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Aerobic biotransformation

Normally, cyanuric acid degrades very slowly under aerobic conditions since the majority of aerobic microorganisms do not possess the genes to produce the specific enzymes required to degrade cyanuric acid. As a result, cyanuric acid shows minimal biodegradation in standard screening tests conducted at atmospheric oxygen levels and with no acclimation.[1]

 

However, cyanuric acid can be degraded much more rapidly if: 1) specific fungal or bacterial strains are present which contain the genes/enzymes required, 2) the microorganisms have been acclimated to cyanuric acid, and 3) organic nutrients are present for the microorganisms. Furthermore, since bacteria degrade cyanuric acid in order to obtain ammonia for use in synthesis of biomass, the biodegradation of cyanuric acid is normally more pronounced if the ambient conditions are nitrogen-limited, but will be less likely to occur if more readily degraded sources of ammonia-nitrogen are present.

 

In the early 1980’s, Zeyer, et al. [2] screened 160 strains of microorganisms isolated from the wastewater of ans-triazine production plant and from soils of fields treated for years with s-triazine herbicides. Only one strain, identified as the fungus Sporothrix schenckii, gave significant degradation of cyanuric acid under aerobic conditions. Using radiolabelled cyanuric acid, they found rapid degradation to CO2and ammonia without accumulation of intermediates and that all of the liberated ammonia was incorporated into biomass. Acclimating the strain to cyanuric acid increased the degradation rate.

 

The soil fungi Stachybotrys chartarum [3],Hendersonula toruloidea [3], Penicillium sp.[4] and Hormodendrum masonii [4] have also been reported to degrade cyanuric acid. Testing at Monsanto Co. confirmed degradation by Hormodendrum masonii, but not by Penicillium, evidently since different strains were used. Myskow, et al. [5] reported degradation of cyanuric acid by fungi belonging to the genera Aspergillus, Penicillium, Fusarium and Pseudogymnoascus. 15 -N-labelling experiments showed that most of the nitrogen from the degraded cyanuric acid was incorporated into fungal biomass (mycelium and proteins). The literature on cyanuric acid degradation by fungi suggests a widespread natural occurrence of cyanuric acid catabolism in fungi. Unfortunately, little work on degradation by fungi or yeast has been published since 1987.

 

Various workers have reported that several types of aerobic bacteria, including Bacillus sp [5], Pseudomonas sp. [6,7], and Achromobacter sp. [6]can degrade cyanuric acid. In all cases, the bacteria grew under aerobic conditions with cyanuric acid as the only source of nitrogen, so growth involved utilization of the cyanuric acid as the nitrogen source. Cyanuric acid was rapidly and completely converted to CO2 and ammonia.

 

While Saldick [1] indicated that aerobic systems are unsatisfactory for biodegradation of cyanuric acid, he noted that biodegradation is possible in typical aerated activated sludge treatment systems. These commonly operate at low (1-3 µg/ml) dissolved oxygen levels, giving rise to local anaerobic conditions in the biomass.

 

Sisodia, et al. [8] reported that cyanuric acid is readily degraded in aerated activated sludge under nitrogen-limited conditions. Cyanuric acid is not an energy source for the bacteria, since the degradation is a hydrolysis, so a separate source of organic nutrient must be present for the bacteria to grow. As in previous reports, they found that cyanuric acid is converted into CO2 and ammonia. The ammonia is then incorporated into the biomass or nitrified.

 

Thus, cyanuric acid can be readily degraded aerobically under nitrogen-limited conditions, but only by a limited number of microorganisms and only if the microorganisms are suitably acclimated to cyanuric acid and another source of organic carbon is present. Biodegradation occurs through the use of cyanuric acid as a nitrogen source for the bacteria or fungi.

 

As shown in the equations below, cyanuric acid is degraded by hydrolysis and no oxygen is used in this reaction. Therefore, the biological oxygen demand (BOD) for degradation of cyanuric acid is expected to be zero. The only expected BOD is due to the nitrification of the ammonia reaction product. Since BOD is not a measurement of the extent of biodegradation of cyanuric acid, a low BOD does not indicate a lack of biodegradation.

 

A recent test [9] on the monosodium salt of cyanuric acid found that the effect of the test material on the respiration of activated sewage sludge gave a 3 hour EC50of > 3402 mg cyanuric acid/L. The no observed effect concentration (NOEC) after 3 hours exposure was 2401 mg cyanuric acid/L.

 

Anaerobic biotransformation

Saldick [1] using radiolabelled cyanuric acid found that: “Cyanuric acid biodegrades readily under a wide variety of natural conditions, and particularly well in systems of either low or zero dissolved oxygen level, such as anaerobic activated sludge and sewage, soils, mud, and muddy streams and river water, as well as ordinary aerated activated sludge systems with typically low (1 to 3 ppm) dissolved oxygen levels. Degradation also proceeds in 3.5% sodium chloride solution. Consequently, there are degradation pathways widely available for breaking down cyanuric acid discharged in domestic effluents. The overall degradation reaction is merely a hydrolysis: CO2 and ammonia are the initial hydrolytic breakdown products. Since no net oxidation occurs during this breakdown, biodegradation of cyanuric acid exerts no primary biological oxygen demand (BOD); however, eventually nitrification of the ammonia released will exert its usual biological oxygen demand.” He also concluded that all of the carbon in cyanuric acid is transformed into CO2and is not incorporated into the biomass.

 

Other reports have also demonstrated that cyanuric acid is readily biodegraded under anaerobic conditions by several strains of bacteria including Pseudomonas [10], Klebsiella pneumoniae[10], and others[11] into CO2 and ammonia. The s-triazine nitrogen was incorporated into cell material. Biodegradation of cyanuric acid occurred in the absence of oxygen and if oxygen was present it was not utilized for degradation.

 

Jessee, et al. [11] claim to have isolated a facultative anaerobic bacterium that used cyanuric acid as the major carbon and energy source. This bacterium was isolated from pond sediment which had been acclimated to cyanuric acid for years. However, it is unlikely that the carbon atoms in cyanuric acid can provide an energy source since these atoms cannot be oxidized further.

 

Cyanuric acid is produced as a common intermediate in the biodegradation of various s-triazine herbicides, such as atrazine, prometryne, and simazine, but does not accumulate since cyanuric acid is degraded more rapidly than the herbicides themselves.[3,7,8].

 

The Genetic Basis of Cyanuric Acid Biodegradation in Bacteria

A number of recent papers have investigated the genetic details of the biodegradation of cyanuric acid. Much of this research is concerned with the aerobic biodegradation of the various triazine herbicides, particularly atrazine, with biodegradation of cyanuric acid being the last three steps in a multistep process.

 

Biodegradation of cyanuric acid occurs in three steps [12], shown in equations 1 - 3 below, with a different enzyme, each produced by a different gene, being required for each step. Thus, either the “trzD” or the “atzD” gene produces the amidohydrolase enzyme responsible for hydrolysis of cyanuric acid to biuret, which is the reaction breaking the cyanuric acid ring. Reactions 2 and 3 are then catalysed by the enzymes produced by the “atzE” and “atzF” genes, respectively.

 

Older literature had suggested that urea was the intermediate produced in reaction 2 and then used in reaction 3. However, allophanate (N-carboxyurea) has now been identified as the correct intermediate.[12] Allophanate is unstable, decomposing to urea in the acidic conditions normally used for the analysis, thus leading to the incorrect identification in the earlier work.

 

Eaton and Karns [13] located and characterized the “trzD” gene in the three strains of bacteria studied by Cook, et al. [22] The “trzD” genes from all three strains had the same restriction patterns, suggesting that there is extensive sequence identity. Karns determined the complete DNA sequence of the fragment containing the “trzD” gene and described the purification and some properties of the corresponding enzyme. When the “trzD” gene was transferred to E. coli, the E. coli was able to hydrolyse cyanuric acid to biuret. The purified enzyme had a fairly low Km for the substrate cyanuric acid (50 µM) and a high turnover rate (15,000 mol of substrate/mol of enzyme/min)at pH 8.0 and 30°C.[14] The enzyme was quite specific for cyanuric acid, was inhibited by barbituric acid, and does not require any metal ions for activity.[14]

 

More recently,Martinez et al. [12] determined the sequence of the “atzD” gene from Pseudomonas sp. strain ADP encoding the enzyme for hydrolysis of cyanuric acid to biuret. The “atzD” gene is similar, but not identical, to the “trzD” gene. When the “atzD” gene was transferred to E. coli, the E. coli was able to hydrolyse cyanuric acid to biuret. The same Pseudomonas strain also contained the “atzE” and “atzF” genes encoding the buret hydrolase and allophanate hydrolase enzymes, respectively. When the “atzE” gene was transferred to E. coli, it was able to hydrolyse biuret to ammonia and allophanate. When the “atzF” gene was transferred to E. coli, it was able to hydrolyse allophanate, but not biuret or urea.

 

Martinez et al. [12] also found that the three genes (“atzD”, “atzE” and “atzF”) for the three steps for degradation of cyanuric acid are closed associated on the catabolic plasmid isolated from this Pseudomonas strain and are co-transcribed as a single mRNA. The authors note that catabolic plasmids can transfer among bacteria, thus disseminating the genes encoding the metabolism of environmentally relevant compounds. In this way, the genes for degradation of cyanuric acid can be readily transferred between different types of bacteria. This is one way that bacteria adapt and can rapidly acquire the ability to metabolize a given compound. This is also why acclimation to a given compound can lead to the ability to metabolize that compound after a lag period, provided at least some of the microorganisms present possess the genes required for metabolism of that compound.

 

Fruchey, et al. [15] characterized the reactivity of the purified atzD enzyme. Out of 22 cyclic amides and s-triazine compounds tested, this enzyme only reacts with cyanuric acid and N-methyl-isocyanuric acid, indicating a high degree of substrate discrimination by this enzyme. Five other cyclic amidases did not hydrolyze cyanuric acid. Ten taxonomically and geographically diverse strains of bacteria capable of utilizing cyanuric acid as a sole source of nitrogen for growth were found to contain either the atzD or the trzD gene, but not both.

 

Twenty years after the work of Zeyer and coworkers discussed above, Rousseaux, et al. [16] were able to isolate and characterize 25 bacterial strains capable of degrading atrazine from a number of French soils. Several strains of the Gram-negative species Chelatobacter heintzii and one strain of the Gram-negative Aminobacter aminovorans contained the trzD gene for cyanuric acid ring cleavage. The kinetics of ring degradation was also reported to give 60 - 90% degradation in 15 days. The authors note that Chelatobacter heintzii is known to be ubiquitous in natural environments, as shown by the fact that they isolated a number of degrading strains from a number of geographically different soils. In contrast, four other isolated strains of Chelatobacter heintzii and four strains of Gram-positive bacteria did not contain the trzD gene and were unable to degrade cyanuric acid.

 

In summary, if a microorganism has the genes to produce the required enzymes then it can readily degrade cyanuric acid. Conversely, if a microorganism does not have these genes then it cannot degrade cyanuric acid. In some cases, one microorganism may not have all three required genes. In this case, a community of microorganisms, with different microorganisms having one of the required genes, is required to complete the biodegradation of cyanuric acid.

 

 

Summary

Cyanuric acid is biodegraded by a limited number of bacteria and fungi which possess the genes encoding the required enzymes. Biodegradation of cyanuric acid proceeds through biuret and allophanate (N-carboxyurea) intermediates to give carbon dioxide and ammonia as the final products, according the following reactions:

 

           C3H3N3O3(cyanuric acid) + H2O → C2H5N3O2(biuret) + CO2                                                (1)

           C2H5N3O2(biuret) + H2O → C2H3N2O3-(allophanate) + NH3 + H+                                     (2)

           C2H3N2O3-(allophanate) + H2O + H+ → 2 CO2 + 2 NH3                                                         (3)

 

The overall net reaction is:

 

           C3H3N3O3(cyanuric acid) + 3 H2O → 3 CO2 + 3 NH3                                                                 (4)

 

This reaction sequence is illustrated in Figure 1 below:

 

Figure 1: Pathway for biodegradation of cyanuric acid12(read right to left).

 

Each step is a hydrolysis reaction, not an oxidation. There is no requirement for molecular oxygen in these reactions, so the reactions proceed under aerobic or anaerobic conditions and the BOD of these reactions is zero. The ammonia produced by the last two reactions does exert its normal BOD, if nitrified. During biodegradation, the ammonia is normally incorporated into the biomass during the bacteria’s growth phase, but some may be nitrified, particularly when the bacteria are in a non-growth phase.

 

Table1 lists the bacteria which have been shown in the studies cited here to metabolize cyanuric acid. Note that atrazine or another s-triazine was the initial substrate in some studies. Since cyanuric acid is an intermediate in the biodegradation of these compounds, the microorganism is included in this table if the data indicates that the cyanuric acid ring was degraded. The classification into aerobic or anaerobic is based on whether the bacteria are aerobic or anaerobic and the reported experimental conditions. However, oxygen is not required for biodegradation of cyanuric acid. 

 

In addition, aerobic fungi which have been found to metabolize cyanuric acid include: Sporothrix schenckii [2], Stachybotrys chartarum [3], Hendersonula toruloidea [3],Penicillium [4,5], Hormodendrum masonii [4], Aspergillus minutus [5], Fusarium [5], and Pseudogymnoascus [5].


Table 1. Some bacteria known to biodegrade cyanuric acid.

Bacteria Strain

Aerobic or

anaerobic

Reference

Achromobacter sp.

aerobic

6 (Ernst and Rehm)

Acidovorax sp. strain JLS4

 

Table 2 in Ref 15

Activated sludge

aerobic

7 (Cook and Hütter)

Alcaligenes sp. strain SG1

 

Table 2 in Ref 15

Aminobacter aminovorans

aerobic

16 (Rousseaux, et al)

Chelatobacter heintzii (several strains)

aerobic

16 (Rousseaux, et al)

Klebsiella planticola 99

 

7 (Cook and Hütter)

Klebsiella pneumoniae 99

aerobic or anaerobic

10 (Cook, et al)

Pseudomonas sp.

anaerobic

10 (Cook, et al)

purified enzyme AtzD from

  Pseudomonas sp. strain ADP

aerobic

15 (Fruchey, et al)

Pseudomonas sp. strain NRRLB-12227

aerobic

10 (Cook and Hütter)

Pseudomonas sp. strain NRRLB-12228

aerobic

10 (Cook and Hütter)

Ralstonia pickettii strainD

 

Table 2 in Ref 15

several anaerobic bacteria

anaerobic

11 (Jessee, et al)

a sulfate reducing bacterium

anaerobic

11 (Jessee, et al)

 

References

1. J. Saldick, “Biodegradation of cyanuric acid”, Applied Microbiology, 1974, 28(6) 1004-1008.

2. J. Zeyer, J. Bodmer and R. Hütter, “Rapid degradation of cyanuric acid by Sporothrix schenckii”, Zentrabl. Bakteriol., Mikrobiol. Hyg., Abt. 1, Orig. C, 1981, 2(2) 99-110.

3. D. Wolf and J. Martin, “Microbial decomposition of ring-14C atrazine, cyanuric acid, and 2-chloro-4,6-diamino-s-triazine”, J. Environmental Quality, 1975, 4(1) 134-139.

4. H. Jensen and A. Abdel-Ghaffar, “Cyanuric acid as nitrogen source for micro-organisms”, Arch. Mikrobiol., 1969, 67, 1-5.

5. W. Myskow, T. Lasota and A. Stachyra, “Cyanuric acid – a s-triazine derivation as a nitrogen source for some soil microorganisms”, Acta Microbiologica Polonica (Poland), 1983, 32(2) 177-183.

6. C. Ernst and H. Rehm, “Degradation of cyanuric acid by immobilized bacteria”, DECHEMA Biotechnology Conferences, 1990, 4(Pt. A), 577-580.

7. A. Cook and R. Hütter, “s-Triazines as nitrogen sources for bacteria”, J. Agricultural and Food Chemistry, 1981, 29(6) 1135-1143.

8. S. Sisodia, A. Weber and J. Jensen, “Continuous culture biodegradation of simazine’s chemical oxidation products”, Water Research, 1996, 30(9) 2055-2064.

9. N. Clarke, “Monosodium salt of cyanuric acid: Assessment of the inhibitory effect on the respiration of activated sewage sludge”, SafePharm Laboratories Ltd., Project No. 2255-0006, 2007.

10. A. Cook, P. Beilstein, J. Grossenbacher and R. Hütter, “Ring cleavage and degradative pathway of cyanuric acid in bacteria”, Biochemical J., 1985, 231, 25-30.

11. J. Jessee, R. Benoit, A. Hendricks, G. Allen and J. Neal, “Anaerobic degradation of cyanuric acid, cysteine, and atrazine by a facultative anaerobic bacterium”, Applied and Environmental Microbiology, 1983, 45(1) 97-102.

12. B. Martinez, J. Tomkins, L. Wackett, R. Wing and M. Sadowsky, “Complete nucleotide sequence and organization of the atrazine catabolic plasmid pADP-1 from Pseudomonas sp. strain ADP”, J. of Bacteriology, 2001, 183(19) 5684-5697.

13. R. Eaton and J. Karns, “Cloning and comparison of the DNA encoding ammelide aminohydrolase and cyanuric acid amidohydrolase from three s-triazine-degrading bacterial strains”, J. of Bacteriology, 1991, 173(3) 1363-1355.

14. J. Karns, “Gene sequence and properties of an s-triazine ring-cleavage enzyme from Pseudomonas sp. strain NRRLB-12227”, Applied and Environmental Microbiology, 1999, 65(8) 3512-3517.

15. I. Fruchey, N. Shapir, M Sadowsky and L. Wachett, “On the origins of CYA hydrolase: purification, substrates, and prevalence of AtzD from Pseudomonas sp. strain ADP“, Applied and Environmental Microbiology, 2003, 69(6) 3653-7.

16. S. Rousseaux, A. Hartmann and G. Soulas, “Isolation and characterization of new Gram-negative and Gram-positive atrazine degrading bacteria from different French soils”, FEMS Microbiology Ecology, 2001, 36(2-3) 211-222.