BART information

Denitrifying Bacteria, DN-BART™ Quality Control

DN is short for denitrification. This activity is extremely important not only in environmental but also in geochemical terms. The reason for this is that the essentially all of the atmospheric nitrogen (N₂) has been derived from the process of denitrification which is driven by the denitrifying bacteria. It is therefore an extremely important stage in the nitrogen cycle in the crust of planet Earth. There is a distinctive cycle in which nitrogen from the atmosphere is fixed, cycles through the biomass, is oxidized to nitrate by nitrification (see N-BART^TM) and reduced back to nitrogen gas by denitrification which is controlled by the denitrifying bacteria.

The denitrifying bacteria are therefore an important indicator group for the decomposition of waste organic nitrogenous materials. These denitrifiers reduce nitrate through to nitrite and some continue the nitrification on down to gaseous nitrogen (complete denitrification). In waters, the presence of an aggressive population of denitrifiers can be taken to indicate that there are significant amounts of nitrate in the water. Such waters are most likely anaerobic (free of oxygen) and relatively rich in organic matter. A common use for the presence of aggressive denitrifying bacteria in waters is that these bacteria signal the latter stages in the degradation of nitrogen-rich sewage and septic wastewater. Aggressive presence of denitrifiers in water can be used to indicate that there is a potential for the water to have been polluted by nitrogen-rich organics from such sources as compromised septic tanks, sewage systems, industrial and hazardous waste sites. It is recommended that, where a high aggressivity is determined, the water should be subjected to further evaluation as a hygiene risk through a subsequent determination for the presence of coliform bacteria. In soils, the presence of an aggressive denitrifying bacterial population may be taken to indicate that the denitrification part of the soil nitrogen cycle is functional.

Denitrification serves as the major route by which complex nitrogenous compounds are returned to the atmosphere as nitrogen gas. There are four steps in the denitrification process:

        (1)     (2)          (3)         (4)
   NO3-  »  NO2-    »    NO          N2     »     N2 GAS

Nitrate   Nitrite  Nitric oxide Nitrous oxide  Nitrogen

Denitrifying bacteria are not necessarily able to perform all four steps in the denitrification process and have been divided into four distinctive groups that can perform one or more of the various steps in the denitrification process. These are listed below:

	Group 1		-	step (1) only
	Group 2		-	steps (1), (2), and (3)
	Group 3		-	steps (2), (3), and (4)
	Group 4		-	steps (1) and (3) only
               

One of the largest groups of denitrifying bacteria are the enteric bacteria which includes the coliform bacteria. All of these bacteria perform denitrification under anaerobic (oxygen-free) conditions in a reductive environment.

Some of the principal genera associated with denitrification are:
Actinomyces, Aeromonas, Agrobacterium, Alcaligenes, Arthrobacter, Bacillus, Bacteroides, Campylobacter, Cellulomonas, Chromobacterium, Citrobacter, Clostridium, Enterobacter, Erwinia, Escherichia, Eubacterium, Flavobacterium, Geodermatophilus, Halobacterium, Halococcus, Hyphomicrobium, Klebsiella, Leptothrix, Micrococcus, Moraxella, Mycobacterium, Nocardia, Peptococcus, Photobacterium, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Shigella, Spirillum, Staphylococcus, Streptomyces, Thiobacillus, Vibrio.

As can be seen from the list, a very wide ranging number of bacteria are capable of denitrification. Their ability to perform denitrification is controlled, in part, by the availability of the nitrate, nitrite, nitrous or nitric oxide substrates.

The patented denitrifying bacterial activity reaction test biodetector (DN-BART) has been designed to detect the aggressivity of the denitrifying bacteria that will reduce the nitrite to gaseous nitrogen (steps 2, 3 and 4). These bacteria are an important part of the nitrogen cycle in soils and waters. In waters, their aggressivity may be used to signal the fact that there is a significant degradation of nitrogenous material occurring.

FO - Foam Formation (formerly reaction five, DN-BART)
Solution usually cloudy but the major positive for FO is the presence of very many bubbles collecting over >50% of the area under and around the ball to form a foam around the ball. This shows that complete denitrification has occurred and the denitrifying bacteria are present. Populations can be assessed by the time lag to the foam formation (Table Fifteen).

There is only one reaction recognized in the DN- BART that occurs when the nitrate is completely denitrified to nitrogen gas that collects as foam (interconnected gas bubbles) around the ball. This is more of a presence/absence test and the foaming usually is generated on the second test of testing at room temperature.

Table Fifteen

The Relationship Between Time Lag and the Population
For Denitrifying Bacteria

Time Lag (days)		Population cfu/ml
	1			1,000,000
	2			  200,000
	3			    50000
	4			    10000
               

The denitrifying bacteria tend either to be aggressive and cause a rapid denitrification, or to be relatively placid. This test now functions through the detection of the complete denitrifiers. These bacteria reduce the nitrate to dinitrogen gas that appears as a foam ring around the ball. Generally, if the test is still negative after a time lag of two days, the population can be considered to be very small and non-aggressive.

Population Assessment of DN using BART Extinction Dilution
To quantify the numbers of denitrifying bacteria in the sample, a dilution (extinction) technique would need to be used. To measure the population of denitrifying bacteria, four tenfold dilutions of the original water sample should be used. These dilutions can be achieved using the following technique:

1. Dispense 14 ml of sterile water into each of four DN-BART™ tests. Label these tubes: "1", "2", "3", and "4.".
2.Charge a DN-BART™ with the water sample (15 ml) and label "0."
3.Withdraw 1 ml of water from tube "0" and transfer into tube "1." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
4.Withdraw 1 ml of water from tube "1" and transfer into tube "2." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
5.Withdraw 1 ml of water from tube "2" and transfer into tube "3." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
6.Withdraw 1 ml of water from tube "3" and transfer into tube "4." Invert and gently shake tube for 10 seconds. Allow to settle (5 seconds).
7. Observe the tubes for FO (foam) after two days of incubation at room temperature.
8. Refer to Table Sixteen below to determine population.

Table Sixteen

Interpretation of the BART Extinction Dilution
For Denitrifying Bacteria

	Tube #			Population Assessment
	"0"		FO	FO	FO	FO	FO
	"1"		FO	FO	FO	FO	--
	"2"		FO	FO	FO	--	--
	"3"		FO	FO	--	--	--
	"4"		FO	--	--	--	--
			____	____	____	____	____
Possible Population:	>5.0	>4.0	>3.0	>2.0	>1.0
(log DN/ml)
               

Hygiene Risk
Denitrifying bacteria flourish in waters that have sources of nitrate and organics. Such sources may involve wastewater that contain some septic material and could therefore present a potential hygiene risk. A coliform test should be considered to assess this risk where there is a detected population of denitrifiers (FO observed). Where the DN population is >3.0 log DN/ml, a coliform test should routinely be used to determine the health risk.

DN-BART™
This test detects bacteria that can reduce nitrate (NO₃) to dinitrogen gas (N₂) by the observation of gassing which occurs when the nitrate as been completely denitrified. While nitrite is an intermediate in the denitrification of nitrates (with dinitrogen gas being the terminal product), it does not remain resident for a significant period of time particularly where nitrifying bacteria are active. Where there is an aggressive population of denitrifying bacteria present in the sample, the liquid sample will show a variable amount of cloudiness but there will be a generation of (dinitrogen) gas bubbles. These usually collect around the ball in the form of foam. This foam will last one to three days and usually not show any color. The presence of a foam or (less commonly) gas bubbles under and around the ball covering at least 50% of the submerged area) cloudiness with gas formation represents a positive detection of denitrifying bacteria.

DN-BART Medium
Opaque beige crystalline deposit extends out towards the walls of the test vial. It has a defined but irregular edge and appears to darken somewhat towards the central peg. Occasionally, crystalline deposits may also be seen at the wall conical base interface and may extend up the sidewall of the inner test vial up to 3mm. Table Thirty-Seven gives the characteristics that would be expected due to medium diffusion into sterile water.

Table Thirty-Seven

Medium Diffusion in a Sterile DN-BART 
Inner Test Vial to Confirm a Negative Reaction

Time (days)				Color			
		Basal		Lower column	Upper column
0.25		Pale yellow	Clear		Clear
0.5		Pale yellow	Clear		Clear
1.0		Pale yellow	Clear		Clear
2.0		Pale yellow	Clear		Clear  
               

Note that the liquid medium is crystal clear and has been generated using sterile distilled or deionized water. Natural water samples can cause minor chemical reactions that may be seen through an intensification of the color in the diffusion front and crystalline deposits may form in the base of the test vial. These crystalline deposits can be differentiated from a basal slime since the crystalline deposits swirl up and have a defined edge, do not have a gel-like appearance, and settle rapidly to the base after shaking. 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. Therefore do not use water taken directly from a refrigerated or cold source but allow the water to rise to room temperature before beginning the test to ensure any surplus saturation of the water with oxygen has vented.

Table Thirty-Eight 

Characterization of Medium Diffusion in a 
Sterile DN-BART Inner Test Vial

Time (day)		Color			Contamination      
		Basal		Mid-column
1.0		Pale yellow 	Clear		Cloudy, may be gassing
2.0		Pale yellow	Clear		Turbid, may be gassing
4.0		Pale yellow	Clear		Turbid, commonly gassing
               

The medium used in the DN-BART can encourage the growth of a range of facultatively anaerobic and nitrate respiring bacteria. If there has been contamination of the test vial, then this test will commonly exhibit a cloudy medium that gradually becomes more turbid with time. If any of the contaminants are complete denitrifiers, gassing may occur. Incubation for this test is normally at 22 to 24^oC but using blood heat (35 to 37^oC) can speed up the growth of many contaminants.

Confirmation of the Selective Media Composition in the DN-BART
In order to confirm the suitability of the selective medium for the biodetection of the various bacteria recognized by this test method (see text above), it is recommended that the following A.T.C.C. (American Type Culture Collection) strains be applied to the DN-BART to determine the standard reaction patterns. Each culture should be prepared as a 48 hour culture incubated at 35^oC to reach the stationary growth phase using Brain Heart Infusion broth. Inoculation of the inner test vial should be with 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 broth culture immediately after the culture had been gently agitated. This inoculated solution should be applied directly over the FID ball as the test vial is filled. Do not shake the vial. Incubate at 22 to 24^oC for one day and observe for activities and reactions after applying the reactant cap following the standard procedure. Typical results are listed below for the recommended A.T.C.C. strains in Table Thirty-Nine.

Table Thirty Nine Cultural Characterization of the DN-BART

A.T.C.C.	Genus/species			Solution          
13048		Enterobacter aerogenes		++, clouding,     
27853		Pseudomonas aeruginosa		++, slight clouding
12228		Staphylococcus epidermidis	-, clouding,      
19606		Acinetobacter calcoaceticus	++, no clouding,     
25922		Escherichia coli		++,clouding
               

* Gassing or Foaming (++) is considered the prime test for complete denitrification that can be recognized as a foam ring or intense bubbles under and around the ball. Clouding is not a confirmation of denitrification and should be considered negative.

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