Bacterial Cyanuric Acid Hydrolase for Water Treatment

Abstract

Di-and trichloroisocyanuric acids are widely used as water disinfection agents, but cyanuric acid accumulates with repeated additions and must be removed to maintain free hypochlorite for disinfection. This study describes the development of methods for using a cyanuric acid-degrading enzyme contained within nonliving cells that were encapsulated within a porous silica matrix. Initially, three different bacterial cyanuric acid hydrolases were compared: TrzD from Acidovorax citrulli strain 12227, AtzD from Pseudomonas sp. strain ADP, and CAH from Moorella thermoacetica ATCC 39073. Each enzyme was expressed recombinantly in Escherichia coli and tested for cyanuric acid hydrolase activity using freely suspended or encapsulated cell formats. Cyanuric acid hydrolase activities differed by only a 2-fold range when comparing across the different enzymes with a given format. A practical water filtration system is most likely to be used with nonviable cells, and all cells were rendered nonviable by heat treatment at 70°C for 1 h. Only the CAH enzyme from the thermophile M. thermoacetica retained significant activity under those conditions, and so it was tested in a flowthrough system simulating a bioreactive pool filter. Starting with a cyanuric acid concentration of 10,000 M, more than 70% of the cyanuric acid was degraded in 24 h, it was completely removed in 72 h, and a respike of 10,000 M cyanuric acid a week later showed identical biodegradation kinetics. An experiment conducted with water obtained from municipal swimming pools showed the efficacy of the process, although cyanuric acid degradation rates decreased by 50% in the presence of 4.5 ppm hypochlorite. In total, these experiments demonstrated significant robustness of cyanuric acid hydrolase and the silica bead materials in remediation.C yanuric acid is a high-volume industrial chemical (1) and is not known to be a product of biosynthesis, but it is nonetheless biodegradable (2). Cyanuric acid is toxic in admixture with melamine, as was recently discovered when crude melamine containing cyanuric acid was added to pet food, and more than 1,000 animals died from kidney failure (3, 4). Cyanuric acid is also formed during the bacterial metabolism of s-triazine herbicides and as a by-product of disinfection processes (5). In the latter example, di-or trichloroisocyanuric acid is frequently used to disinfect outdoor swimming pools, and their decomposition leaves cyanuric acid (6), but high cyanuric acid levels impair the disinfection process, leaving people vulnerable to viral, bacterial, and protozoal infections. The cyanuric acid must be removed to sustain disinfection, and there are currently no efficient means to accomplish this. A typical remedy is to drain and refill pools, leading to downtime for the pool, disposal issues, and increased freshwater usage.Cyanuric acid is very stable below 300°C (7), but it can be degraded at ambient temperature in soil and water via a specific enzyme that has been identified within a limited number of bacteria (8). Those bac...

The Sodium (Iso)Cyanurates Nax[H3–xC3N3O3]·yH2O (x = 1–3, y = 0, 1): A Key‐Series for Understanding the Crystal Chemistry of Metal (Iso)Cyanurates

“…Besides questions of structural chemistry, a thorough characterization of sodium isocyanurates might also be of considerable interest to environmental chemists,, biochemists,, and toxicologists,, as sodium isocyanurates are not only the most common precursors to, but also the main degradation products of chlorinated isocyanuric acid derivatives used as swimming pool disinfectants, as well as likely degradation products of atrazine and related s‐triazine herbicides. The first description of a member of the Na x [H 3– x C 3 N 3 O 3 ] · y H 2 O series is given by Hofmann with Na 3 [C 3 N 3 O 3 ], including synthesis instructions for phase pure products, common impurities, description of crystal morphologies and chemical analysis.…”

Ancient Evolution and Recent Evolution Converge for the Biodegradation of Cyanuric Acid and Related Triazines

“…Yeom et al showed that CAH activity declined as a function of increasing chlorine levels in the municipal pool waters that were treated (89). A recent study showed that properly prepared 3-aminopropyltriethoxysilane (APTES)-encapsulated cells could withstand hypochlorite concentrations up to 10 ppm, providing evidence that this could be an effective biohybrid filter medium for treating chlorinated swimming pool and other waters containing cyanuric acid (91).…”

“…A natural solution to this problem is the use of CAH to degrade cyanuric acid. With that purpose in mind, CAH has been encapsulated in a porous silica matrix (89) and experimentally tested in a bioreactive pool filter. The M. thermoacetica ATCC 39073 CAH gene was cloned into Escherichia coli and heated to ensure cell nonviability while maintaining CAH functionality.…”

“…Either by stabilizing hypochlorous acid and its conjugate base, hypochlorite (chlorine bleach), directly or by adding trichloroisocyanuric acid, cyanuric acid can be used to maintain chlorine disinfection (9,89).…”

Structure of the Cyanuric Acid Hydrolase TrzD Reveals Product Exit Channel

“…The use of more than 100 million pounds of cyanuric acid as a chlorine stabilizer in outdoor pools, spas and fountains has provided new impetus to study the cyanuric acid hydrolases that can enzymatically remove the compound from those waters34. In waters requiring disinfection, chlorine dissipates over time but cyanuric acid is extremely stable, accumulates, and with increasing concentration, it sequesters all of the free chlorine and diminishes its effectiveness in destroying viruses, bacteria and protozoa5.…”