BART information

Iron Related Bacteria, IRB-BART™ Quality Control

Iron is well known to be a critical substance for all life. In animals, it is a common part of the mechanisms for moving oxygen throughout the living body. Because iron plays such an important function in the energy metabolism, there is considerable biological competition for iron. Microorganisms also compete for iron and the use of various types of proteins called siderophores (e.g., hydroxamates and catechols). Additionally, many bacteria can also bind ferric (Fe⁺⁺⁺) ions into chelating structures know as ligands. This means that many bacteria are able to bind and hold iron in many forms to make large iron-rich structures that are sometimes seen as encrustations, tubercles and bog iron ore deposits. Little is known of the possible use of this iron to generate electro-motive forces (EMF) as a part of the growth of these iron-related bacteria. There is one group of bacteria, called the magnetotactic bacteria, which actually posses small magnet-like structures ( magnetosomes) and are able to sense magnetic fields.

So complex are these various biochemical systems for holding onto iron, the precise nature of these events remain only partially understood. However, there are many bacteria which can continue to accumulate iron to the point that the growth becomes almost saturated with oxidized iron and forms a hardening clog or encrustation. Such mineralizing growths may also incorporate carbonates and sulfides with a high iron content (going from 1% up to as high as 40% dry weight) and reducing organic content (declining to as low as <1% organic carbon). The formation of hardening clogs/encrustations can seriously impair the designed hydraulic characteristics of the infested region, causing degenerated water quality and production capacities.

In using the IRB-BART to examine waters for the presence of iron-related bacteria, it has to be remembered that iron bacteria grow predominantly on surfaces and not directly in the water. When testing water, the BART user has to assume that the IRB have detached, are suspended, and possibly are active in the water. As a consequence of this problem, there is a potential for an IRB-BART to give a "false" negative since the IRB are absent from the water but are present on the surfaces over which the water is flowing towards the sampling site. To get IRB to release and enter the flowing waters, it is necessary to cause a shift in the local environment that will make the conditions more hostile to the IRB. This is easily done by changing the pumping conditions (e.g., turn the pump off for a day if it is an active well) or applying a mild chemical shock using something like a low-dosage hypochlorite.

IRB infestations usually occur in the presence of oxygen and so may be more readily seen as slimes, clogs or encrustations. Over the century, these growths have had two common features: the presence of high concentration of ferric (Fe⁺⁺⁺) and of high populations of IRB (either as stalked Gallionella, the sheathed IRB or the heterotrophic IRB). The seriousness of these growths in engineered structures has led to the use of the term "Iron Bacteria." Recent research has shown that these bacteria are able to shunt the iron through oxidative and reductive states through ferric (Fe⁺⁺⁺) and ferrous (Fe⁺⁺) forms respectively. The ^BART biodetector is designed for the detection of these bacteria and is able to perform both the oxidative and reductive based reactions involving iron. This comprehensive group is known as the "Iron-related Bacteria" (IRB).

The medium selected for the culture of the IRB is based on an original formulation developed by Sergei Winogradsky in which the major form of iron is presented as ferric ammonium citrate. The IRB-BART thus provides the major carbon (citrate), nitrogen (ammonium) and iron (ferric) from the same complex chemical form. When the crystallized pellet in the base of the test vial begins to dissolve after the sample has been added, a complex series of reactions occur. These reactions are influenced by both the chemical and biological composition of the sample and the redox and nutrient gradients created in the BART test. Under sterile conditions, a sample may be expected to cause a gradual dissolving of the nutrients from the pellet with the formation of a colored transparent diffusion front which gradually ascends through the fluid column until all of the liquid medium has a similar color. Where there has not been any major chemical reaction and the sample contains some oxygen (oxidative), the resultant color can generate yellow. If the sample is reductive (devoid of oxygen) and contains a relatively high calcium-magnesium concentration, the diffusion front may become a transparent green color.

RPS (reaction pattern signatures) revolve around a complex pattern of signals which are generated when the IRB in the water sample begin to utilize the nutrients and manipulate the ferric form of iron present in the base of the inner BART test vial. Common events range from:

- gas formation (common where anaerobic conditions exist),
- clouding (commonly at the REDOX (reduction-oxidation) front),
- slime formations (commonly starting at the base or around the FID ball in the test vial),
- color changes (which can pass through various shades of yellow, red, brown, to black, or through shades of green).

Careful QC is employed during manufacturing to ensure that the ferric ammonium citrate yields a consistent reproducible response to the various test cultures.

Iron-related bacteria (IRB) are difficult to enumerate since they are subdivided into a number of groupings (e.g., iron oxidizing and iron-reducing bacteria). These bacteria function under different Redox conditions and utilize a variety of substrates for growth. By the routine (e.g., monthly) testing of water or wastewater using this technique, the levels of ggressivity, possible population and community structure (RPS) can all be determined. The status of an iron-related bacterial population within a given sample can be determined and related to any biofouling in the surrounding environment.

To conduct the test, it is necessary to add 15ml of the sample to the biological activity reaction test biodetector. The ball floats up and restricts the entry of oxygen into the liquid medium. At the same time, components in the modified Winogradsky selective culture medium for IRB begin to diffuse upwards into the sample from a dried medium pellet in the base of the biodetector. Two gradients form within the fluid column: nutrients diffusing upwards, and oxygen diffusing downwards. These gradients form a variety of different habitats in which IRB can flourish. The color displayed by microbial activity may be a result of the form into which the ferric iron becomes modified in the medium.

It should be noted that, in a biologically active BART tester, the ferric form of the iron added with the selective Winogradsky medium will revert to the ferrous form along the reductive (lower) part of the redox gradient. Commonly, where there is a radical reduction of the ferric form to the ferrous during the early phase of an IRB-BART test, the color of the diffusing medium in the bottom of the BART tester may shift from a yellow to a green. This should be considered negative unless this "greening" at the base of the inner test vial is accompanied with clouding.

Reaction Patterns

There is a range of reactions that can occur in the IRB-BART, all of which can be observed. It is recommended that the BART tester be held up to a diffuse light to confirm some of these reactions which may be difficult to see against a dark background. (Note that traditional numbered reactions are shown in parentheses and some reactions have been discounted as unreliable):

	BC	Brown Cloudy		(Reaction 4)
	BG	Brown Gel		(Reaction 3)
	BL	Blackened Liquid	(Reaction 10)
	BR	Brown Ring		(Reaction 4)
	CL	Cloudy Growth		(Reaction 2)
	FO	Foam			(Reaction 5)
	GC	Green Cloudy		(Reaction 8 & 9)
	RC	Red Cloudy		(Reaction 7)
               

Each of the reactions has been produced in a unique manner by the various species and consortia of bacteria becoming active in the test. There is, therefore, no specific form of any reaction pattern because these are controlled by the form of bacterial growths. Below is listed the descriptions for each of the IRB-BART test reactions.

CL - Clouded Growth
When there are populations of aerobic bacteria, the initial growth may be at the redox front that commonly forms above the medium diffusion front. This growth usually takes the form of lateral or "puffy" clouding which is most often grey in color. It should be noted that if the observer tips the BART slightly, the clouds will move to maintain position within the tube. Commonly, the medium will be darker beneath the zone of clouding and lighter above.

BG - Brown Gel
In this reaction, a basal, gel-like brown growth forms that maintains structure and position even when gently rotated or tilted. This brown gel can occupy the whole of the basal cone of the inner test vial and also extend up the sidewall of the inner test vial to a height of <15 mm. The solution above the gel is commonly clear and colorless. Over time it is often noticed that the size of the gel mass will grow and later shrink. Detachment sometimes happens so that a single brown gel-like mass can be seen floating in the test vial.

BC - Brown Cloudy
Unless there is a very large population of IRB in the sample, this reaction is normally a secondary reaction (often following reactions CL, FO, or RC) and may be recognized as a dirty brown solution that may have a brown ring around the ball.

FO - Foam
This is a very easy reaction to recognize since gas bubbles around the ball form a foam ring or sometimes the bubbles collect over greater than 50% of the underside of the ball. On some occasions, bubbles will collect on the walls of the inner test vial but this is not significant until the bubbles collect around the ball. The solution usually remains clear but commonly has a yellow or greenish-yellow color. The bubbles can sometimes be seen in the foam to be individually coated with slime that may give the bubbles a color ranging from brown through to orange, yellow or grey. Sometimes when integrated together into a foam, this foam is tough enough to either "lift" the FID out of the liquid solution or submerge the FID below the surface of the liquid solution.

Do not confuse this reaction with the generation of bubbles (usually randomly) when oxygen supersaturates as the sample temperature comes up from a lower temperature (of the sample's source). These bubbles are recognized as being reflective and not bound in any slime and dispersed within the inner test vial under the ball and on the walls. They usually disappear within two days.

This FO reaction is most commonly related to a sample in which many microbes are functioning anaerobically. It can often be "harmonized" with the presence of SRB (reactions BB, BT or BA). In other words, the occurrence of a FO in the IRB-BART can often be followed by a positive detection of SRB in the SRB-BART if that test has been performed on the same sample.

RC - Red, Slightly Clouded
The liquid medium remains a clear to a dark reddish solution. The solution will cloud fairly quickly and shift to a BC reaction generally after a BR has formed around the ball.

BR - Brown Ring
A reddish- brown to dark brown slime ring forms around the ball. This ring is entire and tight and usually <3 mm in width. Generally, the brown slime ring will sit between the liquid surface and the equator of the ball and commonly intensifies over time. On some occasions this reaction possesses an unusual feature in that the slime ring can "bio-lock" the ball to the walls of the test vial. In these cases, when the test vial is turned upside down, the ball remains (glued) in-place and the liquid remains above the ball. What has happened is that the ring has become formed biologically into an hydraulic barrier.

GC - Green Clouded
Solution goes to a shade of green and becomes cloudy without necessarily, the formation of defined clouds or gel-like forms. No slime ring is formed around the FID. This cloudiness will gradually increase and often this reaction will shift to a dark green very cloudy solution. As the solution becomes a darker green and cloudier, a BR reaction may form but this is usually fairly thin.

BL - Blackened Liquid
This is commonly a secondary or tertiary reaction rather than an initial reaction. It is recognized as a clear, often colorless, solution surrounded by large blackened zones in the basal cone and up the walls of the inner test vial.

Other reactions not coded are described below. These reactions occur less than 1% of the time in water testing using the IRB-BART.

If "fuzzy" growths form around the ball in the IRB-BART, then it is probable that the a water sample had traveled through a semi-saturated zone where there was fungal activity. These create reaction 13 in which a white, grey or speckled "fuzzy" mat forms around and even over the ball. The upper surface of the mat often forms into a tight mass with an irregular surface. The lower surface of the mat can often be seen to be extending into the liquid medium by thread-like processes 2 to 5 mm in length. These growths may bio-lock the ball to the wall of the inner test vial for a period of time. Solution usually remains fairly clear but globular-like deposits may be present. Solution may cloud over time. This reaction is caused by the presence of large populations of fungal spores in the water.

RPS (Reaction Pattern Signatures)

Because of the complex communities that form the iron bacteria, the reaction patterns can develop some very distinctive sequences. In the last ten years, the meaning of the sequences (RPS) has been determined. The common characterizations are listed below:

• BC - WB - BR
  IRB with carbonate deposition and some slime formers present
  
• CL - GC
  Mixed heterotrophic IRB dominated by Pseudomonads
  
• CL - BG
  Mixed heterotrophic IRB with some Enteric bacteria (possibly Enterobacter)
  
• CL - BC
  Mixed heterotrophic IRB
  
• CL - BC - BR
  Mixed heterotrophic IRB with some slime formers
  
• CL - FO
  IRB with mixed aerobes and some anaerobic activity
  
• CL - BC
  Where a white deposit forms in the vial. Aerobic IRB with carbonate deposition
  
• FO - CL
  Anaerobic bacteria with some aerobic heterotrophic IRB
  
• FO - CL - RC
  Anaerobic bacteria with some aerobic heterotrophic IRB and Enteric bacteria (possibly Enterobacter, Citrobacter or Serratia)
  
• FO - CL - BC - BR
  Mixed anaerobic and Enteric bacteria with some slime-forming IRB
  
• FO - BR - BC
  Mixed anaerobic and IRB with some aerobic slime-forming bacteria
  
• FO GC
  Mixed anaerobic and aerobic bacteria dominated by Pseudomonads
  
• FO - GC - BL
  Mixed anaerobes, Pseudomonads and Enteric bacteria
  
• GC
  Most of the bacteria present are Pseudomonads
  
• GC - BL
  Pseudomonads dominate with some IRB and Enteric bacteria present
  
• RC - CL - BR
  Enteric bacteria dominate
  

  The IRB are generally slow growing and often will display the first reaction as either a foam (FO) or a cloudy plate (CP). The consortium is complex and involves a mixture of stalked and sheathed bacteria along with heterotrophic and slime-forming bacteria. Because of the complex nature of this consortium, it takes longer to become established and is more likely to show a succession of secondary reactions as the consortium stabilizes.

  Time Lag (days of delay) to IRB-BART Populations
  
  The populations of IRB can be determined using the time lag to the observation of first reaction. This relationship is shown in Table Four.

  
  Table Four
  
  The Relationship Between Time Lag and the Population
  For Iron-related Bacteria
  
  Time Lag (days)		Population cfu/ml
  	1		  540,000
  	2		  140,000
  	3		   35,000
  	4		     9000
  	5		     2300
  	6		      500
  	7		      150
  	8		       25
  

  
  

  Risk Potential Assessment
  
  The IRB are a complex of many bacteria that possess a common ability to utilize iron. As a result this test has a complex set of reactions which can be displayed. The shorter the time lag to the IRB displaying a reaction, the greater the aggressivity and the need to treat. Not all reactions are equally important in determining the aggressivity of the IRB (and therefore the need to treat). Below is a list of the reactions described previously and the relative importance in relation to the need to treat. Concern can be expressed through the shortness of the time lag (in days) as:

  1-2. Very aggressive (treatment should be started as early as convenient)
  2-4. Aggressive (treatment should be considered in the near future before the condition degenerates further)
  5-8. Moderately Aggressive (treatment may not be required but vigilance through ongoing testing should be practiced)
  >8. Normal Background Levels (routine testing is recommended)
  

  Table Five Relationship Between the Time Lag to the First Reaction in an 
                       IRB-BART and the Aggressivity of the Iron-related Bacteria
                           
  				     Aggressivity (significance)
  				Very	Sign.	Moderate	Not
  BC	Brown Cloudy		<2	3	4-8		>8
  BG	Brown Gel		<1	2-6	7-8		>8
  BL	Blackened Liquid	<2	3-6	7-8		>8
  BR	Brown Ring		<1	2	3-6		>6
  CL	Cloudy Growth		<0.5	0.5-2	3-4		>4
  FO	Foam			<0.5	0.5-1	2-4		>4
  GC	Green Cloudy		<1	2-4	5-8		>8
  RC	Red Cloudy		<1	2-3	4-8		>8
  

  Some remedial treatments should be considered urgently where the time lag (in days) shows aggressivity to be at the 1 or 2 level. Where there has been a RPS (sequence of reactions to form a signature), then the aggressivity should be considered to be equivalent to the most aggressive of the reactions using the above table.

  Hygiene Risk
  
  Four of the possible reactions can indicate a potential hygiene risk. These include:

  BG, BL, GC and RC.
  

  
  Where these are found to have a time lag that would project an aggressivity of 1 (very aggressive) or 2 (aggressive), then a fecal coliform test should be performed to ensure that there were no fecal coliform bacteria present. Note that the use of the total coliform test could yield a positive since some of the bacteria causing these reactions could be environmental enterics. If the RPS includes GC, a test for the presence of fluorescing pseudomonads should also be performed.

  IRB-BART™
  
  The iron-related bacteria (IRB) are recognized as being those bacteria that are iron utilizing. The form of this utilization can range from simply a passive bioaccumulation in the slime (biofilm) growths through to active use in metabolism. This test specifically uses the ability of bacteria to use ferric and ferrous iron in many ways to create various reactions, some of which are colored. It is now understood that some of the IRB may be able to derive respiratory or energy functions out of the reduction (ferrous) to oxidation (ferric) manipulations which occur through the activity of the IRB.

  IRB-BART Medium
  A brownish green opaque crystal-like hardened mass extends outwards from the central peg towards the walls of the test vial. The center of the mass appears darker than the perimeter. The outer edge of the medium pellet is sharply defined and appears to be yellow and contain brown fibers extending out from the central deposit. A transparent film may extend up the walls for 1 to 2 mm and have a yellow color. When charged, the liquid medium remains clear and generates to a greenish yellow color. However, there are sometimes reactions between the chemicals present in a water sample that will change the color generated usually through to a yellow or a green. The green color appears to be darkened in and near the basal cone region of the test vial and will diffuse to a lighter green or yellowish green. These green colors appear to be associated with higher calcium and/or magnesium levels in the water sample. If there is microbial activity in the test, this will be displayed by color shifts, gassing, cloudiness or the formation of slime. These events reflect a reaction involving IRB.

  
  Table Twenty-One
  
  Medium Diffusion in a Sterile IRB-BART
  Inner Test Vial to Confirm a Negative Reaction
  
  				Color                                              
  Time (days)	Basal		Lower column	Upper column
  
  0.25		Green-yellow	Clear		Clear
  0.5		Green-yellow	Clear		Clear
  1.0		Greenish-yellow	Greenish-Yellow	Clear
  2.0 - 10.0*	Greenish-yellow	Greenish-yellow	Green-yellow
  

  *Note that these colors are crystal clear and have been generated using sterilized distilled or deionized water. Natural water samples can cause secondary chemical reactions that may be seen through an intensification of the green color in the diffusion front and crystalline deposits forming in the base of the test vial. Water saturated with oxygen stored at low temperatures can, when used in this test, cause bubbles to form as oxygen comes out of solution as the temperature rises to room temperature.

  
  Table Twenty-Two
  
  QC Characterization of Medium Diffusion in a 
  Sterile IRB-BART Inner Test Vial
  					Color                                   
  Time (hr.)		Basal		Column*		Contamination***
  0.5**		Diffusion begins	Clear		Clear
  2.0		Green-yellow		Clear		Clear
  24		Greenish-yellow		Clear		Clouding or gassing
  48 - 240***	Greenish-yellow		Green-yellow	Cloudy or Slime
  

  *The column reflects the whole length of the water column and should stay crystal clear even as the diffusion front created by the dissolving medium passes through.
  **Incubation is commonly at a (room) temperature of 22 to 24^oC.
  ***An acceptable test should remain crystal clear, have no gassing or slime deposits. Generally, evidence of microbial contamination is not immediately observable but may appear within 24 hours commonly as either a clouding just above the diffusion front, or as an excessive gassing. These contaminants will cause turbidity, color shifts and may involve slime formations by 240 hours. Reference to the BART reaction chart may aid in the determination of the causative microbes.

  Confirmation of the Selective Media Composition in the IRB-BART
  
  In order to confirm the suitability of the selective medium for the biodetection of the various iron-related bacteria recognized by this test method, it is recommended that the following A.T.C.C. (American Type Culture Collection) strains be applied to the biodetectors to determine the standard reaction patterns (Table Twenty). Each culture should be prepared as a 48 hour broth culture incubated at 30^oC to reach the stationary growth phase. Inoculation of the inner test vial should use a cell 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 broth culture immediately after the culture had been gently agitated. This inoculum should be applied directly over the ball as the test vial is filled. Do not shake the inoculated inner vial. Incubate at 22 to 24^oC for seven days and observe for activities and reactions. Typical results are listed below for the recommended A.T.C.C. strains in Table Twenty-Three.

  Table Twenty-Three Cultural Characterization of the IRB-BART
  
  A.T.C.C.	Genus/species			Characterization
   8090		Citrobacter freundii		GC	Reaction 6
  13048		Enterobacter aerogenes		BR	Reaction 4
  27853		Pseudomonas aeruginosa		GC	Reaction 8&9
  19606		Acinetobacter calcoaceticus	GC	Reaction 8
  23355		Enterobacter cloacae		CL-BG	Reaction 2&3 
  13315		Proteus vulgaris		CL-BC	Reaction 2&4 
  13883		Klebsiella pneumoniae		RC-BC	Reaction 7&4 
  25922		Escherichia coli		FO	Reaction 5
  

  Note: Some of these culture tests will shift from one reaction type to another as the growth in the IRB-BART matures. For example, Citrobacter freundii may cause after 5 to 8 days a bio locking of the ball so that when the test vial is turned upside down, the ball remains "glued" into position with the liquid medium held above the ball. The first reaction normally precedes the second reaction. The test using E.coli is performed at 35^oC.

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