Polygenic analysis of ammonia-oxidizing bacteria for completely autotrophic nitrogen removal

This study investigated the functional microbial community in a sequencing batch biofilm reactor (SBBR) for completely autotrophic nitrogen removal over nitrite (CANON), and analyzed the phylogenetic relationships of the key bacteria, the aerobic ammonia oxidizing bacteria (AOB), with three different markers genes: 16S rDNA, amoA and hao. The resulting tree topologies from all the markers were inclined to consistence with minor discrepancy. 16S rDNAand hao-based phylogenetic analyses revealed that AOB for completely autotrophic nitrogen removal were related most closely to Nitrosomonas sp., and amoA-based phylogenetic analysis demonstrated AOB had highest similarity with a group of uncultured bacteria, while 88 to 94% similarity with Nitrosomonas sp. was also achieved. Generally, the detection of three different markers revealed that AOB were closely related to the bacteria of Proteobacteria Nitrosomonas. This study shows that each of the functional markers (16S rDNA, amoA and hao) could be used to trace AOB for completely autotrophic nitrogen removal and it is more accurate if more than two markers are used at a time.


INTRODUCTION
Biological nitrogen removal is generally used for elimination of nitrogen from wastewater since these components can be toxic to aquatic life, and cause oxygen depletion and eutrophication in receiving water (Brauer and Eitzer, 1997).Ammonia is especially abundant in many wastewater streams and its removal is often achieved using nitrification/denitrification systems.Most nitrification/ denitrification systems consist of two steps: aerobic nitrification and anaerobic denitrification, and require two separate reactors (Ahn, 2006).Furthermore, heterotrophic denitrification, widely used in wastewater treatment processes, requires an external organic carbon source to provide electron donors when treating low C/N ratio wastewater, or high internal recycle ratios between aerobic reactors when treating high C/N ratio wastewater (Aoi et al., 2005).Although the recently developed anaerobic ammonia oxidation (Anammox) reaction has the potential to achieve high ammonia-removal rates at low cost, it requires specific operating conditions and adjustment of the ratio between NH 4 + -N and NO 2 − -N before the Anammox reaction occurs (Aoi et al., 2005).
Recently, completely autotrophic nitrogen removal system, in which simultaneous nitrification and denitrification under aerobic conditions in a single reactor is achieved, has been described and investigated in the laboratories (Gong et al., 2008;Helmer et al., 2001;Olav Sliekers et al., 2003;Strous, 2000;Third et al., 2005).This is performed in a single reactor at oxygen limited conditions, without the production of N 2 O or NO and with production of N 2 .In such a system, autotrophic nitrogen removal process was achieved by the close cooperation between aerobic ammonia-oxidizing bacteria (AOB) and Anammox bacteria.AOB oxidize ammonia to nitrite, consuming oxygen and creating anoxic conditions that the Anammox bacteria need (Olav Sliekersa et al., 2002).The oxidation of ammonia to nitrite by AOB in wastewater treatment plants (WWTPs) is often considered the most sensitive step in the nitrification process (Purkhold et al., 2000).The oxidation of NH 3 is a two-step process catalyzed by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO), the key enzymes from AOB (Arp et al., 2002).AMO catalyzes the oxidation of NH 3 to NH 2 OH and HAO catalyzes the oxidation of NH 2 OH to NO 2 -.AOB are obligate chemolithotrophs that derive all of the reductant required for energy and biosynthesis from the oxidation of NH 3 to nitrite (NO 2 -).In the NH 4 + -rich environment, AOB play an important role by initiating the conversion of NH 3 to N 2 .AOB also have potential applications in the bioremediation of polluted soils and waters through the indiscriminate action of the monooxygenase that initiates nitrification (Painter, 1986).In view of the significance of AOB in nitrogen removal process, they have been investigated by the molecular technique (Aoi et al., 2005;Gong et al., 2008;Rowan et al., 2003).The 16S rDNA sequences are suitable for providing a comprehensive long-term evolutionary view of prokaryotic taxonomy because of their conservative characters (Rossello-Mora and Amann, 2001), and have proven useful in the discrimination among organisms (Beja et al., 2002;Hollibaugh et al., 2002;Stephen et al., 1996;Utaker et al., 1995;Weisburg et al., 1991), However, the outcome is confusing when examining a single genus by using 16S rDNA (Rotthauwe et al., 1997).For this reason, genes encoding protein, such as amoA which codes for the active site of AMO, have been added to the collection of comparative tools used by taxonomists and molecular ecologists for diversity studies (Aoi et al., 2005;Calvo et al., 2005;Gong et al., 2008;Purkhold et al., 2003), and amoA has been extensively used for the detection and study of ammonia oxidizers, particularly in natural environments (Aakra et al., 2001a;b;Horz et al., 2000;Juretschko et al., 1998;Rotthauwe et al., 1997).In addition, another protein encoding gene, hao, has been considered as a new molecular biological marker for AOB (Shinozaki and Fukui, 2002), because methane-oxidizing bacteria have the particulate methane monooxygenase (pmo) gene which resembles the amo gene (Holmes et al., 1995).
AOB plays a significant role for completely autotrophic nitrogen removal (Aoi et al., 2005;Gong et al., 2008;Guo et al., 2008), however they are very sensitive to the natural environment (pH, temperature etc.) and are hard to isolate (Hermansson and Lindgren, 2001).In our previous study, the sequencing batch biofilm reactor (SBBR) for completely autotrophic nitrogen removal over nitrite (CANON) was developed and the complete conversion of ammonia into N2 in a single reactor was achieved (Fang et al., 2007;Guo et al., 2009), and the mechanism responsible for completely autotrophic nitrogen removal was studied primarily (Yang et al., 2009), but still knowledge about the community of AOB for SBBR completely autotrophic nitrogen removal is very limited.Of the two groups of important functional bacteria for completely autotrophic nitrogen removal, the dominant Anammox bacteria community has already been identified (Huang et al., 2010).The purpose of this study, therefore, was to investigate the community of AOB for completely autotrophic nitrogen removal and to analyze the phylogenetic relationships.The phylogenetic tree topologies based on 16S rDNA, amoA and hao from AOB for the process were used for phylogenetic relationship analysis, respectively.The phylogenetic trees constructed from each gene were then weighted against the composite sequence dataset to identify the marker that best reproduced the information resulting from the polygenic tree.

Total DNA extraction from the sludge
Details of the SBBR for completely autotrophic nitrogen removal system operation have been described (Guo et al., 2009).The seed sludge samples used for total DNA extraction were obtained from the bottom of the reactor, which had been in a stable operation with 90% ammonia conversion ration and 80% total nitrogen removal ration.The total genomic DNA extraction was performed as previously described (Huang et al., 2009) with minor modification.To isolate purified total genomic DNA from the enriched sludge, polyvinylpoly-pyrrolidone was used to remove the humic acid contents.The total genomic DNA was obtained by lysing microbes in the soil sample through a series of lysozyme, sodium dodecyl sulfate, and rapid freeze-thaw treatments.

PCR amplification of 16S rDNA, amoA and hao
Primers aobF/aobR, amoA-F/amoA-R, and hao-F/hao-R for 16S rDNA, amoA and hao, respectively were designed according to the reported nine ammonia oxidizing strains within the β subdivision of the Proteobacteria (Head et al., 1993;Woese et al., 1984), usin g the Primer Express 1.0 (PE Applied Biosystems, Foster City, CA, USA).For 16S rDNA, aobF of 5'-CGAAAGATGTGCTAATACCG-3' and aobR of 5'-TGTGAAGCCCTACCCATAA-3' were used.The amplification of 16S rDNA partial sequence of AOB was performed as follows.The reaction volumes (25 μl) consisted of 1 × PCR buffer, 0.2 mmol l -1 dNTPs, 0.2 μmol l -1 of each of the primers, 0.5 U of pfu polymerase (Promega Corp., Wisconsin, USA) and 1 μl of the total DNA from the sludge as the templates.The amplification was conducted with an initial denaturing step at 94°C for 4 min, followed by 30 cycles (94°C for 30 s, 62°C for 30 s, 72°C for 2 min) and 72°C for 5 min and a final elongation step at 72°C.DNA sequence of amoA was amplified using the following primers: amoA-F (5'-GTGAGTATATTTAGAACGGAAGA-3') and amoA-R (5'-TTTATTTGATCCCCTCTGG-3').Reactions were performed as described above.The standard thermal profile used for the amplification of the amoA target sequence was as follows: 3 min at 94°C, and then 30 cycles consisting of 30 s at 94°C, 30 s at 48°C, 2 min at 72°C, and a final cycle 5 min at 72°C.
The full length sequence of the hao was amplified by using the following primers: hao-F (5'-CGGAGGAGAGAGATGAGAATAG-3') and hao-R (5'-CGGGTCGGTTGTCAGTGCGGT-3').PCR reactions were performed in 20 µl containing 1 × GC buffer (TaKaRa Shuzo, Shiga, Japan), 0.4 mmol l -1 dNTPs, 0.1 μmol l -1 of each primer, 0.5 U of Pfu polymerase (Promega Corp., Wisconsin, USA) and 0.8 µl of template DNA.The initial denaturation at 94°C for 3 min was  Burrell et.al., 2001 followed by 30 cycles consisting of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 2 min and the final extension at 72°C for 10 min.Aliquots (5 µl) of the PCR products of 16S rDNA, amoA and hao were electrophoresed and visualized in 1% agarose gels by using standard electrophoresis procedures.

Phylogenetic analyses
The nucleotide sequences for 16S rDNA, amoA and hao genes have been submitted to GenBank database under accession numbers HQ144199, HM473177 and HQ174563, respectively.For phylogenetic analysis, the homologues of 16S rDNA (Table 1), amoA (Table 2) and hao (Table 3) genes were retrieved from DDBJ/EMBL/GenBank International Nucleotide Data Banks using 16S rDNA (HQ144199), amoA (HM473177) and hao (HQ174563) as queries, respectively.The collected data was used to construct the phylogenetic trees.Neighbor-joining (NJ) phylogenetic trees for 16S rDNA, amoA, and hao genes were generated from the corresponding matrix of nucleotide divergence between sequences using the program MEGA5.0, respectively.Confidence in the branching points was obtained with 100 bootstrap replicates.The GenBank accession numbers for the sequences determined in this study are HQ144199 (sequence for 16S rDNA), HM473177 (sequence for amoA) and HQ174563 (sequence for hao).

AOB phylogeny inferred from 16S rDNA
A unique DNA fragment of approximately 1100 bp was amplified by primers aobF/aobR from the total DNA extracted from the sludge in the reactor for completely autotrophic nitrogen removal system (Figure 1).DNA sequence analysis of the fragment of 1074 bp revealed that it was partial sequence of 16S rDNA from AOB.Only sequences of more than 98% in similarity were concerned for phylogenetic analysis (Table 1).The phylogenetic tree based on the similarity of sequences of 16S rDNA was constructed (Figure 2).The phylogenetic inference analysis revealed that AOB for completely autotrophic nitrogen removal formed a monophyletic group with Nitrosomonas sp., multiple sequence alignments that showed highest similarities to Nitrosomonas sp.(99%).AOB strains having more than 60% DNA-DNA similarity are considered as members of the same species (Purkhold et al., 2003).According to DNA-DNA similarity data, AOB for completely autotrophic nitrogen removal are members of the same species of Nitrosomonas sp.

AOB phylogeny inferred from amoA
During the past few years, the gene encoding the active site subunit of amoA has been exploited increasingly as a marker molecule for AOB diversity research in natural and engineered systems.In this study, the full length of amoA was used to characterize the AOB for completely autotrophic nitrogen removal.The primers amoA-F/ amoA-R produced a fragment of approximately 830 bp (Figure 3).The Phylogenetic trees for amoA were calcu-lated from the nucleotide datasets and the amoA sequence of AOB from completely autotrophic nitrogen removal system showed the highest similarity (99%) to FJ577880 and FJ577884 (Table 2).The organisms from completely autotrophic nitrogen removal system probably represent a novel lineage in the amoA-based topology.In addition, 88 to 94% similarity with the amoA gene of Nitrosomonas  sp. was achieved, which demonstrated AOB for completely autotrophic nitrogen removal were also closely related to the bacteria of the β subclass of the Proteobacteria, Nitrosomonas.

AOB phylogeny inferred from hao
With the extending dataset and an increasing number of closely related amoA sequences, the limitation of the amoA approach as applied now becomes more apparent.
Although AOB pure cultures or AOB in environmental samples can be assigned rapidly to some phylogenetic subgroups within this guild by using the amoA approach, the amoA fragment analysed does provide less resolution.In this study, therefore, AOB for completely autotrophic nitrogen removal were further analysed by a new marker, the hao gene.The protein coding gene, hao, is 1713 bp in length and is expressed as a monocistronic transcript (Sayavedra-Soto et al., 1994).In this study, the full length sequence of hao, approximately 1700 bp, was produced by primers hao-F/hao-R (Figure 5).The hao-based phylogenetic tree demonstrated that AOB for completely autotrophic nitrogen removal are related most closely to "Nitrosomonas sp.ENI-11" (Table 3 and Figure 6).The close relationship  16S rDNA-based phylogenetic tree of the ammoniaoxidizing bacteria (AOB) for completely autotrophic nitrogen removal and its relatives.The tree includes all isolates for which 16S rDNA gene sequences have higher than 98% of similarities with that of AOB (HQ144199).GenBank accession numbers in the tree represent different organisms (Table 1) .Confidence in the branching points was obtained with 100 bootstrap replicates.Most of the nodes are supported with high bootstrap values (>60%).
between AOB for completely autotrophic nitrogen removal and Nitrosomonas sp. is also reflected by their high DNA-DNA similarity.

DISCUSSION
The autotrophic ammonia-oxidizing bacteria (AOB) do not represent a monophyletic clade but are members of at least two phylogenetically different groups.The first group is characterized by the members of Nitrosomonas-Nitrosospira clade in the β subclass of the Proteobacteria, and the second group is characterized by the strains of the species Nitrosococcus oceanus, and probably, Nitrosococcus halophilus in the γ subclass of the Proteobacteria (Rotthauwe et al., 1997).The first group exists widely in the (Belser, 1979), and in this study, the results show that the "Nitrosomonas" clade of the β proteobacteria AOB existed in completely autotrophic nitrogen removal system.
The phylogenetic tree is a tool which analyzed the phylogenetic relationship of unknown samples in environment.Distance-based neighbor-joining method was used to construct the phylogenetic tree in this study.Though maximum parsimony trees were found to be more accurate than distance-based neighbor-joining method to analyze the AOB (Calvó et al., 2005), the neighbor-joining method was more widely used (Aakra et al., 2001a;Purkhold et al., 2000;Purkhold et al., 2003;Tabei and Ueno, 2010), and neighbor-joining method is proposed for reconstructing phylogenetic trees from evolutionary distance data (Saitou and Nei, 1987).In this study, the 16S rDNA, amoA and hao-based phylogenetic trees were constructed.The 16S rDNA and hao genes sequences determined showed highest similarities (99%) to sequences of Nitrosomonas sp., and the amoA gene sequences determined demonstrated highest similarity (99%) to sequences of a new group of "uncultured bacterium", and was also closely related to Nitrosomonas sp.(88 to 94% of similarity).
The phylogenetic relationship of AOB in the environment has been investigated based on the 16S rDNA and amoA previously (Purkhold et al., 2000;Purkhold et al., 2003), while this is the first report to characterize AOB for completely autotrophic nitrogen removal with three differ- .aomA-based phylogenetic tree of the ammonia-oxidizing bacteria for completely autotrophic nitrogen removal and its relatives.The tree includes all isolates for which amoA has higher than 88% of similarity with that of AOB (HM473177).GenBank accession numbers in the tree represent different organisms (Table 2).Confidence in the branching points was obtained with 100 bootstrap replicates.Most of the nodes are supported with high bootstrap values (>50%).rent markers 16S rDNA, amoA and hao genes.Taken together, topologies of 16S rDNA and hao-based trees were similar, but inconsistent affiliations were also found in the amoA -based tree (Figures 2, 4 and 6).The 16S rDNA and hao-based phylogenetic analysis revealed that AOB for completely autotrophic nitrogen removal belong to Nitrosomonas sp., while the amoA-based topology showed the members are related most closely to uncul-tured bacteria, while higher similarity (88 to 94%) with Nitrosomonas sp. was also achieved.Previous results demonstrated a superior resolution of 16S rDNA and amoA analysis (Purkhold et al., 2003).Comparison of 16S rDNA, amoA and hao gene in chemolithotrophic AOB showed that the hao gene is more useful than the amoA gene as a molecular biological marker for AOB detection from environmental samples (Shinozaki and Figure 6.hao-based phylogenetic tree of the ammonia-oxidizing bacteria for completely autotrophic nitrogen removal and its relatives.The tree includes all isolates for which hao has higher than 81% of similarities with that of AOB (HQ174563).GenBank accession numbers in the tree represent different organisms (Table 3).Confidence in the branching points was obtained with 100 bootstrap replicates.Most of the nodes are supported with high bootstrap values (>70%).Fukui, 2002), because methane-oxidizing bacteria have the particulate methane monooxygenase (pmo) gene which resembles the amo gene (Holmes et al., 1995) and a sequence similar to the hao gene has not been reported in other organisms.Therefore, the hao gene may be used in the future if the database of this gene is expanded appropriately.In this study, the hao gene was first used as a marker to identify AOB for completely autotrophic nitrogen removal and proved its practicability.

Figure 2 .
Figure 2. 16S rDNA-based phylogenetic tree of the ammoniaoxidizing bacteria (AOB) for completely autotrophic nitrogen removal and its relatives.The tree includes all isolates for which 16S rDNA gene sequences have higher than 98% of similarities with that of AOB (HQ144199).GenBank accession numbers in the tree represent different organisms (Table1) .Confidence in the branching points was obtained with 100 bootstrap replicates.Most of the nodes are supported with high bootstrap values (>60%).
Figure 4. aomA-based phylogenetic tree of the ammonia-oxidizing bacteria for completely autotrophic nitrogen removal and its relatives.The tree includes all isolates for which amoA has higher than 88% of similarity with that of AOB (HM473177).GenBank accession numbers in the tree represent different organisms (Table2).Confidence in the branching points was obtained with 100 bootstrap replicates.Most of the nodes are supported with high bootstrap values (>50%).

Table 1 .
Sequences for 16S rDNA used in this study.

Table 2 .
Sequences for amoA used in this study.

Table 3 .
Sequences for hao used in this study.