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

Katz, Lynn E.

Kirisits, Mary Jo

Speitel, Gerald E.

Wahman, David G.

Applied and Environmental Microbiology

PubMed

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.AwwaRF Civil, Architectural, and Environmental Engineerin

Texas ScholarWorks

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2011, p. 2537–2540 Vol. 77, No. 70099-2240/11/$12.00 doi:10.1128/AEM.02464-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.Ammonia-Oxidizing Bacteria in Biofilters Removing TrihalomethanesAre Related to Nitrosomonas oligotrophaDavid G. Wahman,1* Mary Jo Kirisits,2 Lynn E. Katz,2 and Gerald E. Speitel, Jr.2United States Environmental Protection Agency, Office of Research and Development, Cincinnati, Ohio 45268,1 and University ofTexas at Austin, Department of Civil, Architectural, and Environmental Engineering, Austin, Texas 787122Received 18 October 2010/Accepted 23 January 2011Ammonia-oxidizing bacteria (AOB) in nitrifying biofilters degrading four regulated trihalomethanes—trichloromethane, bromodichloromethane, dibromochloromethane, and tribromomethane—were related toNitrosomonas oligotropha. N. oligotropha is associated with chloraminated drinking water systems, and itspresence in the biofilters might indicate that trihalomethane tolerance is another reason that this bacteriumis dominant in chloraminated systems.No evidence indicates that trihalomethanes (THMs) supportmicrobial growth, but previous research demonstrated thatfour regulated THMs—trichloromethane (TCM), bromodi-chloromethane (BDCM), dibromochloromethane (DBCM),and tribromomethane (TBM)—were cometabolized in batchculture (15) by Nitrosomonas europaea (an ammonia oxidizer)and in batch culture (14) and laboratory-scale biofilters (16,17) by mixed-culture nitrifiers. The current research extendedsuch process evaluation by analyzing the ammonia-oxidizingbacteria (AOB) present in biofilters through quantitative real-time PCR (qPCR) and amoA gene sequence analysis.Duplicate anthracite biofilters (1-min empty-bed contacttime; 1.3 gallons min1 ft2 surface loading rate) were seededwith one of two mixed cultures (14). Trains A and B wereseeded with a sample collected from Lake Austin in Austin,TX, and trains C and D were seeded with an enriched nitrifierculture initially dominated by Nitrosomonas oligotropha (N.oligotropha enrichment, provided by D. R. Noguera, Universityof Wisconsin). The trains were fed 4 mg N/liter TOTNH3 (sumof ammonia-nitrogen [NH3-N] and ammonium-nitrogen[NH4-N]). The three operational periods were based on in-fluent THM concentrations as follows: period I, TCM-DBCM(50 g/liter TCM and 25 g/liter DBCM); period II, DBCM(25 g/liter DBCM); and period III, all THMs (15 g/litereach of TCM, DBCM, BDCM, and TBM). THM removals aresummarized in Table 1 for each period; all biofilters removedTHMs with removals ranging from 7 to 24% (Table 1).At the end of period II, backwash water was collected forbatch kinetic tests conducted as described previously (16). Be-cause backwash water may have contained extracellular mate-rial, attachment media, or nonnitrifying organisms, a biomass-independent analysis was required. To accomplish this, apreviously described (16) simplified THM cometabolismmodel (equation 1) was used, as follows:STHM1STHM0 eTOTNH3k1THMkTOTNH3 (1)where STHM0 is the initial THM concentration (g/liter THM);STHM1 is the final THM concentration (g/liter THM);TOTNH3 is initial TOTNH3 minus final TOTNH3 (mg N/li-ter); and k1THM/kTOTNH3 is the ratio of THM and TOTNH3 rateconstants (liter/mg TOTNH3). The k1THM/kTOTNH3 ratio repre-sents the THM removal efficiency, relating THM to ammoniaremoval. For purposes of comparison, the biofilter k1THM /kTOTNH3 ratios were calculated by pooling operational data forduplicate trains (where biofilter THM removal was not statis-tically different between duplicate trains, based on Tukey’spaired comparison method with a two-sided 95% confidenceinterval of the studentized range statistic [2, 4]). The backwashbatch kinetic test k1THM/kTOTNH3 ratios (Fig. 1) provided an-other confirmation that the biofilters were able to degradeTHMs (initial concentrations, 70 to 120 g/liter each THMand 5 mg N/liter TOTNH3).At the end of period III, biofilter DNA was isolated using anUltraClean soil DNA isolation kit (Mo Bio Laboratories, Inc.,Carlsbad, CA). The biofilters were divided into 4 equallyspaced vertical sections (influent end is designated section 1,and effluent end is designated section 4). The amoA qPCRmethod of Regan et al. (7) was used to assess the relativeabundance of AOB in three general classes: (i) non-Oligotro-pha nitrosomonas, (ii) Nitrosospira, and (iii) N. oligotropha.Only N. oligotropha was detected (data not shown), indicatingthat this is the dominant AOB present.Subsequently, amoA genes from trains A and C were clonedand sequenced to supplement the qPCR results. For cloning,amoA was amplified (7), and the amplicon purified using aQIAquick PCR purification kit (Qiagen, Valencia, CA) andcloned using a TOPO TA cloning kit for sequencing (Invitro-gen, Carlsbad, CA). The clones were screened for inserts byPCR with M13 forward and reverse primers. The amplicon wassequenced using BigDye sequencing chemistry (Applied Bio-systems, Foster City, CA).Using MEGA 4 (12), 452-bp sequences (primer sequencesremoved) were generated, and DOTUR was used to sortamoA gene sequences with more than 98% similarity (11) intooperational taxonomic units (OTUs). Analyses of cloned* Corresponding author. Mailing address: United States Environ-mental Protection Agency, 26 W. Martin Luther King Dr., Cincinnati,OH 45268. Phone: (513) 569-7733. Fax: (513) 487-2543. E-mail:wahman.david@epa.gov. Published ahead of print on 28 January 2011.2537 on July 24, 2015 by Univ of Texas Austinhttp://aem.asm.org/Downloaded from amoA gene sequences found two OTUs (GenBank accessionnumbers HQ399455 and HQ399456) that were present in bothtrains, hereinafter designated Biofilter OTU 1 and 2 (Table 2).These OTUs were incorporated into a phylogenetic tree(Fig. 2) constructed in MEGA4 by the neighbor-joiningmethod using 10,000 bootstrap replicates. The sequence anal-ysis confirmed the qPCR results, as these two OTUs wererelated to N. oligotropha whether the phylogenetic tree wasgenerated based on amoA gene sequences (data not shown) oron deduced amino acid sequences from the amoA gene se-quences (Fig. 2). Considering deduced amino acid sequences,the Biofilter OTU 1 consensus sequence was a 100% match tosamples taken from a wastewater treatment plant activatedsludge and membrane bioreactor and to Nitrosomonas NL7and NM47, and the Biofilter OTU 2 consensus sequence was a100% match to an environmental sample taken from a chlo-raminated drinking water distribution system. Taken together,the qPCR and gene sequence analyses show that the dominantAOB in the biofilters used for THM removal were related to N.oligotropha. AOB related to N. oligotropha have been reportedas the dominant AOB in chloraminated drinking water systems(7–9). Because these systems contain THMs at levels similar tothose used in our biofilter experiments, it is possible that onereason for the presence of AOB related to N. oligotropha inchloraminated drinking water systems is their ability to tolerateTHMs. This is supported by the results of Bayer and Speitel(1), who found that the transformation capacities (includingthose of an N. oligotropha-dominated culture) were greaterthan those for N. europaea (15).TABLE 1. Biofilter performanceaTrain Period No. ofsamples0-1TOTNH3(mg N/liter)b% Removal (mean  standard deviation)cTCM BDCM DBCM TBMA I 6 2.7  0.29 9.7  6.4 12  5.3II 3 3.4  0.26 24  2.3III 4 3.1  0.38 16  7.6 18  6.5 19  6.3 16  6.2B I 7 2.1  0.36 7.7  5.2 13  8.3II 3 3.0  0.23 14  2.0III 4 2.5  0.37 13  6.2 14  6.0 15  5.2 13  3.8C I 6 2.6  0.74 6.9  6.0 12  5.0II 2 2.5  0.62 17  2.0III 4 2.8  0.67 14  5.7 21  8.1 23  6.0 18  3.8D I 7 2.0  0.56 6.5  6.5 13  7.3II 3 1.7  0.22 10  3.2III 4 2.4  1.1 16  6.9 16  7.6 17  8.1 15  7.6a Trains were nominally fed 4 mg N/liter TOTNH3 (sum of ammonia-nitrogen NH3-N and ammonium-nitrogen NH4-N). The three operational periods werebased on influent THM concentrations as follows: period I, TCM-DBCM (50 g/liter TCM and 25 g/liter DBCM); period II, DBCM (25 g/liter DBCM); and periodIII, all THMs (15 g/liter each of TCM, BDCM, DBCM, and TBM).b 0-1TOTNH3, TOTNH3 removed through biofilter (mean  standard deviation).c TCM, trichloromethane; BDCM, bromodichloromethane; DBCM, dibromochloromethane; TBM, tribromomethane.FIG. 1. Backwash batch kinetic test k1THM/kTOTNH3 ratios and asso-ciated 95% confidence limits. A, B, C, and D correspond to experi-ments conducted with backwash from trains A, B, C, and D, respec-tively. For comparison purposes, k1THM/kTOTNH3 ratios were alsocalculated using equation 1 and biofilter performance data. LA, LakeAustin; NO, N. oligotropha enrichment.TABLE 2. amoA gene sequences and OTU biofiltersample summarySampleaNo. of amoA gene sequences in:Biofilter OTU 1 Biofilter OTU 2A1 (influent end) 105 25A2 67 3A3 8 1A4 (effluent end) 14 0Total for A 194 29C1 (influent end) 73 0C2 74 0C3 73 1C4 (effluent end) 65 0Total for C 285 1Total for A and C 479 30a The influent end of the biofilter was designated section 1, with the numberingprogressing down the length of the biofilter in equal lengths such that the effluentend was designated section 4. Each section was divided into three subsamplesthat were used for DNA extraction, resulting in triplicate extractions for eachsection. For the extractions, the mass of dry anthracite used was 0.60  0.06 g(mean  standard deviation) per extraction (wet anthracite moisture content,32%  0.74% [mean  standard deviation]).2538 WAHMAN ET AL. APPL. ENVIRON. MICROBIOL. on July 24, 2015 by Univ of Texas Austinhttp://aem.asm.org/Downloaded from Overall, four regulated THMs found in treated drinkingwater were degraded in biofilters seeded with Lake Austinand N. oligotropha enrichment cultures. The ability of thebiofilter biomass to remove THMs was verified in batchkinetic tests. amoA qPCR and gene sequence analyses dem-onstrated that the biofilter AOB were related to N. oligo-tropha. Their presence in biofilters removing THMs suggeststhat one potential reason for their dominance in these sys-tems is their THM tolerance. Future research shouldbroaden the nitrifier community analysis to include ammo-nia-oxidizing archaea, since they are being reported indrinking water systems (6, 13).We thank John M. Regan for plasmids used in constructing qPCRstandard curves and Daniel R. Noguera for a mixed culture source.This research was funded by AwwaRF.Any opinions expressed are those of the authors and do not neces-sarily reflect the views of U.S. Environmental Protection Agency orAwwaRF; therefore, no official endorsement should be inferred. Anymention of trade names or commercial products does not constituteendorsement or recommendation for use.REFERENCES1. Bayer, B. M., and G. E. Speitel, Jr. 2007. Significance of trihalomethanes inpreventing distribution system nitrification. 2007 Am. Water Works Assoc.Annu. Conf. Proc. American Water Works Association, Denver, CO.2. Berthouex, P. M., and L. C. Brown. 2002. Statistics for environmental engi-neers. Lewis Publishers, Boca Raton, FL.3. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach usingthe bootstrap. Evolution 39:783–791.4. Harter, H. L. 1960. Tables of range and studentized range. Ann. Math. Stat.31:1122–1147.5. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generationof mutation data matrices from protein sequences. Comput. Appl. Biosci.8:275–282.6. Kasuga, I., H. Nakagaki, F. Kurisu, and H. Furumai. 2010. Predominance ofammonia-oxidizing archaea on granular activated carbon used in a full-scaleadvanced drinking water treatment plant. Water Res. 44:5039–5049.7. Regan, J. M., A.-Y. Cho, S. Kim, and C. D. Smith. 2007. Monitoring ammo-nia-oxidizing bacteria in chloraminated distribution systems. AwwaRF Re-port 91162. Water Research Foundation, Denver, CO.8. Regan, J. M., G. W. Harrington, H. Baribeau, R. D. Leon, and D. R. Nogu-era. 2003. Diversity of nitrifying bacteria in full-scale chloraminated distri-bution systems. Water Res. 37:197–205.9. Regan, J. M., G. W. Harrington, and D. R. Noguera. 2002. Ammonia- andnitrite-oxidizing bacterial communities in a pilot-scale chloraminated drink-ing water distribution system. Appl. Environ. Microbiol. 68:73–81.10. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new methodfor reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406–425.FIG. 2. Phylogenetic tree based on deduced amino acid sequences of the amoA gene sequences of AOB. The evolutionary history was inferredusing the neighbor-joining method (10). The percentage of replicate trees (those 50%) in which the associated taxa clustered together in thebootstrap test (10,000 replicates) are shown next to the branches (3). The tree is drawn to scale, with branch lengths in the same units as thoseof the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the JTT matrix-based method(5), and the unit of measure is the number of amino acid substitutions per site, shown by the bar. The number of sequences belonging to theOTU/total number of sequences is given in parentheses.VOL. 77, 2011 N. OLIGOTROPHA IN NITRIFYING BIOFILTERS REMOVING THMs 2539 on July 24, 2015 by Univ of Texas Austinhttp://aem.asm.org/Downloaded from 11. Schloss, P. D., and J. Handelsman. 2005. Introducing DOTUR, a computerprogram for defining operational taxonomic units and estimating speciesrichness. Appl. Environ. Microbiol. 71:1501–1506.12. Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: MolecularEvolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol.Evol. 24:1596–1599.13. van der Wielen, P. W. J. J., S. Voost, and D. van der Kooij. 2009. Ammonia-oxidizing bacteria and archaea in groundwater treatment and drinking waterdistribution systems. Appl. Environ. Microbiol. 75:4687–4695.14. Wahman, D. G., A. E. Henry, L. E. Katz, and G. E. Speitel, Jr. 2006.Cometabolism of trihalomethanes by mixed culture nitrifiers. Water Res.40:3349–3358.15. Wahman, D. G., L. E. Katz, and G. E. Speitel, Jr. 2005. Cometabolism oftrihalomethanes by Nitrosomonas europaea. Appl. Environ. Microbiol. 71:7980–7986.16. Wahman, D. G., L. E. Katz, and G. E. Speitel, Jr. 2011. Performance andbiofilm activity of nitrifying biofilters removing trihalomethanes. Water Res.45:1669–1680.17. Wahman, D. G., L. E. Katz, and G. E. Speitel, Jr. 2006. Trihalomethane cometabo-lism by a mixed-culture nitrifying biofilter. J. Am. Water Works Assoc. 98:2–20.2540 WAHMAN ET AL. APPL. ENVIRON. MICROBIOL. on July 24, 2015 by Univ of Texas Austinhttp://aem.asm.org/Downloaded from 

Ammonia-Oxidizing Bacteria In Biofilters Removing Trihalomethanes Are Related To Nitrosomonas Oligotropha

ABSTRACT
          
            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.
          </jats:p

David G. Wahman

Mary Jo Kirisits

Lynn E. Katz

Gerald E. Speitel

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Ammonia-Oxidizing Bacteria in Biofilters Removing Trihalomethanes Are Related to
            <i>Nitrosomonas oligotropha</i>

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3067439

Ammonia-Oxidizing Bacteria in Biofilters Removing Trihalomethanes Are Related to Nitrosomonas oligotropha ▿

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