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Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofi


APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2006, p. 2896–2904 0099-2240/06/$08.00 0 doi:10.1128/AEM.72.4.2896–2904.2006 Copyright ? 2006, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 4

Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Bio?lters of a Marine Recirculating Aquaculture System?
Yossi Tal,1 Joy E. M. Watts,2 and Harold J. Schreier1,3*
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, Maryland 212021; Department of Biological Sciences, Towson University, 8000 York Road, Towson, Maryland 212522; and Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 212503
Received 30 August 2005/Accepted 17 January 2006

Microbial communities in the biological ?lter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic ?xed-?lm bio?lters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of ampli?ed PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse bio?lter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying bio?lters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-speci?c probe con?rmed the presence of anammox Planctomycetales in the microbial bio?lm from the denitrifying bio?lters, and anammox activity was observed in these bio?lters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the ?rst identi?cation of anammox-related sequences in a marine recirculating aquaculture ?ltration system, and our ?ndings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems. The continuing decline of marine ?sheries and the increased demand for seafood by consumers have created a gap that endangers the world’s seafood supply. In order to ?ll this gap while the pressures on wild ?sheries stocks are eased, marine species must be farmed through aquaculture. A major drawback of the traditional open pond and marine net pen aquaculture technology is their negative impact on the marine environment, which includes organic and inorganic pollution of coastal areas, the spread of disease to wild populations, and “genetic pollution” as a result of farmed ?sh mixing with wild stocks (53). In response to these concerns there is a trend to shift marine ?sh farming inland using environmentally friendly closed recirculating aquaculture systems. Elimination of waste in these systems is generally managed through mechanical ?ltration for removal of solids and bio?ltration for the conversion of toxic ammonia produced by the ?sh to nitrate through nitri?cation. Nitrate is typically maintained at low levels by daily replacement of a portion of the water (48). However, strict environmental regulations for both nitrate levels and salt concentrations in discharge water are motivating the recirculating aquaculture industry to integrate a denitrifying bio?ltration stage, which results in the conversion of nitrate to nitrogen gas. Denitri?cation is performed mainly by facultative anaerobic bacteria that utilize organic (heterotrophic denitri?cation) or inorganic (autotrophic denitri?cation) compounds as electron sources to reduce nitrate (40). So far, dif?culties associated with the denitri?cation process, such as the high potential for toxic sul?de production, as well as the requirement for addition of an external electron source, have prevented its use in full-scale commercial marine recirculating systems (51). One option for closing the nitrogen cycle without exclusive reliance on denitri?cation is to integrate the anaerobic ammonium oxidation (anammox) process in recirculating systems (39, 51). An important pathway of the nitrogen cycle, anammox allows ammonia to be oxidized by nitrite under anoxic conditions (36) and is performed by autotrophic bacteria that are members of the order Planctomycetales. The anammox reaction has been shown to occur in an organelle-like compartment called the anammoxosome, where ammonia is oxidized via hydrazine (N2H4) and hydroxylamine (NH2OH) intermediates (47). Anammox bacteria have been found in the anoxic water column of the Black Sea (22), the Costa Rica shoreline (5), an oceanic oxygen-minimum zone (21), and sediments from the Baltimore inner harbor (38) and several estuaries (24, 43, 44). They have also been shown to play a role in the nitrogen cycle associated with the microbial consortia of activated sludge from wastewater treatment plants (6, 8, 18, 37, 42). Stimulation of anammox activity for use in wastewater puri?cation has been examined by several research groups (6, 12, 18). Utilization of the process for complete removal of nitrogen from contaminated water is economically viable since ammonia oxidation requires, at a minimum, 50% less oxygen than that required by the conventional nitri?cation-denitri?cation process (17). Moreover, compared to heterotrophic denitri?cation, the anammox process results in complete autotrophic nitrogen removal with no organic electron source requirement. Previously, we characterized the microbial community and the nitrogen transformation processes in a moving bed biore2896

* Corresponding author. Mailing address: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt St., Baltimore, MD 21202. Phone: (410) 234-8874. Fax: (410) 234-8896. E-mail: schreier@umbi.umd.edu. ? Paper no. 05-119 from the Center of Marine Biotechnology.

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FIG. 1. Schematic diagram of the marine recirculating aquaculture system used in this study. The system included aerobic and anaerobic loops for complete removal of nitrogen by nitrifying and denitrifying processes, respectively. Triangles represent pumps, and arrows indicate the direction of circulating seawater; the high-?ow aerobic loop (16 m3 h 1) is indicated by solid arrows, and the low-?ow anaerobic side loop (0.1 m3 h 1) is indicated by dotted arrows. Stippling indicates compartments containing bio?lter beads. The inset is a photograph of top and side views of representative polystyrene beads, as well as a fourfold enlargement of a side area. (A) Sterile bead. (B) Bio?lm-containing bead. See Materials and Methods for additional details.

actor connected to a marine recirculating aquaculture system and obtained preliminary evidence for both the presence of Planctomyces spp. and the potential for anammox activity (39). In this study, we identi?ed anammox bacteria as part of the microbial consortia of ?xed-?lm bio?lters of a closed marine recirculating aquaculture system and measured anammox-related activity in enrichment cultures obtained from these ?lters. Moreover, we compared anammox and denitri?cation activities, and we discuss ways to implement the anammox process as an alternative to denitri?cation in such systems.
MATERIALS AND METHODS System con?guration. A schematic diagram of the marine recirculating system used in this study is shown in Fig. 1. Two 4.2-m3 tanks containing gilthead seabream (Sparus aurata) were operated at a density of 10 to 50 kg m 3 and a feeding rate of 1% to 1.6% body weight day 1. The tanks were connected to a 2-m3 nitrifying moving bed bioreactor ?lled with 1 m3 of polyethylene beads (39) that were 1 cm in diameter and had a surface-to-volume ratio of 500 m2 m 3. A ?ow rate of 16 m3 h 1 was used to obtain two exchanges of tank water per hour through the nitrifying bio?lter. Attached to each tank as a denitri?cation side loop was a 0.3-m3 cylindrical up-?ow ?xed-bed bio?lter ?lled with 0.2 m3 of polyethylene beads. The ?ow rate for this anaerobic bio?ltration component was 0.1 m3 h 1, and the water was supplied by two sources, direct water from the ?sh tank and water recovered from the sludge tank. The average concentrations of ammonia, nitrite, and nitrate for this bio?lter component during 350 days of operation were 1.5 0.2, 1.2 0.2, and 121 20 mg of N liter 1, respectively, for input water and 0.2 0.04, 0.1 0.02, and 61.6 12 mg of N liter 1, respectively, for ef?uent water (Tal and Schreier, unpublished data). Sludge was collected with a drum screen ?lter using a backwash system in which tank water was used. Sludge and backwash water were collected in a 0.3-m3 rectangular tank with 0.1-m3 beads that provided a means for solids removal, as well as a substrate for bacterial colonization. Water from the sludge tank was pumped back into the system via the anaerobic bio?lter, and high-density sludge was collected and removed. Anammox enrichments. Ninety polyethylene beads from aerobic (nitrifying) and anaerobic (denitrifying) bio?lters were incubated in 125-ml glass bottles with 85 ml of autoclaved seawater. The bottles were supplied with 0.7 mmol ammonia and nitrite, ?ushed with 99.99% nitrogen gas, sealed with gas-tight butyl rubber stoppers, and placed in the dark in a shaking incubator at 37°C for 3 months; once the ammonia and nitrite were consumed, periodic additions were necessary to replenish these substrates. After 3 months of incubation, anammox activity was evaluated by addition of ammonia and nitrite or hydrazine and nitrite in

different combinations, and the liquid phase was periodically collected and analyzed to determine the ammonia, nitrite, and hydrazine contents (see below). Analysis of ammonia, nitrite, and hydrazine. Total ammonia (NH3 and NH4 ) contents were determined by the hypochlorite oxidation reaction, as described by Scheiner (31). Nitrite was measured by the sulfanilamide reaction as described by Strickland and Parsons (35). Hydrazine contents were determined spectrophotometrically using the American Society for Testing and Materials standard methods (3) by adding 1 ml of p-dimethylaminobenzaldehyde reagent (4 g of p-dimethylaminobenzaldehyde dissolved in 200 ml of methyl alcohol and 20 ml of concentrated HCl) and 0.1 ml concentrated HCl to 5 ml of sample and, after 10 min of incubation at room temperature, determining the absorbance at 458 nm. Standard curves for establishing the hydrazine concentration were determined using stock solutions containing between 5 and 200 g hydrazine liter 1. All measurements were carried out in triplicate, and the values were 10%. Bio?lm DNA extraction. Five polyethylene beads from aerobic or anaerobic bio?lters and the sludge tank were sliced into small fragments and were suspended in 2 ml of buffer (0.15 M NaCl, 0.1 M Na2EDTA, pH 8). One gram of silica beads (diameter, 0.1 mm) was mixed with the polyethylene bio?lter bead fragments in 5-ml tubes vigorously for 5 min. After centrifugation at 15,000 g for 5 min, each supernatant fraction was collected, and bacterial DNA was extracted as described previously (39). PCR ampli?cation. To identify anammox bacteria, a sequential PCR approach was employed, as described by Tal et al. (38). Ampli?cation of Planctomycetalesspeci?c 16S rRNA genes was performed using the Pla46 oligonucleotide primer (5 -GAC TTG CAT GCC TAA TCC-3 ) (25) with the universal bacterial primer 1392r (5 -ACG GGC GGT GTG TAC-3 ) (10). The product of this reaction was then cloned into Escherichia coli, and selected transformants were examined further by PCR ampli?cation using primer Pla46 and anammox-speci?c primer Amx820 (5 -AAA ACC CC TCT ACT TAG TGC CC-3 ) (33). Ampli?cation of 16S rRNA gene sequences used for denaturing gradient gel electrophoresis (DGGE) was performed using universal bacterium-speci?c 16S rRNA gene primer 1055f (5 -TGG CTG TCG TCA GCT-3 ), universal bacterial GC clamp primer 1392rGC (5 -CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG CCC ACG GGC GGT GTG TAC-3 ) (10), and either bio?lter DNA or the ampli?ed anammox-related Planctomycetales-related clones as the template. The PCR mixtures (50 l) contained 5 l of PCR buffer, 4 l of deoxynucleoside triphosphates (25 mM each), 3 l of MgCl2 (25 mM), 0.1 l of Taq polymerase (250 U) (Applied Biosystems, CA), 2 l of DNA template (10 to 100 ng), and 1 l of each primer (100 to 200 ng). The reaction cycle parameters included an initial denaturation step consisting of 5 min at 95°C, followed by seven touchdown PCR cycles (7) (30 s of denaturation at 94°C, 30 s of annealing at 62, 60, 59, 58, 57, 56, and 55°C, and 30 s of elongation at 72°C) and then 30 cycles consisting of 30 s of denaturation at 94°C, annealing for 30 s at 54°C, and elongation for 30 s at 72°C and a ?nal extension step consisting of 5 min at 72°C.

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The sizes and yields of PCR products were determined by using a 0.8% (wt/vol) agarose gel in TAE buffer (20 mM Tris-HCl, 10 mM sodium acetate, 0.5 mM Na2EDTA, pH 8.0) (29). DGGE. Analysis of PCR products by DGGE was performed as described by Tal et al. (39), using the D-Code universal mutation detection system (Bio-Rad, Hercules, CA). Products from triplicate PCRs were combined and applied directly to 10% (wt/vol) polyacrylamide gels containing a 35 to 50% gradient of denaturants (100 ml of 100% denaturant contained 42 g of urea, 40 ml of deionized formamide, 25 ml of a 40% acrylamide-bisacrylamide solution [37.5:1], and 1 ml of 50 TAE buffer). Electrophoresis was performed for 5 h at 60°C in TAE buffer at 200 V (constant voltage). After electrophoresis, nucleic acids were stained with SYBR Green II (1:10,000 dilution; Molecular Bio-Probes, Eugene, OR), and the staining pattern was immediately recorded using a ?uoroimager (Molecular Dynamics, Sunnyvale, CA). DNA fragments were isolated after gels were stained with ethidium bromide (0.5 g/ml) and visualized using a UV transilluminator. Gel sections containing fragments were excised, placed in TE buffer (50 mM Tris-HCl [pH 8.0], 1 mM Na2EDTA), and incubated at 4°C overnight. The eluted DNA was used as a template for the PCR, as described above, and was checked for purity by DGGE. Cloning, sequencing, and database analysis. PCR-ampli?ed DNA and DNA fragments obtained from DGGE gel slices were introduced into a pCR2A vector and transformed into E. coli using a TOPO TA cloning kit according to the manufacturer’s instructions (Invitrogen, California). The presence of cloned inserts was veri?ed by PCR ampli?cation, and sequence analysis was carried out using an ABI 3100 automated sequencer (PE Applied Biosystems, Foster City, CA). DNA sequences were examined and edited using DNAman (Lynnon Biosoft, Quebec, Canada) and then checked for chimera formation using the Check Chimera program of the Ribosomal Database Project (23). Sequences were subjected to the National Center for Biotechnology Information’s Basic Local Alignment Search Tool (BLAST) (1) and the Ribosomal Database Project (23) to determine the levels of similarity with other 16S rRNA genes. Partial 16S rRNA gene sequences were manually compiled and aligned using the PHYDIT software (4). Evolutionary trees were generated using the neighbor-joining (28), Fitch-Margoliash (11), and maximum-parsimony (20) algorithms in the PHYLIP software (9). Evolutionary distance matrices for the neighbor-joining and FitchMargoliash methods were generated as described by Jukes and Cantor (19). The robustness of the inferred tree topologies was evaluated after 1,000 bootstrap resamplings of neighbor-joining data. FISH analysis. Bio?lms extracted from polyethylene beads (?ve beads from aerobic bio?lters and ?ve beads from anaerobic bio?lters) were suspended in 1 ml of phosphate-buffered saline (PBS) (pH 8.0) (29) and vortexed gently for 2 min. Material was then ?xed with 4% (vol/vol) paraformaldehyde in PBS for 1 h at room temperature, and this was followed by two washes with PBS. After ?xing, the samples were suspended in a 50% (vol/vol) PBS–ethanol solution and stored at 20°C. Aliquots (10 l) were used for ?uorescence in situ hybridization (FISH) analysis as described previously (34). Texas Red-labeled anammox-speci?c probe Amx820 (Sigma Genosys) was used for detection of anammox bacteria, and counterstaining was performed using SYBR Gold (Molecular Probes, Eugene, OR). Texas Red-labeled nonsense probe NONEUB (52) was used to examine background ?uorescence. Image acquisition was performed with a BioRad Radiance 2100 laser confocal microscope equipped with the appropriate ?lter set and a digital camera. Densities were determined by comparing the number of cells stained with the Amx820 probe to the number of cells stained with SYBR Gold, and the average number was obtained by counting 100 to 200 cells in each of 10 random ?elds (100 m by 100 m) on ?ve slides of bio?lm samples (before and after anammox enrichment). Nucleotide sequence accession numbers. Sequences of the partial 16S rRNA genes of the predominant restriction fragment length polymorphism types have been deposited in the GenBank database under accession numbers AY769988, DQ146408, and DQ146409.

FIG. 2. Comparison of the aquaculture system Planctomyces species sequences to anammox and nonanammox Planctomycetales sequences in anammox signature regions. Clone sequences in regions that are around 770, 1,200, and 350 bp from the 5 (Pla46) end are compared to the Amx820 (A), Amx1240 (B), and Amx368 (C) oligonucleotide sequences and corresponding B. anammoxidans (accession number AF375994), K. stuttgartiensis (accession number AF375995), S. brodae (accession number AY254883), S. sorokinii (accession number AY257181), S. wagneri (accession number AY254882), and P. marina (accession number X62912) sequences. Bases that differ from the bases in oligonucleotide probe sequences are underlined. The GenBank accession number for clones 3-8b6, 9, and YC are AY769988, DQ146408, and DQ146409, respectively.

RESULTS Isolation and examination of anammox-speci?c 16S rRNA genes from bio?lter components. Our initial survey of the microbial consortium associated with nitrogen transformation processes in a marine recirculating aquaculture system resulted in identi?cation of two 16S rRNA gene segments that exhibited high levels of sequence similarity to members of the order Planctomycetales (39). However, both of these segments were most closely related to Pirullela spp., which have not been

associated with anammox (39). To enhance our ability to amplify anammox-related 16S rRNA gene sequences, we utilized a sequential PCR approach that we previously used to successfully identify analogous sequences from an environmental sample (38). DNA extracted from aerobic and anaerobic bio?lters, as well as the system sludge tank, was subjected to PCR ampli?cation using the Planctomycetales-speci?c primer Pla46 and the general bacterial primer 1392r as described in Materials and Methods. Under stringent conditions, ampli?cation produced libraries of partial ( 1,350-bp) Planctomycetales 16S rRNA genes, which were then cloned into E. coli. From several hundred transformants isolated for each library, we chose between 30 and 50 transformants that contained the cloned Pla46-1392r insert. Screening of these clones using Pla46 and the anammox-speci?c Amx820 primer resulted in identi?cation of three clones which yielded an approximately 750-bp PCR product, clone 3-8b6 from the denitrifying bio?lter, clone 9

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FIG. 3. Consensus phylogenetic tree generated by neighbor-joining (23), Fitch-Margoliash (11), and maximum-parsimony (19) analysis methods for a comparison of 1,010 bp of 16S rRNA genes. The phylogenetic reconstruction shows the relationship of the 16S rRNA gene sequences retrieved from the aquaculture system to Planctomycetales sequences. The numbers at the nodes are percentages that indicate the levels of bootstrap support based on 1,000 resampled data sets (only values greater than 50% are shown). Thermatoga maritima was used as an outgroup. Scale bar 10 substitutions per 100 nucleotide positions. The numbers in parentheses are accession numbers.

from the nitrifying bio?lter, and clone YC from the sludge tank. DNA sequence analysis of the three Pla46-1392r segments (GenBank accession numbers are shown in the legend to Fig. 2 and in Fig. 3) revealed that clones 9 and YC were 89% similar to each other over the entire sequence but were only 78 to 79% similar to clone 3-8b6. A comparison of the anammox signature Amx820, Amx1240, and Amx368 (33) regions indicated that clone 3-8b6 was identical to Amx820 and Amx368 sequences (Fig. 2A and C) and nearly identical (with a two-base

difference) in the Amx1240 region (Fig. 2B). In addition to the high levels of sequence identity in these regions, clone 3-8b6 was also found to exhibit between 89 and 93% sequence similarity to several anammox Planctomycetales spp., including Brocadia anammoxidans, Kuenenia stuttgartiensis, Scalindua brodae, and Scalindua wagneri, as well as the marine bacterium Scalindua sorokinii, but only 78% similarity to Pirullela marina, a nonanammox Planctomycetales sp. On the other hand, clones 9 and YC were found to differ at several positions in the anammox signature regions, and the sequences

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FIG. 4. DGGE analysis of PCR-ampli?ed 16S rRNA gene fragments. DNA was extracted from bio?lter and sludge tank samples, and PCR ampli?cation with the universal 1055 and 1392rGC clamp primers followed by electrophoresis was performed as described in Materials and Methods. Lane 1, denitrifying bio?lter after enrichment in the presence of ammonia and nitrite; lane 3, nitrifying bio?lter; lane 4, sludge tank sample; lane 5, denitrifying bio?lter before anammox enrichment. The same primer set was used to amplify plasmid DNA of clones 3-8b6 (lane 7), 9 (lane 8), and YC (lane 9). Lanes 2 and 6 contained all three clones together. The arrowheads indicate fragments that were subjected to DNA sequence analysis.

in these regions were more closely related to P. marina sequences (Fig. 2). Furthermore, clones 9 and YC were 88 to 89% similar to P. marina sequences but only 77 to 79% similar to the anammox Planctomycetales sequences. A phylogenetic tree of the 16S rRNA gene sequences was assembled, and this tree included representative members of the order Planctomycetales, the known anammox bacteria, and the three new clones (Fig. 3). Consistent with our sequence analyses, clone 3-8b6 was found to fall deep within the anammox cluster, branching along with B. anammoxidans. Similarly, clones 9 and YC branched with the nonanammox Pirullela group. Thus, not only did the microbial consortium in the main aquaculture wastewater treatment compartments include Planctomycetales spp., as we have demonstrated previously (39), but the system also included one organism whose 16S rRNA gene sequence was characteristic of the sequences associated with the anammox Planctomycetales members. DGGE analysis. The presence of Planctomycetales spp. within the bacterial consortium of the bio?ltration system was examined by DGGE analyses (Fig. 4). Electrophoresis was performed using PCR products generated with universal bacterial primers and microbial community DNA from nitrifying and denitrifying bio?lter beads, as well as heavily colonized beads obtained from the sludge tank. As shown by the band patterns in Fig. 4, lanes 3 to 5, there were signi?cant differences in microbial diversity for each compartment. In addition, bands that comigrated with two of the Planctomycetales clones could be identi?ed in at least one of the compartments. A fragment that migrated with clone 3-8b6 was detected in the denitri?ying bio?lter (lane 5), as well as in the sludge tank (lane 4). This fragment was extracted from the gel, cloned, and puri?ed as described in Materials and Methods, and

it was found to have a DNA sequence identical to that of the clone 3-8b6 fragment (data not shown). Similarly, the denitrifying bio?lter yielded a product that comigrated with the fragment from clone YC, which was initially ampli?ed from the sludge tank (lanes 4 and 5). Bands from either of the bio?lters or the sludge tank that comigrated with the ampli?ed clone 9 fragment were not detected. Moreover, clone 3-8b6 was not detected in samples from the nitrifying bio?lter (lane 3). To examine the occurrence of the Planctomycetales clones under conditions favorable for anammox activity, we incubated beads from the denitrifying bio?lter under anammox-enriching conditions as described in Materials and Methods and examined the microbial population by DGGE analysis. As noted above, the anaerobic denitrifying bio?lter had been found to contain sequences that coincided with both clone YC and 3-8b6 sequences (Fig. 4, lane 5). As shown in Fig. 4, lane 1, while there was a signi?cant difference in the DGGE banding patterns before and after enrichment (compare lanes 1 and 5), bands that comigrated with clones 3-8b6 and YC were clearly evident in the enrichment population. These bands were extracted and were found to have DNA sequences identical to the sequences of the fragments in clones 3-8b6 and YC (data not shown). FISH analysis. The presence of anammox-related Planctomycetales in the bacterial consortia of the bio?lm from the denitrifying bio?lter before and after anammox enrichment was examined by FISH using the anammox-speci?c Amx820 probe labeled with Texas Red and SYBR Gold as a counterstain. As shown in Fig. 5, the inner bio?lm of beads from the denitrifying bio?lter contained SYBR Gold-staining bacteria (Fig. 5A, C, and E) that were also labeled with the Amx820 probe (Fig. 5B, 5D, and 5F) and exhibited the characteristic spherical morphology of the Planctomycetales. In no case were we able to detect any cells that hybridized with the Texas Red-labeled nonsense NONEUB primer (data not shown). By determining the number of cells that hybridized with the Amx820 probe and comparing the results to the number of cells stained by SYBR Gold, we found that anammox-related bacteria accounted for 1.1% 0.1% of the total bacterial population before enrichment (compare Fig. 5A to Fig. 5B and Fig. 5C to Fig. 5D) and 12.5% 1.6% of the total bacterial population after anammox enrichment (compare Fig. 5E and F). Assessment of anammox and denitri?cation activity in bio?lters. In the presence of nitrite, anammox bacteria are able to oxidize either hydrazine or ammonia under anaerobic conditions (30, 33). To examine whether the bacterial consortia from nitrifying and denitrifying bio?lters were capable of anammox activity, samples from these bio?lters were cultured under anammox-enriching conditions, and their abilities to utilize either ammonia and nitrite or hydrazine and nitrite were determined under anaerobic conditions. After 3 months of incubation under anammox-enriching conditions (see Materials and Methods), beads from the denitrifying bio?lter were assayed for the ability to oxidize hydrazine as described in Materials and Methods. As shown in Fig. 6A, hydrazine oxidation occurred at a rate of approximately 0.16 M bead 1 h 1 over a 50-h period. However, no signi?cant hydrazine removal was observed for beads from the nitrifying bio?lter that were treated under the same conditions (Fig. 6B). Although nitrifying bac-

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FIG. 5. Identi?cation of anammox bacteria in denitrifying bio?lter material by FISH analysis. Aggregates of bio?lm before (A, B, C, and D) and after (E and F) enrichment were stained with SYBR Gold (A, C, and E) and visualized with Texas Red ?lters after hybridization with anammox probe Amx820 (B, D, and F) as described in Materials and Methods. The arrows indicate representative SYBR Gold-stained cells that also reacted with the Amx820 probe.

teria are capable of utilizing hydrazine (2, 27), the hydrazine removal rates for nitrifying sludge have been shown to be as much as 10-fold lower than the hydrazine removal rates for anammox sludge (30), which might explain the absence of detectable activity under our experimental conditions. Hydrazine removal could be attributed to biological activity as no signi?cant uptake was detected using beads taken from either bio?lter that was autoclaved prior to incubation (data not shown). Beads from the anammox-enriched denitrifying cul-

ture were also able to completely remove 0.66 mM ammonia in the presence of 0.76 mM nitrite (Fig. 6D) over 90 h of incubation under anaerobic conditions, which corresponded to an ammonia removal rate of 0.098 M NH3 bead 1 h 1, which is approximately 1.6-fold lower than the hydrazine removal rate of the same culture (Fig. 6A). In the absence of nitrite, however, ammonia removal could not be detected (data not shown). These results indicated that the anaerobic denitrifying bio?lter was able to carry out anammox activity, which is con-

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FIG. 6. Anammox and nitrate-reducing activities of denitrifying and nitrifying bio?lter beads. Denitrifying (A and D) and nitrifying (B) bio?lter beads were incubated under anammox enrichment conditions for 3 months, and the abilities to utilize hydrazine ( ) or ammonia ( ) and nitrite (?) were measured as described in Materials and Methods. (C) Nitrate uptake by denitrifying bio?lter beads that were not incubated under anammox enrichment conditions.

sistent with the presence of anammox-related Planctomycetales in such bio?lters. Moreover, the molar ratio of ammonia to nitrite removal for the denitrifying bio?lter (Fig. 6D) was between 1.1 and 1.3, which is characteristic of anammox activity (16). To distinguish between anammox and denitri?cation activities, nitrate reduction rates were determined using beads from the denitri?cation bio?lter that were incubated under anoxic conditions in the presence of only nitrate (Fig. 6C). Under these conditions, the organic carbon incorporated into the bio?lm served as an electron donor to support denitri?cation (39). The nitrate removal rate was determined to be 1.08 M NO3 bead 1 h 1 over 15 h of incubation. Thus, the capacity for nitrogen removal via denitri?cation was as much as 11-fold greater than the ammonia consumption rate. DISCUSSION Our ability to measure anammox activity in enrichment cultures established from a denitrifying bio?lter of a recirculating aquaculture system and our identi?cation of anammox-related bacteria both microscopically and by isolation of associated 16S rRNA gene sequences constitute the ?rst report of the occurrence of anammox-related bacteria in marine recirculating aquaculture systems. Moreover, this study provides a foundation for use of the anammox process for removing inorganic nitrogen waste from recirculating systems.

Identi?cation of anammox-related Planctomycetales. Using an approach for detecting anammox Planctomycetales in environmental samples, we isolated three new Planctomycetalesrelated 16S rRNA gene sequences from different components of a marine aquaculture system bio?lter. One of the clones, 3-8b6, exhibited high levels of sequence similarity to known anammox bacterial 16S rRNA gene sequences in signature anammox-speci?c regions (Amx820, Amx1240, and Amx368), as well as across the entire sequence. Interestingly, clone 3-8b6 sequences were found to fall in the Brocadia branch of anammox Planctomycetales rather than the Scalindua branch, which includes the marine Planctomycetales (22), and additional studies are necessary to determine the relationship between 3-8b6 and the other anammox Planctomycetales members. We hypothesize that clone 3-8b6 represents a new anammox-capable Planctomycetales taxon that was part of the bacterial community of the denitrifying bio?lter of the marine recirculating system. The ability to detect anammox activity in enriched cultures from the bio?lters, the presence of an Amx820-hybridizing population as determined by the FISH analysis, and the identi?cation of clone 3-8b6 sequences in both untreated and anammox-enriched bio?lter preparations are consistent with the notion that 3-8b6 is involved in anammox activity. Whether the anammox activity that we measured can be directly attributed to this clone or was due to some other uncharacterized Planctomycetales sequence could not be determined from our studies and requires future isolation and characterization of the microorganisms. However, while the Amx820 probe used for library screening is selective for a subgroup of anammox Planctomycetales (33), PCR ampli?cation of the Planctomycetales clone library created from the denitrifying ?lter before and after anammox enrichment using the universal anammox Amx368 (33) and 1392r primers resulted in identi?cation of clones having the same sequence as 3-8b6 (Tal and Schreier, unpublished), suggesting that 3-8b6 is the predominant anammox Planctomycetales clone in this system. In addition to the anammox-related clone, we identi?ed two 16S rRNA sequences, clone 9 and clone YC, that were closely related to each other, as well as to sequences that we identi?ed previously (reference 39 and data not shown), and also exhibited high levels of sequence similarity to the nonanammox Planctomycetales, which include the Pirellula spp. Although clones 9 and YC were obtained by the anammox-speci?c PCR approach, a phylogenetic and 16S rRNA gene sequence analysis of these clones did not indicate that there is a strong relationship to known anammox bacteria. It is conceivable that these sequences were detected due to the bias of Planctomycetales PCR clone libraries toward anammox 16S rRNA (33). Alternatively, the wide genus diversity of the anammox Planctomycetales (32) may indicate that these clones may represent new anammox bacteria that are not related to known anammox bacteria. The presence of clone YC sequences in the anammox enrichment culture from the denitrifying bio?lter is consistent with the detection of related Pirellula-like Planctomycetales spp. in the autotrophic nitrogen-removing bio?lms in wastewater systems (26), and the nature of the participation of these organisms has not been determined. Aquaculture bio?lters and wastewater treatment environments. With the exception of natural environments and wastewater treatment plants, there have been no reports of anam-

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mox-related Planctomycetales in water treatment systems that have characteristics of the organisms encountered in recirculating aquaculture systems. The important differences between water quality parameters of aquaculture systems and wastewater plants include the concentrations of nutrients and the organic loads. While the concentration of ammonia in raw sewage may reach 20 to 40 mg liter 1 (ammonia nitrogen, NH3-N) (51), the maximum ammonia concentrations allowed in aquaculture systems are less then 1 mg NH3-N liter 1, as higher concentrations of ammonia are lethal to ?sh (41). Moreover, the biological oxygen demand values for organic loads in raw sewage reach between 200 and 500 mg liter 1 (13), while they are usually less than 50 mg liter 1 in recirculating aquaculture systems (15). Furthermore, the growth structures of the bacterial communities are different. While activated sludge suspensions of wastewater treatment plants promote granule formation, a thin ?xed ?lm is the main structure of bacterial growth in moving bed bio?lters of recirculating aquaculture systems. The ability of anammox bacteria to populate these distinct synthetic environments is consistent with the belief that the anammox process is amenable to many wastewater treatment applications with minimal apparent restrictions with respect to nutrient and salt concentrations, organic loads, or bacterial growth con?gurations (37). Anammox versus denitri?cation. Anaerobic incubation of beads from the denitrifying bio?lter showed that the removal rates were 1.08 M nitrate nitrogen bead 1 h 1 under denitrifying conditions and 0.098 M ammonia nitrogen bead 1 h 1 and 0.16 M hydrazine bead 1 h 1 under anammox conditions. Although the fraction of anammox bacteria in the consortia of the anammox-enriched beads was 12.5% 1.6%, as determined by FISH, the nitrogen removal rates were between 7- and 11-fold lower than the denitri?cation rates of untreated beads. This discrepancy was likely due to the low activity and growth rate (11 days) of the anammox bacteria and indicates that there is a major limitation in using the anammox process for nitrogen removal in these systems. Ammonia oxidation rates for cultures of anammox bacteria with nitrite as the electron acceptor have been determined to be 55 nmol min 1 mg protein 1 (16), compared to, for example, 400 nmol min 1 mg protein 1 for nitrate reduction by the heterotrophic denitrifyer Pseudomonas stutzeri (50). This more-than-sevenfold difference suggests that denitrifying bacteria may outcompete anammox bacteria, especially in an organic carbon-rich environment, and could explain the relatively low numbers of anammox bacteria in the denitrifying bio?lter, which was populated predominantly by fast-growing heterotrophic denitri?ers (51). Recent studies have shown that anammox bacteria are capable of myxotrophic growth in the presence of fatty acids, such as acetate and propionate (14), which are common organic waste products of aquaculture ?ltration systems (49), supporting the notion that anammox and denitrifying bacteria can share the same environment. To further assess the speci?c contributions of anammox and denitrifying bacteria in the nitrogen removal process of the recirculating system will require the use of 15N-labeling experiments in which isotope pairing distinguishes between these two processes (16, 45). Applying the anammox process. Development of anammox as the major nitrogen removal process in recirculating aquaculture systems is advantageous due to the reduced oxygen demands and the autotrophic nature of the process, which

allow complete nitrogen removal without a need for organic carbon. As noted by other workers (26, 46), the low growth rates and activity exhibited by anammox bacteria should be re?ected in two major features of anammox-based bio?lters, inoculation time and biomass retention ef?ciency. Long initiation periods under conditions favorable for anammox are required in order to establish a stable anammox population. Shortening this period may be possible by inoculation of labenriched anammox cultures, although the success of such an approach will depend largely on the type and origin of the anammox culture used. Inoculation of marine recirculating systems with anammox cultures that are already a component of the natural bacterial consortium of the anaerobic bio?lters should improve the likelihood of success. High biomass retention ef?ciency of an anammox-based bio?lter is essential to overcome the low activity of the anammox bacteria. High surface-to-volume ratios of the bio?lter substrate should increase the anammox bacterial biomass and improve the overall activity per volume of bio?lter. Successful operation of an anammox bio?lter requires a constant supply of ammonia and nitrite along with minimal input of organic carbon. Such an environment is necessary to maintain the anammox consortium, while reducing competition from denitrifying bacteria. One approach for enhancing anammox activity in the recirculating system used in this study would be to partially aerate the sludge tank to stimulate incomplete denitri?cation in order to promote nitrite accumulation. The nitrite-containing water would then be supplied to the anaerobic bio?lter along with ammonia-rich wastewater from the ?sh tank. We are presently addressing whether this combination promotes growth and activity of the anammox bacteria in an anaerobic bio?lter. Conclusion. Stricter environmental regulations for ef?uent discharge from aquaculture facilities are currently motivating the aquaculture industry to reduce ef?uent volumes and nitrogen loads. Closing the nitrogen cycle in the water treatment system by utilizing the anammox process is a desirable alternative to heterotrophic denitri?cation, especially in marine recirculating systems. Our ?nding that bacteria capable of anammox and their activity are associated with the consortia of a denitrifying bio?lter makes this approach feasible for implementation and will direct future advances in building an environmentally sustainable land-based mariculture industry.
ACKNOWLEDGMENTS We thank Sabeena Fasiludeen and COMB’s Bioanalytical Services Lab for DNA sequence analysis and Steve Rodgers and Eric Evans of COMB’s Aquaculture Research Center for technical support. Ammar Zafar’s assistance in screening the Planctomycetales libraries is also appreciated. This work was supported in part by research grant IS-3424-03 from the United States-Israel Binational Agricultural Research and Development (BARD) Fund.
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