Reviews - Multi-Compound - THMs - HAAs - HANs - HAMs - HNMs - Lab Culture - Enzyme Studies

Measuring Degraders - Single DBP - DS Biogrowth

 

Major Reports & Review Papers on DBP Biodegradation
Citation Notes Abstract
Hozalski, R.M., Zhang, P., LaPara, T.M., Grigorescu, A., Leach, L.H., Camper, A.K., Goslan, E.H., Parsons, S.A. and Xie, Y.F. (2010) Biodegradation of HAAs in Distribution Systems, WRF #3122, Denver CO.
   
Baribeau, H., Boulos, L., Haileselassie, H., Crozes, G., Singer, P.C., Nichols, C., Schlesinger, S.A., Gullick, R.A., Williams, S.L., Williams, R.L., Fountleroy, L., Andrews, S.A. and Moffat, E. (2006) Formation and Decay of Disinfection Byproducts in the Distribution System, AWWARF #2770, Denver, CO.    

 

Multi-Compound Biodegradation Studies
Citation Notes Abstract

Hozalski, R.M., Arnold, W.A., Chun, C.L., LaPara, T.M., Lee, J.Y., Pearson, C.R. and Zhang, P. (2008) Degradation of Halogenated Disinfection Byproducts in Water Distribution Systems, In Disinfection by-Products in Drinking Water: Occurrence, Formation, Health Effects, and Control. Karanfil, T., Krasner, S.W. and Xie, Y. (eds), pp. 334-348.

  Water distribution systems are complex environments frequently containing corroded iron pipes and biofilms. To thoroughly understand the fate of halogenated disinfection byproducts (DBPs) in these systems, two degradation processes were investigated: abiotic degradation (i.e. hydrolysis and reductive dehalogenation) and biodegradation. DBPs were selected from 6 different compound classes representing both regulated DBPs (i.e. trihalomethanes or THMs, and haloacetic acids or HAAs) and non-regulated or "emerging" DBPs. Batch experiments were conducted to investigate the pathways and kinetics of DBP degradation. As expected, the relative importance of hydrolysis, abiotic reductive dehalogenation, and biodegradation depends on the DBP structure and on the environmental conditions (i.e. pH, temperature, dissolved oxygen, Fe minerals present, bacteria present, etc.). From our results, chloropicrin (i.e. trichloronitromethane) and most brominated DBPs are highly susceptible to abiotic reductive dehalogenation, trichloracetonitrile and trichloropropanone are the most susceptible to hydrolysis, and HAAs are readily biodegraded under aerobic conditions. Knowledge of DBP degradation mechanisms and rates in distribution systems is important for selecting DBP monitoring locations, modeling DBP fate, and for predicting exposure to these compounds. Such information could also be useful for developing treatment systems for DBP removal.
Hozalski, R.M., Arnold, W.A., Chun, C.L., LaPara, T.M., Lee, J.Y., Pearson, C.R. and Zhang, P. (2008) Degradation of Halogenated Disinfection Byproducts in Water Distribution Systems, In: Disinfection by-Products in Drinking Water: Occurrence, Formation, Health Effects, and Control. Karanfil, T., Krasner, S.W. and Xie, Y. (eds), pp. 334-348.   Water distribution systems are complex environments frequently containing corroded iron pipes and biofilms. To thoroughly understand the fate of halogenated disinfection byproducts (DBPs) in these systems, two degradation processes were investigated: abiotic degradation (i.e. hydrolysis and reductive dehalogenation) and biodegradation. DBPs were selected from 6 different compound classes representing both regulated DBPs (i.e. trihalomethanes or THMs, and haloacetic acids or HAAs) and non-regulated or "emerging" DBPs. Batch experiments were conducted to investigate the pathways and kinetics of DBP degradation. As expected, the relative importance of hydrolysis, abiotic reductive dehalogenation, and biodegradation depends on the DBP structure and on the environmental conditions (i.e. pH, temperature, dissolved oxygen, Fe minerals present, bacteria present, etc.). From our results, chloropicrin (i.e. trichloronitromethane) and most brominated DBPs are highly susceptible to abiotic reductive dehalogenation, trichloracetonitrile and trichloropropanone are the most susceptible to hydrolysis, and HAAs are readily biodegraded under aerobic conditions. Knowledge of DBP degradation mechanisms and rates in distribution systems is important for selecting DBP monitoring locations, modeling DBP fate, and for predicting exposure to these compounds. Such information could also be useful for developing treatment systems for DBP removal.

 

THMs
Citation Notes Abstract
Wahman, D.G., Kirisits, M.J., Katz, L.E. and Speitel, G.E. (2011) Ammonia-Oxidizing Bacteria in Biofilters Removing Trihalomethanes Are Related to Nitrosomonas oligotropha. Applied and Environmental Microbiology 77(7), 2537-2540.   Ammonia-oxidizing bacteria (AOB) in nitrifying biofilters degrading four regulated trihalomethanes-trichloromethane, bromodichloromethane, dibromochloromethane, and tribromomethane-were related to Nitrosomonas oligotropha. N. oligotropha is associated with chloraminated drinking water systems, and its presence in the biofilters might indicate that trihalomethane tolerance is another reason that this bacterium is dominant in chloraminated systems.
Wahman, D.G., Katz, L.E. and Speitel, G.E. (2011) Performance and biofilm activity of nitrifying biofilters removing trihalomethanes. Water Research 45(4), 1669-1680.
biofilters Nitrifying biofilters seeded with three different mixed-culture sources removed trichloromethane (TCM) and dibromochloromethane (DBCM) with removals reaching 18% for TCM and 75% for DBCM. In addition, resuspended biofilm removed TCM, bromodichloromethane (BDCM), DBCM, and tribromomethane (TBM) in backwash batch kinetic tests, demonstrating that the biofilters contained organisms capable of biotransforming the four regulated trihalomethanes (THMs) commonly found in treated drinking water. Upon the initial and subsequent increased TCM addition, total ammonia nitrogen (TOTNH(3)) removal decreased and then reestablished, indicating an adjustment by the biofilm bacteria. In addition, changes in DBCM removal indicated a change in activity related to DBCM. The backwash batch kinetic tests provided a useful tool to evaluate the biofilm's bacteria. Based on these experiments, the biofilters contained bacteria with similar THM removal kinetics to those seen in previous batch kinetic experiments. Overall, performance or selection does not seem based specifically on nutrients, source water, or source cultures and most likely results from THM product toxicity, and the use of GAC media appeared to offer benefits over anthracite for biofilter stability and long-term performance, although the reasons for this advantage are not apparent based on research to date.
Speitel, G.E., Jr, Bayer, B.M. and Kannappan, R. (2010) Significance of Trihalomethanes in Preventing Distribution System Nitrification in Chloraminated Waters, WRF #3173, Denver CO.    

HAAs
Citation Notes Abstract
Grigorescu, A.S. and Hozalski, R.M. (2010) Modeling HAA biodegradation in biofilters and distribution systems. Journal American Water Works Association 102(7), 67-80.   Using a plug-flow reactor model and literature values for haloacetic acid (HAA) biodegradation kinetic parameters, the authors evaluated HAA removal efficiency in blofilters and distribution systems. The simulations performed indicated that HAA removal through bacterial activity is possible both within biofilters and along the pipe walls from the distribution system. Because large numbers of bacteria are needed to be able to reduce HAA levels below US Environmental Protection Agency limits, only the biofilters where relatively large bacterial densities can accumulate on the filter media-proved efficient systems for removing HAAs. Although previous research showed that HAAs could be degraded in biologically active carbon filters and along distribution systems, none of these studies correlated HAA degradation efficiency with physical and biological parameters (e.g., pipe diameter, filter grain diameter, flow velocity, biodegradation kinetic constants). The current research fills in these gaps and demonstrates why a biofilter is more efficient than the distribution system at removing HAAs.
Tung, H.H. and Xie, Y.F. (2009) Association between haloacetic acid degradation and heterotrophic bacteria in water distribution systems. Water Research 43(4), 971-978.   The occurrences of trihalomethanes (THMs), haloacetic acids (HAAS) and heterotrophic bacteria were monitored in five small water systems over a nine-month period to investigate the association between HAA degradation and heterotrophic bacteria populations. The sampling sites were chosen to cover the entire distribution network for each system. An inverse association between heterotrophic bacteria and HAA concentrations was found at some locations where chlorine residuals were around or less than 0.3 mg L-1. At other sample locations, where chlorine residuals were higher (over 0.7 mg L-1), no HAA reduction was observed. A high heterotrophic bacteria count accompanied with a low chlorine residual could be used as an indicator for HAA degradation in distribution systems.
Bayless, W. and Andrews, R.C. (2008) Biodegradation of six haloacetic acids in drinking water. Journal of Water and Health 6(1), 15-22. bench-scale system Haloacetic acids (HAAs) are produced by the reaction of chlorine with natural organic matter and are regulated disinfection by-products of health concern. Biofilms in drinking water distribution systems and in filter beds have been associated with the removal of some HAAs, however the removal of all six routinely monitored species (HAA(6)) has not been previously reported. In this study, bench-scale glass bead columns were used to investigate the ability of a drinking water biofilm to degrade HAA(6). Monochloroacetic acid (MCAA) and monobromoacetic acid (MBAA) were the most readily degraded of the halogenated acetic acids. Trichloroacetic acid (TCAA) was not removed biologically when examined at a 90% confidence level. In general, di-halogenated species were removed to a lesser extent than the mono-halogenated compounds. The order of biodegradability by the biofilm was found to be monobromo > monochloro > bromochloro > dichloro > dibromo > trichloroacetic acid.
Hozalski, R.M. (2007) DBP Fate in Distribution Systems, ACS, Chicago, IL. ppt to pdf; 2/pg  
     

 

 

Haloacetonitiles
Citation Notes Abstract
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Haloacetamides
Citation Notes Abstract
     

 

Halonitromethanes
Citation Notes Abstract
     

 

Laboratory Studies with Isolates
Citation Notes Abstract
Lin, C.J., Yang, L.R., Xu, G. and Wu, J.P. (2011) Enhancement of Haloacetate Dehalogenase Production by Strain Mutation and Condition Optimization. Biotechnology and Bioprocess Engineering 16(5), 923-929.   Enhancement of the activity of an inducible chloroacetate dehalogenase was carried out by efficient and safe mutation with UV and microwave irradiation along with optimization of culture conditions. First, a stable mutant of Pseudomonas sp. CGMCC 3267-MW6 with chloroacetate dehalogenase activity of 2.77 U/mL (3-fold higher activity than the wild strain) was produced by mutation. The maximum activity of this inducible enzyme was measured as 29.41 U/mL when Pseudomonas sp. CGMCC 3267-MW6 was cultured with 4 g/L 3-hydroxybutyrate for 12 h followed by 40 mM 3-chlorobutyrate for an additional 20 h. Production of the enzyme was found to be associated with growth of the bacterium. According to these results, we determined that the optimum inducer of chloroacetate dehalogenase activity would be a hard degradable substrate. The optimum auxiliary carbon source would be the primary metabolite of the substrate or the precursor of the metabolite. The optimum time of inducer supplementation would be during the middle stage of exponential phase. The optimum concentration of substrate would be sufficient but would not induce inhibition. Finally, the optimum collection time would be at the later stage of exponential phase. This work provides further knowledge of chloroacetate dehalogenase and the optimization of inducible enzyme production.
Horisaki, T., Yoshida, E., Sumiya, K., Takemura, T., Yamane, H. and Nojiri, H. (2011) Isolation and characterization of monochloroacetic acid-degrading bacteria. Journal of General and Applied Microbiology 57(5), 277-284.   Five Burkholderia strains (CL-1, CL-2, CL-3, CL-4, and CL-5) capable of degrading monochloroacetic acid (MCA) were isolated from activated sludge or soil samples gathered from several parts of Japan. All five isolates were able to grow on MCA as the sole source of carbon and energy, and argentometry and gas chromatography-mass spectroscopy analyses showed that these five strains consumed MCA completely and released chloride ions stoichiometrically within 25 h. The five isolates also grew on monobromoacetic acid, monoiodoacetic acid, and L-2-monochloropropionic acid as sole sources of carbon and energy. In addition, the five isolates could not grow with DCA but dehalogenate single chlorine from DCA. Because PCR analyses revealed that all five isolates have an identical group II dehalogenase gene fragment and no group I deh gene, only strain CL-1 was analyzed further. The partial amino acid sequence of the group II dehalogenese of strain CL-1, named DehCL1, showed 74.6% and 65.2% identities to corresponding regions of the two MCA dehalogenases, DehC1 from Pseudomonas sp. strain CBS-3 and Hdl IVa from Burkholderia cepacia strain MBA4, respectively. The secondary-structure motifs of the haloacid dehalogenase (HAD) superfamily and the amino acid residues involved in substrate binding, catalysis, and hydrophobic pocket formation were conserved in the partial amino acid sequence of DehCL1.
Zhang, P., Lapara, T.M., Goslan, E.H., Xie, Y.F., Parsons, S.A. and Hozalski, R.M. (2009) Biodegradation of Haloacetic Acids by Bacterial Isolates and Enrichment Cultures from Drinking Water Systems. Environmental Science & Technology 43(9), 3169-3175.
  Biodegradation is a potentially important loss process for haloacetic acids (HAAS), a class of chlorination byproducts, in water treatment and distribution systems, but little is known about the organisms involved (i.e., identity, substrate range, biodegradation kinetics). In this research, 10 biomass samples (i.e., tap water, distribution system biofilms, and prechlorinated granular activated carbon filters) from nine drinking water systems were used to inoculate a total of thirty enrichment cultures fed monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), or trichloroacetic (TCAA) as sole carbon and energy source. HAA degraders were successfully enriched from the biofilm samples (GAC and distribution system) but rarely from tap water. Half of the MCAA and DCAA enrichment cultures were positive, whereas only one TCAA culture was positive (two were inconclusive). Eight unique HAA-degrading isolates were obtained including several Afipia spp. and a Methylobacterium sp.; all isolates were members of the phylum Protelobacteria. MCAA, monobromoacetic acid (MBAA), and monoiodoacetic acid (MIAA) were rapidly degraded by all isolates, and DCAA and tribromoacetic (TBAA) were also relatively labile. TCAA and dibromoacetic acid (DBAA) were degraded by only three isolates and degradation lagged behind the other HAAs. Detailed DCAA biodegradation kinetics were obtained for two selected isolates and two enrichment cultures. The maximum biomass-normalized degradation rates (V-m) were 0.27 and 0.97 mu g DCAA/mu g protein/h for Methylobacterium fujisawaense strain PAWDI and Afipia felis strain EMD2, respectively, which were comparable to the values obtained for the enrichment cultures from which those organisms were isolated (0.39 and 1.37 mu g DCAA/mu g protein/h, respectively). The half-saturation constant (K-m) values ranged from 4.38 to 77.91 mu g DCAA/L and the cell yields ranged from 14.4 to 36.1 mg protein/g DCAA.
McRae, B.M., LaPara, T.M. and Hozalski, R.M. (2004) Biodegradation of haloacetic acids by bacterial enrichment cultures. Chemosphere 55(6), 915-925.   Haloacetic acids (HAAs) are toxic organic chemicals that are frequently detected in surface waters and in drinking water distribution systems. The aerobic biodegradation of HAAs was investigated in serum bottles containing a single HAA and inoculated with washed microorganisms obtained from enrichment cultures maintained on either monochloroacetic acid (MCAA) or trichloroacetic acid (TCAA) as the sole carbon and energy source. Biodegradation was observed for each of the HAAs tested at concentrations similar to those found in surface waters and in drinking water distribution systems. The MCAA culture was able to degrade both MCAA and monobromoacetic acid (MBAA) with pseudo-first order rate constants of 1.06 x 10(-2) and 1.13 x 10(-2) 1 (mg protein)(-1) d(-1), respectively, for concentrations ranging from 10(-5) to 2 mM. The pseudo-first order rate constant for TCAA degradation by the TCAA culture was 6.52x 10(-3) 1 (mg protein)(-1) d(-1) for concentrations ranging from 5.33 x 10(-5) to 0.72 mM. The TCAA culture was also able to degrade MCAA with the rate accelerating as incubation time increased. Experiments with radiolabeled HAAs indicated that the C-14 was primarily converted to (CO2)-C-14 with minor incorporation into cell biomass. The community structure of the enrichment cultures was analyzed by both cultivation-dependent and cultivation-independent approaches. Denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified 16S rRNA gene fragments showed that each of the two enrichment cultures had multiple bacterial populations, none of which corresponded to HAA-degrading bacteria cultivated on HAA-supplemented agar plates. This research indicates that biodegradation is a potential loss mechanism for HAAs in surface waters and in drinking water distribution systems.
Poelarends, G.J., Wilkens, M., Larkin, M.J., van Elsas, J.D. and Janssen, D.B. (1998) Degradation of 1,3-dichloropropene by Pseudomonas cichorii 170. Applied and Environmental Microbiology 64(8), 2931-2936.   The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source, Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible, The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13063, Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene.
Janssen, D.B., Scheper, A., Dijkhuizen, L. and Witholt, B. (1985) Degradation of Halogenated Aliphatic Compounds by Xanthobacter- Autotrophicus GJ10. Applied and Environmental Microbiology 49(3), 673-677.    

 

Enzyme Studies
Citation Notes Abstract
Hamid, T.H.T., Hamid, A.A.A. and Huyop, F. (2011) A review on non-stereospecific haloalkanoic acid dehalogenases. African Journal of Biotechnology 10(48), 9725-9736.   Haloalkanoic acid dehalogenases remove halides from organic haloacids and have potential as bioremediation agents. DehE from Rhizobium sp. RC1, DehI from Pseudomonas putida PP3 and D,L-DEX 113 from Pseudomonas sp. 113 are non-stereospecific dehalogenases that invert the configurations of D-and L- carbons bound to a halogen. The kinetics of DehE has been partially characterized and brominated compounds have greater specificity constant values than do the corresponding chlorinated compounds. The sequence of DehE is similar to that of DehI; therefore, the two enzymes may have similar structures and functions. The three-dimensional structure of DehI is known and its reaction mechanism was inferred from its structure and a mutagenesis study of D,L-DEX 113. Aspartate residues at positions 189 and 194 in DehI and D,L-DEX 113 were predicted to be involved in catalysis. These residues activate a water molecule that directly attacks the chiral carbon. Because DehE and DehI are sequentially related, delineating the structure of DehE is important to ascertain if the catalytic residues and reaction mechanism are the same for both enzymes. A structural prediction, sequence-homology modeling and a site-directed mutagenesis study of DehE might help achieve this goal.
Kurihara, T. (2011) A Mechanistic Analysis of Enzymatic Degradation of Organohalogen Compounds. Bioscience Biotechnology and Biochemistry 75(2), 189-198.   Enzymes that catalyze the conversion of organohalogen compounds have been attracting a great deal of attention, partly because of their possible applications in environmental technology and the chemical industry. We have studied the mechanisms of enzymatic degradation of various organic halo acids. In the reaction of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of the catalytic aspartate residue nucleophilically attacked the alpha-carbon atom of the substrates to displace the halogen atom. In the reaction catalyzed by DL-2-haloacid dehalogenase, a water molecule directly attacked the substrate to displace the halogen atom. In the course of studies on the metabolism of 2-chloroacrylate, we discovered two new enzymes. 2-Haloacrylate reductase catalyzed the asymmetric reduction of 2-haloacrylate to produce L-2-haloalkanoic acid in an NADPH-dependent manner. 2-Haloacrylate hydratase catalyzed the hydration of 2-haloacrylate to produce pyruvate. The enzyme is unique in that it catalyzes the non-redox reaction in an FADH(2)-dependent manner.
Koudelakova, T., Chovancova, E., Brezovsky, J., Monincova, M., Fortova, A., Jarkovsky, J. and Damborsky, J. (2011) Substrate specificity of haloalkane dehalogenases. Biochemical Journal 435, 345-354.   An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1 bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB similar to DbjA>DhlA similar to DhaA similar to DbeA similar to DmbA>DatA similar to DmbC similar to DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.
Beloqui, A., Polaina, J., Vieites, J.M., Reyes-Duarte, D., Torres, R., Golyshina, O.V., Chernikova, T.N., Waliczek, A., Aharoni, A., Yakimov, M.M., Timmis, K.N., Golyshin, P.N. and Ferrer, M. (2010) Novel Hybrid Esterase-Haloacid Dehalogenase Enzyme. Chembiochem 11(14), 1975-1978.
   
Sharir-Ivry, A., Shnerb, T., Strajbl, M. and Shurki, A. (2010) VB/MM Protein Landscapes: A Study of the S(N)2 Reaction in Haloalkane Dehalogenase. Journal of Physical Chemistry B 114(6), 2212-2218.   QM/MM methods are widely used for studies of reaction mechanisms in water and protein environments. Recently, we have developed the VB/MM method in which the QM part is implemented by the ab initio valence bond (VB) method. Here, we report on further improvement of the VB/MM method which makes it possible to use the method for reactivity studies in systems where the QM and MM parts are connected by covalent bonds followed by first ab initio VB study of reactivity in proteins. We implemented a simple link atom scheme to treat the boundary interactions. We tested the performance of the link atom treatment in combination with the VB/MM method on an S(N)2 reaction and found it to be sufficiently accurate. We then used the VB/MM method to study the S(N)2 reaction in haloalkane dehalogenase (DNA). We show that the predicted reaction barrier heights are in good agreement with estimated experimental values, thereby validating the method. Finally, we analyze the reaction energetics in terms of contributions of the VB configurations and conclude that such analysis is instrumental in pinpointing the essential features of the catalytic mechanism.
Kurihara, T. and Esaki, N. (2008) Bacterial hydrolytic dehalogenases and related enzymes: Occurrences, reaction mechanisms, and applications. Chemical Record 8(2), 67-74.   Dehalogenases catalyze the cleavage of the carbon-halogen bond of organohalogen compounds. They have been attracting a great deal of attention partly because of their potential applications in the chemical industry and bioremediation. In this personal account, we describe occurrences, reaction mechanisms, and applications of bacterial hydrolytic dehalogenases and related enzymes, particularly L-2-haloacid dehalogenase, DL-2-haloacid dehalogenase, fluoroacetate dehalogenase, and 2-haloacrylate reductase. L-2-Haloacid dehalogenase is a representative enzyme of the haloacid dehalogenase (HAD) superfamily, which includes the P-type ATPases and other hydrolases. Structural and mechanistic analyses of this enzyme have yielded important insights into the mode of action of the HAD superfamily proteins. Fluoroacetate dehalogenase is unique in that it catalyzes the cleavage of the highly stable C-F bond of a fluorinated aliphatic compound. In the reactions Of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of Asp performs a nucleophilic attack on the cc-carbon atom of the substrate, displacing the halogen atom. This mechanism is common to haloalkane dehalogenase and 4-chlorobenzoyl-CoA dehalogenase. DL-2-Haloacid dehalogenase is unique in that a water molecule directly attacks the substrate, displacing the halogen atom. The occurrence of 2-haloacrylate reductase was recently reported, revealing a new pathway for the degradation of unsaturated aliphatic organohalogen compounds.
Schmidberger, J.W., Wilce, J.A., Weightman, A.J., Whisstock, J.C. and Wilce, M.C.J. (2008) The crystal structure of Dehl reveals a new alpha-haloacid dehalogenase fold and active-site mechanism. Journal of Molecular Biology 378(1), 284-294.   Haloacid dehalogenases, catalyse the removal of halides from organic haloacids and are of interest for bioremediation and for their potential use in the synthesis of industrial chemicals. We present the crystal structure of the homodimer DehI from Pseudomonas putida strain PP3, the first structure of a group I a.-haloacid dehalogenase that can process both L- and D-substrates. The structure shows that the DehI monomer consists of two domains of similar to 130 amino acids that have similar to 16% sequence identity yet adopt virtually identical and unique folds that form a pseudo-dimer. Analysis of the active site reveals the likely binding mode of both L- and D-Substrates with respect to key catalytic residues. Asp189 is predicted to activate a water molecule for nucleophilic attack of the substrate chiral centre resulting in an inversion of configuration of either L- or D-Substrates in contrast to D-only enzymes. These details will assist with future bioengineering of dehalogenases.
Papajak, E., Kwiecien, R.A., Rudzinski, J., Sicinska, D., Kaminski, R., Szatkowski, L., Kurihara, T., Esaki, N. and Paneth, P. (2006) Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects. Biochemistry 45(19), 6012-6017.   DL- 2- Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of (D)- 2- haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on ( R)-(+)- 2- haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha- carbon of the 2- haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for ( S)-(-)- 2- chloropropionate and 1.0082 +/- 0.0005 for the ( R)-(+)- isomer. Together with solvent deuterium isotope effects on V-max/ K-M, 0.78 +/- 0.09 for ( S)-(-)- 2- chloropropionate and 0.90 +/- 0.13 for the ( R)-(+)- isomer, these values indicate that in the case of the ( R)-(+)- reactant another step preceding the dehalogenation is partly rate- limiting. Under the V-max conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of ( S)-(-)- and ( R)-(+)- reactants
Kurihara, T., Esaki, N. and Soda, K. (2000) Bacterial 2-haloacid dehalogenases: structures and reaction mechanisms. Journal of Molecular Catalysis B-Enzymatic 10(1-3), 57-65.   Microbial dehalogenases have been attracting a great deal of attention because of their possible application to fine chemical synthesis and bioremediation of halo compound-polluted environment. Dehalogenases employ various different mechanisms to cleave the carbon-halogen bond. 2-Haloacid dehalogenases catalyze the hydrolytic dehalogenation of 2-haloalkanoic acids to produce the corresponding 2-hydroxyalkanoic acids. The reaction mechanism of L-2-haloacid dehalogenase from Pseudomonas sp. YL has been clarified by O-18 incorporation experiment, site-directed mutagenesis and X-ray crystallographic analysis. The carboxylate group of Asp10 performs a nucleophilic attack on the alpha-carbon atom of the substrate to displace the halogen atom and produce the ester intermediate, which is subsequently hydrolyzed to produce the corresponding D-2-hydroxyalkanoic acid and regenerate the Asp10 residue. The reaction catalyzed by fluoroacetate dehalogenase from Moraxella sp. B similarly proceeds in two steps: the carboxylate group of Asp105 performs a nucleophilic attack on the substrate alpha-carbon atom to form an ester intermediate, and the intermediate is hydrolyzed by a water molecule activated by His272. In contrast with these two enzymes, a water molecule directly attacks the substrate to displace the halogen atom and produce 2-hydroxyalkanoic acid in the reaction catalyzed by DL-2-haloacid dehalogenase from Pseudomonas sp. 113.
Wahman, D.G., Katz, L.E. and Speitel, G.E. (2005) Cometabolism of Trihalomethanes by Nitrosomonas europaea. Applied and Environmental Microbiology 71(12), 7980-7986. THM The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 mu g/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 (k(1THM)) 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.
Kurihara, T. (2004) Bioconversion of organohalogen compounds with microbial enzymes: Mechanistic analysis of the enzyme reactions and their application. Nippon Nogeikagaku Kaishi-Journal of the Japan Society for Bioscience Biotechnology and Agrochemistry 78(10), 938-943.    
Park, C., Kurihara, T., Yoshimura, T., Soda, K. and Esaki, N. (2003) A new DL-2-haloacid dehalogenase acting on 2-haloacid amides: purification, characterization, and mechanism. Journal of Molecular Catalysis B-Enzymatic 23(2-6), 329-336. haloacid amides DL-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively. We have constructed an overproduction system for DL-2-haloacid dehalogenase from Pseudomonas putida PP3 (DL-DEX 312) and purified the enzyme to analyze the reaction mechanism. When a single turnover reaction of DL-DEX 312 was carried out in (H2O)-O-18 by use of a large excess of the enzyme with D- or L-2-chloropropionate as a substrate, the lactate produced was labeled with O-18. This indicates that the solvent water molecule directly attacked the substrate and that its oxygen atom was incorporated into the product. This reaction mechanism contrasts with that of L-2-haloacid dehalogenase, which has an active-site carboxylate group that attacks the substrate to displace the halogen atom. DL-DEX 312 resembles DL-2-haloacid dehalogenase from Pseudomonas sp. 113 (DL-DEX 113) in that the reaction proceeds with a direct attack of a water molecule on the substrate. However, DL-DEX 312 is markedly different from DL-DEX 113 in its substrate specificity. We found that DL-DEX 312 catalyzes the hydrolytic dehalogenation of 2-chloropropionamide and 2-bromopropionamide, which do not serve as substrates for DL-DEX 113. DL-DEX 312 is the first enzyme that catalyzes the dehalogenation of 2-haloacid amides.
Keuning, S., Janssen, D.B. and Witholt, B. (1985) Purification and Characterization of Hydrolytic Haloalkane Dehalogenase from Xanthobacter-Autotrophicus GJ10. Journal of Bacteriology 163(2), 635-639.    

 

Measuring DBP Degraders
Citation Notes Abstract
Leach, L.H., Zhang, P., LaPara, T.M., Hozalski, R.M. and Camper, A.K. (2009) Detection and enumeration of haloacetic acid-degrading bacteria in drinking water distribution systems using dehalogenase genes. Journal of Applied Microbiology 107(3), 978-988.
  To develop a PCR-based tracking method for the detection of a subset of bacteria in drinking water distribution systems capable of degrading haloacetic acids (HAAs). Methods and Results: Published degenerate PCR primers were used to determine that 54% of tap water samples (7/13) were positive for a deh gene, indicating that drinking water distribution systems may harbour bacteria capable of HAA degradation. As the published primer sets were not sufficiently specific for quantitative PCR, new primers were designed to amplify dehII genes from selected indicator strains. The developed primer sets were effective in directly amplifying dehII genes from enriched consortia samples, and the DNA extracted from tap water provided that an additional nested PCR step for detection of the dehII gene was used. Conclusions: This study demonstrates that drinking water distribution systems harbour microbes capable of degrading HAAs. In addition, a quantitative PCR method was developed to detect and quantify dehII genes in drinking water systems. Significance and Impact of the Study: The development of a technique to rapidly screen for the presence of dehalogenase genes in drinking water distribution systems could help water utilities determine if HAA biodegradation is occurring in the distribution system.
Bachas-Daunert, P.G., Sellers, Z.P. and Wei, Y.N. (2009) Detection of halogenated organic compounds using immobilized thermophilic dehalogenase. Analytical and Bioanalytical Chemistry 395(4), 1173-1178.   Environmental pollutants containing halogenated organic compounds can cause a plethora of health problems. Detection, quantification, and eventual remediation of halogenated pollutants in the environment are important to human well-being. Toward this end, we previously identified a haloacid dehalogenase, L-HAD(ST), from the thermophile Sulfolobus tokodaii. This thermophilic enzyme is extremely stable and catalyzes, stereospecifically, the dehalogenation of L-2-haloacids. In the current study, we covalently linked L-HAD(ST) to an N-hydroxysuccinimidyl Sepharose resin to construct a highly specific sensor with long shelf life for the detection of L-2-haloacids. The enzyme-modified resin was packed into disposable columns. Samples containing L-2-haloacids were first incubated in the column, and were then collected to quantify the chloride produced through the breakdown of the substrate. The optimum pH of the immobilized enzyme is around 9.5, similar to that of the soluble protein. Its catalytic activity increased with temperature up to the highest temperature measured (50 degrees C). The resin could be fully regenerated after multiple reaction cycles and retained 70% of the initial activity after being stored at 4 degrees C for 6 months. The L-HAD(ST)-modified resin could be used to breakdown and quantify L-2-haloacids spiked in the simulated environmental samples, indicating dehalogenases from extremophiles can potentially be employed in the detection and decontamination of L-2-haloacids.

Fundamentals of Biogrowth in Distribution Systems
Citation Notes Abstract
Speitel, G.E., Kannappan, R. and Bayer, B.M. (2011) The nitrification index: a unified concept for quantifying the risk of distribution system nitrification. Journal American Water Works Association 103(1), 69-80.   For drinking water providers using chloramination as a residual disinfectant, nitrification can constitute an ongoing problem, resulting in loss of disinfectant residual and regrowth of heterotrophic bacteria. The laboratory-scale annular reactor experiments in this research advance the understanding of the role trihalomethanes (THMs) play in inhibiting distribution system nitrification as a result of their cometabolism by nitrifying bacteria. In addition, the authors place THMs within the context of the other key factors that determine the risk of nitrification. As practical guidance-for utilities, the authors introduce the nitrification index (NI), a framework for assessing nitrification risks. Developed from the fundamental kinetics of nitrifier growth and inactivation by monochloramine and THMs, the NI offers utilities an easy and convenient way to determine the likelihood of nitrification episodes. By tracking the NI of their distribution systems over time-essentially using it as an early warning system-water providers may be. able to make timely adjustments in their treatment processes before nitrification begins.
Zhang, Y., Edwards, M., Pinto, A., Love, N., Camper, A.K., Rahman, M. and Baribeau, H. (2010) Effect of Nitrification on Corrosion in the Distribution System, WRF #4015, Denver CO.    
Zhang, Y. and Edwards, M. (2010) Nutrients and metals effects on nitrification in drinking water systems. Journal American Water Works Association 102(7), 56-66.   Limiting microbial growth via control of nutrients has been well studied for heterotrophic bacteria, but there are few studies on this approach for control of nitrifiers in drinking water. The objective of this study was to investigate the impact of macronutrients, trace nutrients, and pipe materials on nitrification activity under simulated premise plumbing conditions. Nitrification can vary dramatically in a distribution system, depending on the pipe materials used and the nutrients that are present. Utilities concerned about effects of nitrification should examine extremes of the distribution system, including premise plumbing systems, to characterize the full range of possible effects. Water suppliers can use the results to guide sampling and detect parts of the distribution system that are most susceptible to growth of nitrifying bacteria. Control of nitrification is a complex problem with public health implications, so this knowledge will be helpful in planning holistic and comprehensive management of distribution systems.
Morton, S.C., Zhang, Y. and Edwards, M.A. (2005) Implications of nutrient release from iron metal for microbial regrowth in water distribution systems. Water Research 39(13), 2883-2892.
  Control of microbial regrowth in iron pipes is a major challenge for water utilities. This work examines the interrelationship between iron corrosion and bacterial regrowth, with a special focus on the potential of iron pipe to serve as a source of phosphorus. Under some circumstances, corroding iron and steel may serve as a source for all macronutrients necessary for bacterial regrowth including fixed carbon, fixed nitrogen and phosphorus. Conceptual models and experimental data illustrate that levels of phosphorus released from corroding iron are significant relative to that necessary to sustain high levels of biofilm bacteria. Consequently, it may not be possible to control regrowth on iron surfaces by limiting phosphorus in the bulk water.