

This method is based on specific antibody attachment to SRBcells. Antibodies , tagged with a fluorescent compound, attachonly to specific sites on the surface of SRB . When viewed underan epifluorescence microscope, an antibody-linked cell isidentified by a green fluorescent border.

The procedure involves heat-fixing the sample to a special slideand then incubation it for 20 minutes. With a primary antibodyreagent . After the sample is washed to remove excess antibody ,it is then incubated for 20 min.

Counting is done at 1000X magnification under oil immersion.


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+ result can be obtained in two or three hours.

+ the method is specific for SRB ,yet relatively nonstrain-specificamong SRB.

- the detection threshold for this method is 10 *(4) cells/mL.

- the test is sometimes difficult to interpret , especially with dirtysamples in which fluorescing debris obscures the bacteria . Thismethod requires a high level of training to obtain accurate results.

- this method uses an expensive epifluorescence microscope,which typically costs $10,000 to $25,000.


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APS REDUCTASE ANTIBODY

It involves antibodies developed against adenosine-5-phosphosulfate (APS) reductase, an internal enzyme found in allSRB. The sample is first washed to remove interfering chemicalssuch as hydrogen sulfide. It is then treated ultrasonically in asmall, battery-powered instrument that breaks open the bacteria

cells and release the APS reductase enzyme. The reminder of the test takes place within a polyethylene transfer

pipette. The sample is drawn into the pipette past a treatedporous bead.

It is washed four times, and then a color developing solution isdrawn through the bead.

If the APS reductase enzyme is present in the sample (indicatingthe presence of SRB), the bead turns blue within 10 min.


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+ Results are obtained in about 15 min.

+ Results of the test are easy to interpret. Color reactions indicatepositive tests; no color indicates a negative test.

+ The method is specific for SRB.

+No expensive or sensitive equipment is needed to performthe test.

+ This method is the only one that allows filed interpretation anddoes not involve use of a laboratory facility.

- The detection threshold for this method is 10*3 cells/mL.


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CONCLUSION

Around Sulfate-Reducing bacteria the culturing methods, ingeneral ,were able to detect lower numbers of SRB than the directmethods, although they are somewhat strain specific


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Srb & Tcb Tests Evaluation ,

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