Microbes Environ. Vol. 24, No. 1, 72–75, 2009 http://wwwsoc.nii.ac.jp/jsme2/ doi:10.1264/jsme2.ME08556 Short Communication

Analysis of Bacterial Community Diversity in Anaerobic Fluidized Bed Bioreactors Treating 2,4-Dinitroanisole (DNAN) and n-Methyl-4-nitroaniline (MNA) Using 16S rRNA Gene Clone Libraries

CLINT M. ARNETT1*, GISELLE RODRIGUEZ1, and STEPHEN W. MALONEY1 1US Army Engineer Research and Development Center, Construction Engineering Research Laboratory, 2902 Newmark Dr., Champaign, IL 61822, USA (Received October 27, 2008—Accepted January 21, 2009—Published online February 16, 2009)

Clone libraries were used to evaluate the effects of 2,4-dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA) on bacterial populations within three anaerobic bioreactors. Prior to the addition of DNAN and MNA greater than 69% of the clones in each reactor were identified as a single Desulfuromonales species. However, after 60 days of treatment the Desulfuromonales distribution decreased to less than 13% of the distribution and a clone identified as a Levilinea sp. became the dominant organism at greater than 27% of the clone distribution in each reactor suggesting the species may play an important roll in the reduction of DNAN and MNA. Key words: 2,4-dinitroanisole, DNAN, insensitive munitions, MNA, n-methyl-4-nitroaniline

Many defense agencies have a goal to phase out traditional demonstrated effective treatment of energetic compounds 2,4,6-trinitrotoluene (TNT) based explosives in favor of using bioreactor technologies (2, 3, 9). Although the engi- Insensitive Munitions (IM). IM retain or surpass the per- neering aspects of bioreactors are fairly well understood, formance of traditional TNT based explosives, but are char- very little is known about the microbial communities present acteristically more resistant to heat and shock detonation within the reactor systems. making them safer to manufacture, transport and store. Here we examined the effects of DNAN and MNA on the Picatinny Arsenal eXplosive 21 (PAX-21) is one such IM that microbial diversity in three separate anaerobic fluidized bed is currently in production within the U.S. and has been test reactor (AFBR) systems. A direct cloning and sequencing loaded at select Army ammunition plants. PAX-21 contains methodology using the 16S rRNA gene was used to charac- a mixture of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), terize each reactor prior to the addition of IM constituents ammonium perchlorate, 2,4-dinitroanisole (DNAN), and n- and after a 60 day treatment period to evaluate the effects on methyl-4-nitroaniline (MNA). Although the exact composi- the population dynamics of each AFBR. tion of PAX-21 is proprietary, it is estimated that nearly half Three 9.1 L AFBRs were designed and operated as of the dry weight is composed of DNAN and MNA. described by Atikovic et al. (2) in order to evaluate the effec- As militaries continue to transition from conventional tiveness of biological IM treatment. Bioreactor 1 (AFBR #1) munitions to IM, substantial amounts of DNAN and MNA treated DNAN, bioreactor 2 (AFBR #2) treated MNA, and will begin entering wastestreams. Both DNAN and MNA are bioreactor 3 (AFBR #3) treated a combination of both nitroaromatic compounds having two and one nitro group DNAN and MNA simultaneously. The bioreactor feed con- substituents, respectively. Cyclic nitramines have a strong sisted of nutrient and buffer solutions as described by electron-withdrawing effect, which can result in acute Maloney et al. (Anaerobic treatment of wastewaters con- toxicity in both humans and wildlife (10). Dodd and taining perchlorate from munitions handling and production. McDougal (Recommendation of an occupational exposure ERDC/CERL TR-08-3) and a contaminant feed stock con- level for PAX-21. AFRL-HE-WP-TR-2001-0103) have taining 600 ppm ethanol and varying IM concentrations. The demonstrated that DNAN is in fact more toxic than TNT, the bioreactors were operated at 35°C and pH was maintained at explosive it is to replace. Due to the recalcitrant nature of 7. The total aqueous flow rate was approximately 6 L day−1 nitroaromatic compounds and their generalized toxic effects, consisting of 0.5 L nutrient stock, 0.5 L buffer solution and 5 release into the environment is highly regulated. Thus, L contaminant feed stock. The empty-bed hydraulic retention wastewater generated during production, load and pack, and time was approximately 36 hours. The reactor was seeded demilitarization operations containing DNAN and MNA with 50 mL of anaerobic digester sludge obtained from a must be treated prior to discharge. pilot-scale system at the University of Cincinnati treating Biotransformation is considered an economical treatment RDX. The reactors were allowed to stabilize and fluidization option that possesses the potential to transform explosive was optimized prior to the addition of IM. After a 200 day constituents to innocuous compounds. Many studies have period, DNAN and MNA were added to the feed stocks to achieve targeted influent concentrations of 21 ppm and 4 * Corresponding author. E-mail: [email protected]; Tel: ppm, respectively. After 30 days of acclimation, the concen- +1–217–398–5507; Fax: +1–217–373–3430. trations were increased to 42 ppm DNAN and 8 ppm MNA. Bioreactor Bacterial Diversity 73

Three mL homogenized mixed-liquor samples containing The purified PCR amplicons were cloned using an Invitro- both liquid and sand were taken at day 200 prior to the addi- gen TOPO TA Cloning Kit according to the manufacturer’s tion of IM and after 60 days of IM treatment. instructions. One hundred random, well isolated, colonies Genomic DNA was extracted and 16S rRNA genes were containing interests were picked from the plates and grown amplified as described by Arnett et al. (1) with the exception overnight at 37°C in liquid LB containing 100 µg mL−1 a universal 926 reverse primer was used for gene amplifica- spectinomycin. Random clones were screened for recombi- tion. Three PCR reactions were performed for each sample nation by PCR using M13 primers. Resulting PCR products and pooled prior to cloning. Both DNA extractions and puri- were-sized on a SYBR Safe 1% agarose gel as described fied PCR amplifications were quantified using a Quant-iT above. Plasmid DNA was extracted from the cultures using DNA Assay Kit (Invitrogen, Carlsbad, CA) according to the heat lysis and inserts were sequenced by the W. M. Keck manufacture’s instructions. Purity was confirmed by gel Center for Comparative and Functional Genomics (University electrophoresis using a SYBR Safe (Invitrogen) 1% agarose of Illinois, Urbana, IL) using a Big Dye Terminator reaction gel ran at 70 V for 45 min. Gels were visualized under UV (Applied Biosystems, Foster City, CA). Read sequences light and products were-sized using a 100 bp DNA ladder were manually edited with Sequencher v4.2 computational (Promega, Madison, WI). sequencing software (Gene Codes Corporation, Ann Arbor,

Table 1. Ribosomal DNA clone distribution in three anaerobic fluidized bed bioreactors treating 2,4-dinitroanisole (DNAN) (AFBR #1), n-methyl-4-nitroaniline (MNA) (AFBR #2), and DNAN and MNA (AFBR #3) AFBR #1 AFBR #2 AFBR #3 DNAN DNAN GenBank a DNAN DNAN MNA MNA (0 ppm) (42 ppm) Clone accession Preliminary classification (0 ppm) (42 ppm) (0 ppm) (8 ppm) MNA MNA number (0 ppm) (8 ppm) no.b % no.c % no.d % no.e % no.f % no.g % UC-1 FJ228416 Bacteria/unclassified 3 3.8 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 UC-2 FJ228417 Clostridia/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 0 0.0 UC-3 FJ228418 Incertae Sedis XV/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 0 0.0 UC-4 FJ228419 Ruminococcaceae/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 0 0.0 UC-5 FJ228420 Veillonellaceae/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 0 0.0 UC-6 FJ228421 Bacteria/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 0 0.0 UC-7 FJ228422 Caldilineacea/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 0 0.0 UC-8 FJ228423 Caldilineacea/unclassified 0 0.0 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 UC-9 FJ228424 Bacteria/unclassified 0 0.0 0 0.0 0 0.0 5 5.6 0 0.0 0 0.0 UC-10 FJ228425 Bacteria/unclassified 0 0.0 0 0.0 0 0.0 0 0.0 1 1.1 0 0.0 UC-11 FJ228426 Bacteroidales/unclassified 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 1.1 UC-12 FJ228427 Bacteria/unclassified 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 1 1.1 UC-13 FJ228428 Bacteria/unclassified 5 6.3 0 0.0 3 3.3 7 7.9 0 0.0 6 6.7 UC-14 FJ228429 Levilinea 0 0.0 37 41.1 8 8.8 32 36.0 10 11.1 25 27.8 UC-15 FJ228430 Bacteria/unclassified 7 8.9 0 0.0 1 1.1 4 4.5 3 3.3 9 10.0 UC-16 FJ228431 Bacteroidales/unclassified 1 1.3 3 3.3 0 0.0 2 2.2 1 1.1 8 8.9 UC-17 FJ228432 Bacteria/unclassified 0 0.0 0 0.0 1 1.1 12 13.5 0 0.0 5 5.6 UC-18 FJ228433 Magnetobacterium 1 1.3 2 2.2 0 0.0 1 1.1 1 1.1 4 4.4 UC-19 FJ228434 Desulfuromonales/unclassified 55 69.6 3 3.3 76 83.5 8 8.9 67 74.4 11 12.2 UC-20 FJ228435 Bacteria/unclassified 0 0.0 1 1.1 1 1.1 1 1.1 0 0.0 1 1.1 UC-21 FJ228436 Deltaproteobacteria/unclassified 0 0.0 1 1.1 0 0.0 2 2.2 0 0.0 1 1.1 UC-22 FJ228437 Smithella 1 1.3 1 1.1 0 0.0 0 0.0 2 2.2 0 0.0 UC-23 FJ228438 Deltaproteobacteria/unclassified 1 1.3 0 0.0 0 0.0 1 1.1 1 1.1 0 0.0 UC-24 FJ228439 Bacteria/unclassified 0 0.0 1 1.1 0 0.0 4 4.5 0 0.0 5 5.6 UC-25 FJ228440 Bacteroidetes/unclassified 1 1.3 0 0.0 0 0.0 1 1.1 1 1.1 1 1.1 UC-26 FJ228441 Dechloromonas 2 2.5 0 0.0 0 0.0 0 0.0 0 0.0 2 2.2 UC-27 FJ228442 Bacteria/unclassified 0 0.0 1 1.1 0 0.0 0 0.0 0 0.0 1 1.1 UC-28 FJ228443 Veillonellaceae/unclassified 0 0.0 4 4.4 0 0.0 0 0.0 0 0.0 0 0.0 UC-29 FJ228444 Bacteria/unclassified 0 0.0 1 1.1 0 0.0 1 1.1 0 0.0 0 0.0 UC-30 FJ228445 Deltaproteobacteria/unclassified 1 1.3 0 0.0 0 0.0 0 0.0 1 1.1 0 0.0 UC-31 FJ228446 Fusibacter 0 0.0 0 0.0 0 0.0 8 8.9 0 0.0 2 2.2 UC-32 FJ228447 Spirochaetaceae/unclassified 0 0.0 2 2.2 0 0.0 0 0.0 2 2.2 0 0.0 UC-33 FJ228448 Bacteroidales/unclassified 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 3 3.3 UC-34 FJ228449 Bacteria/unclassified 0 0.0 27 30.0 0 0.0 0 0.0 0 0.0 1 1.1 UC-35 FJ228450 Clostridium 0 0.0 0 0.0 0 0.0 0 0.0 0 0.0 2 2.2 UC-36 FJ228451 Caldilineacea/unclassified 1 1.3 0 0.0 0 0.0 0 0.0 0 0.0 1 1.1 a Determined with RDP Classifier program using naïve Bayesian rRNA classifier (13). b Number of clones out of a total of 79 prior to the addition of DNAN. c Number of clones out of a total of 90 after 60 days of DNAN treatment. d Number of clones out of a total of 91 prior to the addition of MNA. e Number of clones out of a total of 89 after 60 days MNA treatment. f Number of clones out of a total of 90 prior to the addition of DANA and MNA. g Number of clones out of a total of 90 after 60 days DNAN and MNA treatment. 74 ARNETT et al.

MI). Approximately 550 clones were successfully sequenced bution dropped to 6% of the clone distribution and the having an average length of roughly 700 bp. All sequences unclassified Desulfuromonales population decreased to were examined for chimera using CHIMERA_CHECK v2.7 approximately 3%. Three bacteria belonging to the phylum chimera detection program (http://rdp8.cme.msu.edu/cgis/ Chloroflexi were identified in the reactor, two unclassified chimera.cgi?su=SSU) and suspect sequences were removed Caldilineacea species (clones UC-7 and UC-36) and one from the data set. Sequences were aligned with Sequencher Levilinea sp. (clone UC-14). Clone UC-7 was not detected v4.2 resulting in the identification of 36 unique clones. prior to the addition of DNAN and made up only 1% of the The Ribosomal Database Project II (RDP) Release 9.51 distribution after 60 days of treatment. Clone UC-36 com- Classifier program (http://rdp.cme.msu.edu/classifier/ posed 1% of the distribution prior to the addition of DNAN, classifier.jsp) was used to assign each unique clone to princi- but was not detected after 60 day of treatment. The Levilinea pal hierarchical taxa based on a naïve Bayesian rRNA classi- sp. was undetected prior to treatment, yet became the domi- fication (13). All unique clone 16S rRNA gene sequences nant organism in the presences of DNAN at greater than 41% were submitted to GenBank and assigned accession numbers of the distribution. Clone UC-34 (unclassified bacteria), FJ228416–FJ228451 (Table 1). which was also not detected in the reactor prior to the addi- Throughout the 60 day treatment period effluent concen- tion of DNAN, became the second most abundant organism trations of DNAN and MNA remained below HPLC detec- after the 60 day treatment period at 30% of the total clone tion limits of 0.07 ppm and 0.1 ppm, respectively in each of distribution. the three reactors. Liquid samples were analyzed periodically As shown in Table 1, the microbial distribution in AFBR by LC-MS in positive electrospray ionization mode and mass #2 was also dominated by clone UC-19 (unclassified ions representing reduced DNAN and MNA were observed Desulfuromonales) at 84% of the total clone distribution (Fig. 1). DNAN was reduced to diaminoanisole and MNA to prior to the addition of IM. The second most abundant clone, 4-methoxy-m-phenylenediamine. which made up 9% of the clone distribution, was clone UC-14 Similar bioreactor studies conducted with dinitrotoluene (Levilinea sp.). One additional Chloroflexi species was also have demonstrated anaerobic biological reduction to dia- identified, clone UC-8, which comprised 1% of the distribu- minotoluene, which was easily degraded under aerobic tion. The remaining clones were four species of unclassified conditions via electrophilic attack (3). This suggests that bacteria that collectively made up approximately 7% of anaerobically reduced IM could be discharged to aerobic the total profile. After 60 days of MNA treatment, the wastewater treatment plants for complete degradation. Desulfuromonales clone distribution decreased to less than Table 1 shows the bacterial clone distribution within 9% of the population and the Levilinea distribution increased AFBR #1. Prior to the addition of DNAN the clone distribu- to 36% of the distribution. One Firmicutes species (clone tion in AFBR #1 was dominated by Proteobacteria (76%). UC-31) was identified, which composed 9% of the distri- Nearly 70% of the clones sequenced were of a single unclas- bution. Seven species of unclassified bacteria, which collec- sified Desulfuromonales species (clone UC-19). The second tively made up 38% of the total clone distribution, were also most abundant clone, making up 9% of the clone distribu- identified but clone UC-34 (unclassified bacterium) was not tion, was an unclassified bacterium. The third most abundant detected in the presence of MNA. clone was yet another unclassified bacterium, which made up The bacterial phylogenetic distribution within AFBR #3 is 6% of the distribution and all other clones made up less than summarized in Table 1. Eleven percent of the clone distribu- 4% of the total clone distribution. The addition of DNAN to tion was made up of bacteria from the phylum Chloroflexi the reactor had a significant effect on the dynamics of the and 79% from the phylum Proteobacteria. As with the microbial population. The total Proteobacteria clone distri- reactors treating IM separately, UC-19 (unclassified Desulfuromonales) dominated the microbial clone distri- bution at 74% of the distribution prior to IM treatment. How- ever, after the 60 day treatment period clone UC-19 decreased to approximately 12% of the distribution. UC-14 (Levilinea sp.) was the second most abundant clone within the system making up roughly 11% of the clone distribution prior to the addition of IM. After the treatment period, the Levilinea clone distribution increased to 28% of the clone distribution. Seven species of unclassified bacteria, making up 32% of the total clone distribution, were also identified. Clone UC-34 made up only 1% of the total distribution. Published accounts of microbial diversity within nitramine treating bioreactors are limited. Adrian et al. (Characteriza- tion of microbial communities in an anaerobic fluidized bioreactor treating TNT using molecular techniques. ERDC/ CERL TR-01-10) characterized the microbial communities of AFBRs treating TNT using oligonucleotide probes and found that Proteobacteria populations increased as the TNT Fig. 1. Diaminoanisole and 4-methoxy-m-phenylenediamine for- mation in anaerobic fluidized bed bioreactors (AFBR) treating 2,4- loading concentrations increased, which indicated possible dinitroanisole (DNAN) and n-methyl-4-nitroaniline (MNA). involvement in the degradation process. Kimura et al. (5) Bioreactor Bacterial Diversity 75 studied the microbial community in a 2,4-dinitrophenol (2,4- and a single Chloroflexi (Levilinea sp.) became the dominant DNP) digesting bioreactor and based on 16S rRNA gene dis- clone in each reactor system. Due to the metabolic diversity tribution established the culture was also enriched in Proteo- associated with members of the phylum Chloroflexi, it is bacteria, implying involvement in 2,4-DNP degradation. likely the identified Levilinea sp. played an important roll in Despite literature contentions, the Proteobacteria did not the reduction of the IM. Bioreactors designed to enhance proliferate during the treatment period. Prior to the addition Chloroflexi populations may be a viable means to meet of IM each reactor was dominated by a single species of future wastewater treatment needs. Desulfuromonales belonging to the phylum Proteobacteria that grouped in the family Geobacteraceae. Because sulfur Acknowledgements was continually supplied to the reactor systems in the buffer The authors wish to thank Dr. Makram Suidan for supplying solutions, it was hypothesized that clone UC-19 was an bacterial samples from anaerobic reactors operated in his laboratory acetate-oxidizing sulfur reducer (4). In the presence of DNAN, at the University of Cincinnati and for characterizing DNAN the Desulfuromonales clone distribution decreased 66%. In and MNA reduction products. the MNA amended reactor, the clone distribution decreased 75% and in the DNAN and MNA reactor the clone distri- References bution declined 62%. Several Geobacteraceae species have 1. Arnett, C.M., and N.R. Adrian. 2009. Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a been documented to reduce explosives (8). Thus, it does not Desulfovibrio species under anaerobic conditions. Biodegradation preclude the involvement of UC-19 in DNAN and MNA 20:15–26. reduction, but does demonstrate that IM clearly had an 2. Atikovic, E., M.T. Suidan, and S.W. Maloney. 2008. Anaerobic treatment of Army ammunition production wastewater containing inhibitory effect on the organism’s growth. perchlorate and RDX. Chemosphere 72:1643–1648. Clone UC-34 was not detected in AFBR #1 prior to the 3. Berchtold, S.R., S.L. VanderLoop, M.T. Suidan, and S.W. Maloney. addition of DNAN to the system. However, after the treat- 1995. Treatment of 2,4-dinitrotoluene using a two stage system: Fluidized-bed anaerobic GAC reactors and aerobic activated sludge ment period the clone made up roughly one third of the total reactors. Water Environ. Res. 67:1081–1091. bacterial clone distribution. Clone UC-34 was not detected in 4. Caccavo, F., D.J. Lonergan, D.R. Lovley, M. Davis, J.F. Stolz, and M.J. McInerney. 1994. Geobacter sulfurreducens sp. nov., a hydro- the MNA amended reactor and only 1% of the clone distribu- gen- and acetate-oxidizing dissimilatory metal-reducing micro- tion was made up of UC-34 in the DNAN and MNA organism. Appl. Environ. Microbiol. 60:3752–3759. amended reactor. This suggested that MNA may have had an 5. Kimura, N., Y. Shinozaki, T.H. Lee, and Y. Yonezawa. 2003. The microbial community in a 2,4-dinitrophenol-digesting reactor as inhibitory effect on the bacterium. A GenBank BLAST revealed by 16S rRNA gene analysis. J. Biosci. Bioeng. 96:70–75. search revealed the clone had a 99% sequence similarity to a 6. Kittelmann, S., and M.W. Friedrich. 2008. Novel uncultured Chlorobi Chlorobi Chloroflexi dechlorinate perchloroethene to trans-dichloroethene in bacterium. Members of the phylum are tidal flat sediments. Environ. Microbiol. 10:1557–1570. obligate photolithothotrops that possess very limited versa- 7. Kragelund, C., C. Levantesi, A. Borger, K. Thelen, D. Eikelboom, V. tility with regards to degradation of organic compounds Tandoi, Y. Kong, J. van der Waarde, J. Krooneman, S. Rossetti, T.R. Thomsen, and P.H. Nielsen. 2007. Identity, abundance and ecophysi- (12). Therefore, the species was not believed to be directly ology of filamentous Chloroflexi species present in activated sludge involved in IM reduction. treatment plants. FEMS Microbiol. Ecol. 59:671–682. Based on cloning the presence of IM proportionally 8. Kwon, M.J., and K.T. Finneran. 2006. Microbially mediated biodeg- radation of hexahydro-1,3,5-trinitro-1,3,5-triazine by extracellular enhanced the Chloroflexi population relative to the bacterial electron shuttling compounds. Appl. Environ. Microbiol. 72:5933– community in each reactor system. The Levilinea clone dis- 5941. 9. Maloney, S.W., E. Atikovic, and M.T. Suidan. 2008. Treatment of tribution increased to 41% of the clone distribution in the perchlorate contaminated industrial wastewater commingled with presence of DNAN, 36% in the presence of MNA and 28% high explosives during wide concentration variations. Water Practice in the presence of both DNAN and MNA. This demonstrated 2:1–14. 10. Robidoux, P.Y., C. Svendsen, J. Caumartin, J. Hawari, G. Ampleman, the Levilinea sp. was enriched for in the presence of DNAN S. Thiboutot, J.M. Weeks, and G.I. Sunahara. 2000. Chronic toxicity and MNA. Many species of Chloroflexi are commonly found of energetic compounds in soil determined using the earthworm (Eis- enia Andrei) reproduction tests. Environ. Toxicol. Chem. 19:1764– in wastewater treatment plants and are known to possess 1773. very unique metabolic traits (7). Kittelmann and Friedrich 11. Santoh, K., A. Kouzuma, R. Ishizeki, K. Iwata, M. Shimura, T. (6) have demonstrated the ability of filamentous Chloroflexi Hayakawa, T. Hoaki, H. Nojiri, T. Omori, H. Yamane, and H. Habe. 2006. Detection of a bacterial group within the phylum to dechlorinate perchloroethene. Bacteria belonging to Chloroflexi and reductive-dehalogenase-homologous genes in the phyla have also been associated with the reductive pentachlorobenzene-dechlorinating estuarine sediment from the dehalogenation of polychlorinated biphenyls (14) and Arakawa River, Japan. Microbes Environ. 21:154–162. 12. Trüper, H.G. 1981. Versatility of carbon metabolism in the photo- pentachlorobenzene (11). Additionally, Chloroflexi have been trophic bacteria. p. 116–121. In H. Dalton (ed.), Microbial Growth on detected in bioreactors used to treat 4-methylebenzoate (15) C1 Compounds. Hayden Press, London, England. 13. Wang, Q., G.M. Garrity, J.M. Tiedje, and J.R. Cole. 2007. Naïve and terephthalate (16). This metabolic diversity in addition Bayesian classifier for rapid assignment of rRNA sequences into the to the substantial increase in the proportional distribution new bacterial taxonomy. Appl. Environ. Microbiol. 73:5261–5267. of the Levilinea sp. in the presence of DNAN and MNA 14. Watts, J.E.M., S.K. Fagervold, H.D. May, and K.R. Sowers. 2005. A PCR-based specific assay reveals a population of bacteria within suggested possible involvement in IM reduction. the Chloroflexi associated with the reductive dehalogenation of This study demonstrated that IM significantly impacted polychlorinated biphenyls. Microbiology 151:2039–2046. 15. Wu, J., W. Liu, I. Tseng, and S. Cheng. 2001. Characterization of a 4- the microbial clone distribution in each AFBR. All three methylbenzoate-degrading methanogenic consortium as determined bioreactors were dominated by a single Proteobacteria by small-subunit rDNA sequence analysis. J. Ferment. Bioeng. (Desulfuromonales sp.) prior to the addition of IM. 91:449–455. 16. Wu, J., W. Liu, I. Tseng, and S. Cheng. 2001. Characterization of However, in the presence of DNAN and MNA the microbial consortia in a terephthalate-degrading anaerobic granular Desulfuromonales clone distributions dropped significantly sludge system. Microbiology 147:373–382.