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Sulfate Reducing Bacteria, SRB-BART™ Quality Control

Sulfate-reducing bacteria (SRB) are a group of anaerobic bacteria that, as a part of their normal activities, generate hydrogen sulfide (H2S). This product can cause a number of significant problems. These range from "rotten egg" odors, through to the blackening of equipment, waters and slime formations, and the initiation of corrosive processes.

Detection of these microorganisms is made more challenging because they are anaerobic and tend to grow deep within biofilms (slimes) as a part of a microbial community (consortium). Detection of the SRB is therefore made difficult because SRB may not be present in the free-flowing liquid over the site of the fouling but are growing deeper down in the biofilms. Because of this, the symptoms of SRB fouling may precede their detection using the SRB-BART unless a successful attempt is made to disrupt these biofilms and cause the SRB to come up into the liquid.

The sulfate-reducing bacteria are an unusual group in that they utilize hydrogen rather than oxygen as the basic driver for many of the metabolic activities. As a result of this, the SRB are anaerobic and are inhibited by the presence of oxygen. Sulfate reduction appears to be coupled to the formation of ATP (a major energy driver in metabolism) by a proton motive force (PMF) derived from electron transport. The bottom line is that the sulfate is reduced in a step-wise fashion to H2S while releasing energy for growth. It is the H2S which creates the problems through electrolytic corrosion, "rotten" egg smells, bad taste problems and the formation of black slimes.

There is another group of SRB which cause the reduction of sulfur to H2S but these are not detected using the SRB-BART. Usually, these sulfur-reducing bacteria are less common and, hence, have been discounted in the SRB-BART tester. Upon request, there is a tester for the sulfur reducing bacteria (SURB-BART™) which can be made to special order.

SRB activity in the BART tester is easily recognized since the sulfate becomes reduced to hydrogen sulfide. This product now reacts with the diffusing ferrous iron to form black iron sulfides. This sulfide commonly forms either in the base (as black precipitates) and/or around the ball (as an irregular black ring). In the latter event, the SRB may form a part of an aerobic consortium forming around and on the FID ball. Generally, where this happens, the blackening may be seen as granular structures held within the slime ring that is commonly not totally black.

The SRB-BART uses the short chain fatty acids to provide the substrates for the growth of the SRB. On some occasions, heterotrophic anaerobic bacteria can also become very active in the BART test and often grow faster than the SRB. When this happens, the liquid will tend to go cloudy. Usually, this is seen as a gel-like clouding most commonly in the bottom third of the BART inner test vial and shows that anaerobic heterotrophs are present and active. It should be remembered that these bacteria may not necessarily grow in the SLYM-BART™ since the major organic carbon nutrients are not short chain fatty acids.

Under exceptional circumstances, an SRB-BART may display a blackening very quickly (e.g., less than half an hour). In this case it is likely that the sample being tested contains some residual hydrogen sulfide which has rapidly reacted with the iron in the test vial. Where this happens, it is recommended that the water sample be aseptically aerated to drive off the gaseous hydrogen sulfide from the sample before conducting the SRB-BART test. While the aeration would admit oxygen to the sample, the SRB should survive through being protected by the other bacteria within the slime formations.

Reaction Patterns

BB - Blackened Base (Reaction 1)
BT - Blackening around Ball (Reaction 2)
Combination of Blackening in Base and Ball (Reaction 3)

There are three reaction patterns that are positive for the SRB. Detailed descriptions of these is given below:

BB - Blackened Base
The reaction is recognizable by the formation of a blackened deposit in the basal cone of the test vial. It may be first observed by looking up into the underside of the cone of the inner test vial. Blackening frequently starts as a 2 to 3-mm wide ring around the central peg and gradually spreads outwards. Black specking may also occur on the bottom 15 mm of the walls of the test vial immediately above the cone. The liquid medium should be clear (see reaction CG below) and there should be no slime ring around the ball.

BT - Blackening around the Ball
A slime ring may be viewed around the ball with patches of black specking or zones intertwined in the slime growths. The slime itself is not a characteristic of this reaction but the blackening is. The slime usually is either a white, grey, beige, or yellow color and tends to form on the upper side of the ball. The blackening often begins as a specking which gradually expands to patches within the slime.

Combination of BB and BT
Blackening occurs both in the base and around the ball although the length of the inner test vial may not be blackened.

The other recognized reaction is a negative for SRB but commonly occurs where there are aggressive anaerobic bacteria present. Often this reaction will precede a positive reaction for SRB (i.e., BB and BT). This negative SRB reaction is:

CG - Cloudy Gel-Like
While not a positive indication for the presence of SRB, this reaction is recognized since it does indicate the presence of anaerobic bacteria and often precedes the generation reactions BB or BT. It is recognized by the appearance of cloud-like structures in the colorless liquid medium. Usually these form from the bottom up and initially at a height of 20 to 25 mm up the side-wall of the inner test vial. This clouded zone may expand to render the liquid medium turbid. These clouds are relatively stable structures and have defined edges.

RPS (Reaction Pattern Signatures)

    • BB: Deep-seated anaerobic bacteria dominated by Desulfovibrio
    • BT: Dominant aerobic slime-forming heterotrophs include SRB in the consortium
    • BB - BT: Dominant anaerobic consortium including SRB with a fraction able to function aerobically as slime formers incorporating the SRB
    • BT - BB: Aerobic slime formers incorporate SRB and are also able to colonize anaerobic conditions

      Note that the SRB-BART includes another common test reaction, which does not relate to the presence or absence of the SRB in the sample under test. This test reaction is recognized by the development of a cloudy growth that often begins close to the base and gradually fills at least 20% of the liquid volume. Often this growth reaction appears almost gel-like and has a fuzzy but distinct edge. This may be admitted as a reaction:

      CG-Cloudy Gel-like

      This reaction does not mean that SRB are present but that anaerobic bacteria are. Commonly, the CG reaction precedes the blackening or occurs shortly after the commencement of the blackening.

      Time Lag (days of delay) to SRB-BART Populations
      The common relationship between the time lag measured in days of delay and the population of SRB is given in Table Six. Because the SRB commonly are aggressive as a part of a consortium of different species of bacteria, their numbers may be difficult to determine using some of the standard procedures for SRB. This methodology allows the growth of the consortium in the SRB-BART that consequently initiates greater levels of aggressivity. The populations given in Table Six reflect the higher recovery rates, and comparisons with other tests may show the SRB-BART to be the more sensitive.

      Table Six The Relationship between Time Lag and the Population For Sulfate-reducing Bacteria
      Time Lag (days) Population cfu/ml 1 6,800,000 2 700,000 3 100,000 4 18,000 5 5000 6 1200 7 500 8 200

      Sulfate-reducing bacteria (SRB) are a narrow group of bacteria that have the common facility to reduce sulfates to hydrogen sulfide. It is this sulfide which reacts with metals (commonly iron) to form the black sulfides. These black deposits cause an identifiable reaction in the base of the tube (BB) or around the ball (BT); the SRB do function as part of a consortium that is either anaerobic (BB) or aerobic (BT).

      Risk Potential Assessment
      The SRB are a relatively simple consortium in which the SRB tend to either dominate over a facultatively/strictly anaerobic heterotrophic bacterial flora (BB), or become integrated into an aerobic slime-forming heterotrophic bacterial community growing around the ball (BT). Where a more complex and aggressive form of SRB are present (involving both forms of consortial activity, the SRB are usually very aggressive and the BA reaction occurs without being preceded by either of the other two reactions. The risk potential for the severity of a detected SRB event can be expressed through the shortness of the time lag (in days) as follows:

      1. Very aggressive (treatment should be started as early as convenient)
      2. Aggressive (treatment should be considered in the near future before the condition degenerates further)
      3. Moderately Aggressive (treatment may not be required but vigilance through ongoing testing should be practiced)
      4. Normal Background Levels (routine testing is recommended)

      Table Seven Relationship Between the Time Lag to the First Reaction in an SRB-BART and the Aggressivity of the Sulfate-reducing Bacteria
      Aggressivity Very Sign. Moderate Not BB - Black Base <1 2-3 4-8 >8 BT - Black Ball <1 2-4 5-8 >8 BA* - Black All <2 2-5 6-8 >8

      Note that the BA reaction (*) listed above must have occurred without either of the other reactions occurring first. If either the BB or the BT reaction did occur first, then the aggressivity should be based on the first of the reactions that did occur. Some remedial treatments should be considered urgently where the time lag (in days) shows aggressivity to be at the 1 or 2 level.

      A non-SRB reaction can also commonly occur in this test when a cloudy gel (CG) forms. This is indicative of the presence of anaerobic bacteria (not SRB). However, on some occasions, these anaerobic bacteria can also become very aggressive and can cause deep-seated plugging. The aggressivity for these bacteria can be judged using the table below:

      Table Eight Relationship Between the Time Lag to the CG Reaction in an SRB-BART and the Aggressivity of the Anaerobic Bacteria
      Aggressivity Very Sign. Moderate Not CG - Cloudy Growth <0.5 0.5-2 3-4 >4

      SRB-BART™
      Bacteria that are able to reduce sulfate to sulfide are often associated with corrosion (H2S electrolytic), odor problems ("rotten" egg) and black water. This test uses a medium selective for the SRB and provides a source of iron so that the black iron sulfides are produced when the SRB are active. To improve the selectivity of this test, the liquid medium is manipulated to generate an anaerobic (reductive) state that restricts growth to a narrow spectrum of anaerobes.

      SRB-BART™ medium
      Beige crystalline deposits extend from the central peg to the walls of the test vial. The central 5mm of this deposit often appear darker extending outwards to lighter shades of grey. These deposits are very granular. A thin film of white and beige granular deposits extends irregularly up the sidewall of the test vial to a height of up to 6 mm and have an irregular edge. The characterization of the medium is given in Table Twenty-Four.

      Table Twenty-Four Medium Diffusion in a Sterile SRB-BART Inner Test Vial to Confirm a Negative Reaction
      Time (days) Medium characterization Basal Lower column Upper column 0.25 Yellow ppt Clear Clear 0.5 Yellow ppt Clear Clear 1.0 Beige ppt Clear Clear 2.0 - 10.0* Beige ppt Clear Clear

      *Note that liquid medium remains crystal clear when generated using sterile distilled or deionized water. Natural water samples can cause secondary chemical reactions that may be seen as an initial cloudiness that clears rapidly and/or commonly crystalline deposits forming in the base of the test vial. ppt stands for precipitate.

      Table Twenty-Five QC Characterization of Medium Diffusion in a Sterile SRB-BART™ Inner Test Vial
      Time (hrs) Color Contamination Basal Column* 0.5 Beige floc Beige floc Not determinable* 2.0 Beige floc Clear Not determinable* 12.0 Beige ppt Clear Cloudy zones 24.0-240 Beige ppt Clear Cloudy zones/black ppt

      *See note for Table-Twenty-Four.

      Due to the floc-like elements which form within the test vial once the water has been added, it is not possible to determine any contamination until at least twelve hours after the test has been set up. The beige floc forms as the water reacts with the medium pellet and the lipid oxygen barrier impregnated into the pellet. Where contamination occurs, growth is usually slow and tends to form cloudy structures above the basal cone. The presence of blackening in the QC test may reflect contamination by SRB.

      Confirmation of the Selective Media Composition in the SRB-BART
      In order to confirm the suitability of the selective medium for the biodetection of the various SRB recognized by this test method, it is recommended that the following A.T.C.C. (American Type Culture Collection) strains be applied to the SRB-BART biodetectors to determine the standard reaction patterns. Each culture should be prepared as a 48-hour broth culture incubated at 30oC to reach the stationary growth phase. Inoculation of the inner test vial should use a suspension of 0.1 ml of the broth culture in 15 ml of the sterile Ringer's solution. This inoculum should be taken from the midpoint of the Brain Heart Infusion broth culture immediately after the culture had been gently agitated. This inoculum is administered over the ball as the test vial is filled. Do not shake the vial. Incubate at 22 to 24oC for ten days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains cultured in Table Twenty-Six. Note that the Desulfurovibrio desulfuricans strain DSM1924 should be cultured on Sulfate Reducer API agar in accordance with the recommendations of the API (Recommended Practice for Biological Analysis of Subsurface Injection Waters, Volume 38, 2nd. edition, 1965).

      Table Twenty-Six Cultural Characterization of the SRB-BART
      A.T.C.C. Genus/species Characterization 13048 Enterobacter aerogenes CG Reaction 4 27853 Pseudomonas aeruginosa CG Reaction 4 13315 Proteus vulgaris CG Reaction 4 DSM1924 Desulfovibrio desulfuricans BD-BA Reaction 1&3 A + A Ps.aeruginosa + D.desulfuricans BU-BA Reaction 2&3

      Note that some of these cultural tests will shift from one reaction type to another as the growth in the SRB-BART matures.

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