Appl Environ Microbiol 71(12): 7980-7986

Cometabolism of Trihalomethanes by <em>Nitrosomonas europaea</em>

The University of Texas at Austin, Department of Civil, Architectural and Environmental Engineering, 1 University Station, Austin, Texas 78712

^{Corresponding author. Mailing address: The University of Texas at Austin, 1 University Station, Austin, TX 78712. Phone: (512) 471-4996. Fax: (512) 471-0592. E-mail:}ude.saxetu.liam@letieps.

Received 2005 Jun 28; Accepted 2005 Aug 4.

Abstract

The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 μg/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants ( equation M1 ) increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH₃-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (T_c), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.

Abstract

Balancing the competing goals of disinfection and disinfection by-product (DBP) regulations is a challenge for many drinking water utilities. The proposed stage 2 disinfection and DBP regulations (15, 16) will only make this task more difficult. Although chlorine disinfection remains quite popular in the United States (6, 7), many utilities now use combinations of chlorine and chloramines to avoid excessive trihalomethane (THM) and haloacetic acid formation. A typical treatment scheme consists of an initial period of chlorination to help achieve disinfection goals followed by quenching with ammonia at some point in the treatment train to meet DBP goals through the lower DBP formation rates associated with chloramines. Nevertheless, significant formation of THMs and haloacetic acids can occur within treatment plants even during relatively short periods of chlorination (24, 28). Therefore, approaches for minimizing the formation of these DBPs or for removing the DBPs within treatment plants are potentially of much practical value.

Much effort over the past two decades has gone into approaches for minimizing DBP formation through modification of disinfection practices and removal of precursor materials (23), while comparatively little effort has been expended on approaches for removing DBPs formed in treatment plants before finished water is sent into distribution systems. Very early on, both technological and philosophical problems with the removal of THMs through activated carbon adsorption and air stripping were identified (28), and approaches for removing DBPs after formation have been largely ignored since that time. Recent developments in biological treatment, however, strongly suggest that revisiting treatment processes for THM removal is worthwhile.

No evidence indicates that THMs can support microbial growth. Considerable evidence is available, however, for cometabolism of chloroform, or trichloromethane (TCM), by bacteria growing on other chemicals (2, 5, 9). Of particular interest is the observation that nitrifying bacteria can cometabolize chloroform at a reasonable rate (0.03 to 0.1 liter/mg/day). The premise of this research is that THM removal should be possible within drinking water treatment plants by introducing a biological treatment step based on THM cometabolism by nitrifying bacteria. In this way, cometabolism would be sustained by adding a chemical commonly used in drinking water treatment while avoiding the addition of simple organic chemicals (i.e., methanol), which is highly undesirable in drinking water treatment.

Most of the cometabolism research with nitrifiers has been done with the soil bacterium Nitrosomonas europaea, which has been used as an example of the ubiquitous soil- and water-dwelling nitrifying bacteria. Vannelli et al. (30) showed that this organism could cometabolize various halogenated methanes, ethanes, and ethenes including chloroform, dichloromethane, and dibromomethane. THMs other than chloroform were not studied. Chloroform cometabolism by N. europaea was subsequently confirmed by Rasche et al. (17) and Ely (8), who also conducted detailed kinetic experiments. Melin et al. (14) and Ginestet et al. (10) showed that a mixed culture of nitrifiers from a marine sediment and activated sludge, respectively, could cometabolize chloroform, thereby providing some evidence that nonspecific ammonia monooxygenase (AMO) enzymes may be widely distributed in the environment, which would be advantageous for easy implementation of the proposed THM cometabolism process.

This research extended the previous work on TCM cometabolism kinetics to the other three regulated THMs to provide a basis for assessing the feasibility of the proposed treatment process. The key question was whether or not nitrifying bacteria can reliably cometabolize all four THMs at a sufficient rate to make the process attractive to utilities that practice (or want to practice) prechlorination, in particular, utilities practicing a combination of chlorination and chloramination. Information was developed on TCM, bromodichloromethane (BDCM), dibromochloromethane (DBCM), and tribromomethane (TBM) cometabolism kinetics as well as the toxicity of their intermediate by-products. N. europaea was chosen as a starting point for this evaluation to build on the large body of literature on this organism and to provide a pure-culture baseline for evaluating the performance of the mixed cultures that are likely to dominate in practice.

Acknowledgments

This research was funded by the American Water Works Association Research Foundation and the Texas Advanced Technology Research Program, which we thank for their financial, technical, and administrative assistance with the project.

The comments and views detailed herein may not necessarily reflect the views of the American Water Works Association Research Foundation or its officers, directors, affiliates, or agents.

Acknowledgments

REFERENCES

References

1. Alvarez-Cohen, L., and P. L. McCarty. 1991. A cometabolic biotransformation model for halogenated aliphatic-compounds exhibiting product toxicity. Environ. Sci. Technol.25:1381-1387. [PubMed]
2. Alvarez-Cohen, L., and P. L. McCarty. 1991. Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells. Appl. Environ. Microbiol.57:1031-1037.
3. Alvarez-Cohen, L., and G. E. Speitel. 2001. Kinetics of aerobic cometabolism of chlorinated solvents. Biodegradation12:105-126. [[PubMed]
4. Arcangeli, J. P., and E. Arvin. 1997. Modeling of the cometabolic biodegradation of trichloroethylene by toluene oxidizing bacteria in a biofilm system. Environ. Sci. Technol.31:3044-3052. [PubMed]
5. Aziz, C. E., G. Georgiou, and G. E. Speitel. 1999. Cometabolism of chlorinated solvents and binary chlorinated solvent mixtures using M. trichosporium OB3b PP358. Biotechnol. Bioeng.65:100-107. [[PubMed]
6. Connell, G. F., J. C. Routt, B. Macler, R. C. Andrews, J. M. Chen, Z. K. Chowdhury, G. F. Crozes, G. B. Finch, R. C. Hoehn, J. G. Jacangelo, A. Penkal, G. R. Schaeffer, C. R. Schulz, and M. P. Uza. 2000. Committee report: disinfection at large and medium-size systems. J. Am. Water Works Assoc.92:32-43. [PubMed]
7. Connell, G. F., J. C. Routt, B. Macler, R. C. Andrews, J. M. Chen, Z. K. Chowdhury, G. F. Crozes, G. B. Finch, R. C. Hoehn, J. G. Jacangelo, A. Penkal, G. R. Schaeffer, C. R. Schulz, and M. P. Uza. 2000. Committee report: disinfection at small systems. J. Am. Water Works Assoc.92:24-31. [PubMed]
8. Ely, RL. 1996. Effects of substrate interactions, toxicity, and bacterial response during cometabolism of chlorinated solvents by nitrifying bacteria. Ph.D. dissertation. Oregon State University, Corvallis, Oreg.
9. Ely, R. L., K. J. Williamson, M. R. Hyman, and D. J. Arp. 1997. Cometabolism of chlorinated solvents by nitrifying bacteria: kinetics, substrate interactions, toxicity effects, and bacterial response. Biotechnol. Bioeng.54:520-534. [[PubMed]
10. Ginestet, P., J. M. Audic, and J. C. Block. 2001. Chlorinated solvents cometabolism by an enriched nitrifying bacterial consortium. Water Sci. Technol. Water Supply1:95-102. [PubMed]
11. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1998. Standard methods for the examination of water and wastewater, 18th ed. American Public Health Association, Washington, D.C.
12. Hooper, A. B., and K. R. Terry. 1974. Photoinactivation of ammonia oxidation in Nitrosomonas. J. Bacteriol.119:899-906.
13. Keener, W. K., and D. J. Arp. 1993. Kinetic studies of ammonia monooxygenase inhibition in Nitrosomonas europaea by hydrocarbons and halogenated hydrocarbons in an optimized whole-cell assay. Appl. Environ. Microbiol.59:2501-2510.
14. Melin, E. S., J. A. Puhakka, S. E. Strand, K. J. Rockne, and J. F. Ferguson. 1996. Fluidized-bed enrichment of marine ammonia-to-nitrite oxidizers and their ability to degrade chloroaliphatics. Int. Biodeter. Biodeg.38:9-18. [PubMed]
15. Pontius, FW. 2001. Regulatory update for 2001 and beyond. J. Am. Water Works Assoc.93:66-80. [PubMed][Google Scholar]
16. Pontius, FW. 2003. Update on USEPA's drinking water regulations. J. Am. Water Works Assoc.95:57-68. [PubMed][Google Scholar]
17. Rasche, M. E., M. R. Hyman, and D. J. Arp. 1991. Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: cometabolic inactivation of ammonia monooxygenase and substrate specificity. Appl. Environ. Microbiol.57:2986-2994.
18. Rittmann, B. E., and P. L. McCarty. 2001. Environmental biotechnology: principles and applications. McGraw-Hill, Boston, Mass.
19. Robinson, JA. 1985. Determining microbial kinetic parameters using nonlinear regression analysis. Adv. Microb. Ecol.8:61-114. [PubMed][Google Scholar]
20. Schwarzenbach, R. P., P. M. Gschwend, and D. M. Imboden. 2003. Environmental organic chemistry, 2nd ed. Wiley, Hoboken, N.J.
21. Segar, R. L., S. L. Dewys, and G. E. Speitel, Jr. 1995. Sustained trichloroethylene cometabolism by phenol-degrading bacteria in sequencing biofilm reactors. Water Environ. Res.67:764-774. [PubMed]
22. Shears, J. H., and P. M. Wood. 1985. Spectroscopic evidence for a photosensitive oxygenated state of ammonia monooxygenase. Biochem. J.226:499-507.
23. Singer, PC. 1999. Formation and control of disinfection by-products in drinking water. American Water Works Association, Denver, Colo.
24. Singer, P. C., G. W. Harrington, G. A. Cowman, M. E. Smith, D. S. Schecter, and L. J. Harrington. 1999. Impacts of ozonation on the formation of chlorination and chloramination by-products. Report no. 90766. American Water Works Association, Denver, Colo.
25. Smith, L. H., P. K. Kitanidis, and P. L. McCarty. 1997. Numerical modeling and uncertainties in rate coefficients for methane utilization and TCE cometabolism by a methane-oxidizing mixed culture. Biotechnol. Bioeng.53:320-331. [[PubMed]
26. Smith, L. H., P. L. McCarty, and P. K. Kitanidis. 1998. Spreadsheet method for evaluation of biochemical reaction rate coefficients and their uncertainties by weighted nonlinear least-squares analysis of the integrated Monod equation. Appl. Environ. Microbiol.64:2044-2050.
27. Suzuki, I., U. Dular, and S. C. Kwok. 1974. Ammonia or ammonium ion as substrate for oxidation by Nitrosomonas europaea cells and extracts. J. Bacteriol.120:556-558.
28. Symons, JM., et al. 1982. Treatment techniques for controlling trihalomethanes in drinking water. American Water Works Association, Denver, Colo.
29. Uemoto, H., A. Ando, and H. Saiki. 2000. Effect of oxygen concentration on nitrogen removal by Nitrosomonas europaea and Paracoccus denitrificans immobilized within tubular polymeric gel. J. Biosci. Bioeng.90:654-660. [[PubMed]
30. Vannelli, T., M. Logan, D. M. Arciero, and A. B. Hooper. 1990. Degradation of halogenated aliphatic compounds by the ammonia-oxidizing bacterium Nitrosomonas europaea. Appl. Environ. Microbiol.56:1169-1171.
31. Wong-Chong, G. M., and R. C. Loehr. 1975. The kinetics of microbial nitrification. Water Res.9:1099-1106. [PubMed]