Microbes Environ. Vol. 22, No. 3, 232–242, 2007 http://wwwsoc.nii.ac.jp/jsme2/

Detection of Activity and Diversity of Anammox - Related 16S rRNA Genes in Coastal Marine Sediment in Japan

TERUKI AMANO1, IKUO YOSHINAGA1*, KENTARO OKADA1, TAKAO YAMAGISHI2, SHINGO UEDA3, AKIRA OBUCHI2, YOSHIHIKO SAKO1, and YUICHI SUWA2

1 Laboratory of Marine Microbiology, Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606–8502, Japan 2 Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, 16–1, Onogawa, Tsukuba, Ibaraki 305–8569, Japan 3 College of Bioresource Sciences, Nihon University, 1866, Kameino, Fujisawa, Kanagawa 252–8510, Japan

(Received April 16, 2007—Accepted May 10, 2007)

A first assessment of anammox activity, which is a unique N2 emission process, was conducted in samples of coastal marine sediment from Japan with a 15N tracer. The occurrence and diversity of bacteria possibly responsi- ble for the anammox process were also evaluated by selective PCR-amplification of the 16S rRNA gene for known anammox bacteria. Anammox activity, detected by measuring 14N15N gas production, was only found in samples collected at the intertidal sand bank located at the Yodo River . In the Yodo River samples, 16S rRNA gene fragments affiliated with the known anammox bacterial lineage were also recovered, and the two major phylotypes were both “ wagneri” relatives with 95% and 98% sequence similarity. Even from the other samples in which no recognizable anammox activity was detected, 16S rRNA gene frag- ments related to known anammox bacteria, but not to “Ca. S. wagneri”, were detected. This is the first report of anammox-mediated N2 emission in coastal marine environments in Japan. Notably, the PCR-based analysis allowed us to discover unexpected phylogenetic diversity of anammox bacteria-related 16S rRNA gene sequences. The selective PCR primer set developed in this study could be a powerful tool to unveil the of anammox bacteria in natural environments.

Key words: anammox, 16S rRNA gene, cycle, marine sediment, 15N tracer

Coastal and enclosed sea areas, into which excess fication. A specific group of bacteria belonging to the order amounts of terrestrial nitrogen flow, can easily become Planctomycetales in the phylum is consid- eutrophic and suffer from harmful algal blooms. For the ered to mediate the anammox reaction under anoxic sustainable management of such marine environments, a conditions28). Since anammox has a different mechanism better understanding of the nitrogen cycle, especially the from in N2 formation, the presence of anam- emission of N2 gas as a mechanism of removing nitrogen mox will cause one to overestimate true N2 production when 15 from the fixed nitrogen pool, is essential. N2 gas emission in the nitrogen isotope ( N) pairing technique (IPT), which marine environments may also be a key regulator of ecosys- has been widely used for measuring N2 production by deni- tem function and global biogeochemistry2). trification in natural samples, is employed19). Therefore, Recently, it was demonstrated that anaerobic investigating the distribution of anammox activity and oxidation (anammox) was also involved in the emission of microorganisms responsible in marine environments is 12,30) N2 gas from marine environments in addition to denitri- required for a precise estimation of N2 gas emission. Most of the information on anammox has been obtained * Corresponding author. E-mail address: [email protected]. from enriched cultures with high concentrations of inor- jp; Tel.: +81–75–753–6219; Fax: +81–75–753–6226. ganic nitrogen substrates. In the anammox reaction, the two Anammox in Coastal Marine Sediment 233

+ − nitrogen atoms in N2 are derived from NH4 and NO2 Sea, Osaka Bay, Ise Bay and the intertidal sand bank at the 36) respectively , while denitrification forms N2 gas from two Yodo River estuary in 2005 and 2006, using a grab sampler − − 37) molecules of NO2 (NO3 ) through a stepwise reduction. (Rigo Co. Ltd., Tokyo, Japan) (for samples S1, S2, S3, IB1, This biochemical difference allows us to distinguish and IB2), a KK core sampler (Hashimoto Kagaku Co. Ltd., 15 between N2 production by the two processes by using N as Kyoto, Japan) (for samples OB1 and OB2), or a plastic core a tracer. Regarding microorganisms responsible for the sampler (for samples YR-M and YR-J) (Fig. 1 and Table 1). anammox reaction, four genera of Planctomycetales (“Bro- The surface layer of each sediment sample was collected cadia”, “Kuenenia”, “Scalindua” and “Anammoxoglobus”) into plastic containers, tightly sealed, and stored at 4°C in are proposed as anammox bacteria10,24,25,28), although no iso- the dark until experiments. The measurement of anammox late has been established so far. activity was performed within a week at longest after sam- However, there has been limited information available pling except for the samples S1, S2, and S3 which were about the contribution of anammox to the nitrogen cycling stored for one and a half months at 4°C in the dark. and diversity of anammox-related bacteria in natural envi- ronments. At present, the activity and related bacteria at Measurement of anammox and denitrification activity several sites has been investigated with a 15N tracer and The occurrence of the anammox process was examined 12,13,20,26,27,29) 14 15 29 molecular techniques . A PCR amplification of by quantifying N N ( N2) emissions using gas chroma- the 16S rRNA gene related to known anammox bacteria tography-quadrupole mass spectrometry (GC-MS) in a was carried out in these investigations, however, sequences strictly anaerobic incubation of sediment slurries amended 15 affiliated with the anammox bacterial lineage were recov- with N-labeled or non-labeled NH4Cl and NaNO2 (Suwa et ered at a relatively low efficiency. For instance, when a al. in preparation). Each sediment sample was suspended at Planctomycetales-specific primer was used for PCR ampli- a concentration of 8–25% (wt/vol) in seawater that was pre- fication, only 4 out of 50 clones were recovered as anam- liminarily filtrated through a 0.22 µm cellulose acetate filter mox bacteria-related sequences in a library constructed system (Corning Inc., NY, USA), and the suspension was from the sediment sample showing anammox activity29). thoroughly purged with Ar to remove oxygen. The salinity This could be explained by the inadequacy of the primer in the slurry was adjusted to in situ levels with an oxygen- sets previously available, including nucleotide sequences free inorganic medium developed previously35) except that targeting a broader range of phyla as bacterial universal the medium used in this study contained 7.15 mM NaHCO3 primers or a primer for all Planctomycetales. The low instead of 5 mM KHCO3. Portions (40 ml) of the slurry recovery of anammox bacteria-related 16S rRNA gene frag- were transferred to 67-ml glass vials, and capped tightly ments limits information on the diversity and community with a butyl rubber stopper and an aluminum seal. These structure of anammox bacteria in natural environments, and procedures were carried out inside a glove box under an prevents us from understanding the ecology of the anam- Ar+H2 (anoxic) atmosphere. Subsequently, the headspace mox bacterial community. gas of a vial was replaced with He by repeatedly vacuuming The ultimate goal of our project is to elucidate the eco- and purging with high-purity He gas. The final pressure of logical significance of the anammox process in marine envi- ronments. As a first step to achieving this, here we demon- strate the occurrence of anammox activity and anammox bacteria-related 16S rRNA gene fragments in coastal marine sediment in Japan. Our research areas, the Seto Inland Sea, Osaka Bay and Ise Bay are representative enclosed sea areas in Japan; however, no report has been available on anammox activity there up to now. We also propose effec- tive PCR primer sets to detect anammox bacteria-related 16S rRNA genes with a relatively high recovery efficiency.

Materials and Methods Sampling Sediment samples were collected from the Seto Inland Fig. 1. Sampling sites in western Japan. 234 AMANO et al.

Table 1. Sample characteristics

Sample Location Water depth (m) Date Sediment layer (cm) Seto Inland Sea S1 34°04'N, 133°17'E 28 10 Aug. 05 0–5 S2 34°25'N, 134°30'E 40 9 Aug. 05 0–5 S3 34°30'N, 134°31'E 42 8 Aug. 05 0–5 Osaka Bay OB1 34°39'N, 135°22'E 10.5 18 Apr. 06 0–5 OB2 34°38'N, 135°23'E 11 18 Apr. 06 0–5 Ise Bay IB1 34°43'N, 135°44'E 36 20 Jul. 06 0–2 IB2 34°39'N, 135°55'E 21 20 Jul. 06 0–2 Yodo River estuary YR-M 34°41'N, 135°27'E intertidal 12 May. 06 0–1 YR-J 13 Jun. 06 0–2

29 15 + − the headspace gas with He was 0.15 MPa. The vials con- of N2 gas production in vials amended with NH4 +NO2 . 30 + 15 − taining anoxic sediment slurries were pre-incubated for 1–4 Increases in N2 accumulated in vials with NH4 + NO2 hours in the dark at 20°C before substrates were added, in were expressed as potential denitrification activity. A time order to eliminate the remaining and as well as course was used for determining rate constants of both reac- 29 30 dissolved oxygen. tions. Abundances of N2 and N2 gas in vials for a nega- 15 15 Oxygen-free stock solutions of N-labeled NH4Cl tive control experiment with NH4Cl but without nitrite (Shoko-Co., Ltd., Tokyo, Japan, 15N atom%: >99) and were monitored to see if and denitrification 15 29 NaNO2 (ISOTEC Inc, OH, USA, N atom%: >99) or non- coupled to produce N2. labeled substrates (Wako Ltd., Osaka, Japan) were added 15 + + 4 with a gas-tight syringe. The final concentrations of NH4 N labeling of the NH pool in sediment slurries − 15 + and NO2 were 400 µM and 1 mM, respectively. Labeled/ The N labeling of the NH4 pool in the vials at the non-labeled substrates were added in three combinations as beginning of incubation was calculated upon samples YR- 15 15 + follows: (1) NH4Cl+NaNO2, (2) NH4Cl+Na NO2, (3) M and YR-J from NH4 concentrations in pore water of the 15 15 + NH4Cl but without nitrite (negative control). If anammox slurries before and after the addition of NH4 (final conc. activity was detected in a sediment sample, autoclaved sam- 400 µM). One milliliter of slurry was sampled with a dis- 15 + − ples were anoxically incubated with NH4 +NO2 to con- posable syringe and needle. Subsequently, pore water was firm that the activity is microbiologically mediated. The extracted by centrifugation at 10,000×g for 20 min and the + slurries in vials were incubated with stirring at 150–350 rpm supernatant was stored at 4°C for analysis. NH4 concentra- on a magnetic stirrer at 20°C. The headspace gas (100 µl) of tions were measured by ion chromatography (HIC-6A Shi- each vial was collected periodically with a gas-tight syringe madzu. Co., Kyoto, Japan). under a He stream, and 50 µl of the gas was immediately loaded into a GC-MS (Agilent 6890N/5973 GC/MSD sys- DNA extraction from sediment tem, Agilent Technologies, Inc., CA, USA) equipped with a Approximately 1.2 g (wet weight) of each sediment was 2 m stainless steel column packed with a molecular sieve centrifuged at 10,000×g for 20 min, and the precipitates 28 (80/100 mesh) for quantification of N2 isotopomers ( N2, were frozen at −30°C. Total DNA was extracted from the 29 30 N2, N2). Ultra pure (99.99995%) He gas was used as a frozen sediment using ISOIL (Nippon Gene, Tokyo, Japan) carrier gas, the flow rate of which was 12.0 ml min−1. The according to the manufacturer’s directions. This DNA abundance of each N2 isotopomer was calibrated with a extraction kit was more effective than any other DNA 29 standard curve, which was prepared with standard N2 and extraction methods or kits we tried. 30 N2 gases, to determine their quantity. Anoxic incubations were performed in duplicate, and potential anammox activities were calculated from a slope Anammox in Coastal Marine Sediment 235

the Ribosomal Database Project3) were used to determine PCR amplification of anammox bacteria-related 16S phylogenetic affiliations. The Bellerophon program8) was rRNA gene fragments used to identify possible chimeric artifacts. Multiple align- To detect the presence of anammox bacteria, PCR ampli- ments were carried out using the CLUSTALW program32). fication targeting the 16S rRNA gene of the known anam- A phylogenetic analysis was performed by the neighbor- mox bacterial lineage was performed with total community joining method22) using the CLUSTALW program. DNA directly extracted from sediment samples. The PCR Kimura’s two-parameter correction was used, with all other primers used in this study are listed in Table 2. The reaction settings set by default. mixture (100 µl in total) contained dNTPs (200 µM each), each of the forward and reverse primers (0.25 µM), 25 U of Calculation of DNA sequence similarity and defining EX TaqTM DNA polymerase (TaKaRa Bio Inc., Shiga, OTUs Japan), and 100 ng of template DNA. The thermal cycle The DOTUR program23) was used to assign determined program employed here was an initial denaturation step at sequences to operational taxonomic units (OTUs). Deter- 94°C for 4 min, followed by 30 cycles of denaturation at mined sequences were aligned with the sequences of the 94°C for 30 s, annealing at 56°C for 30 s, elongation at corresponding region of known anammox bacterial candi- 72°C for 1 min, and a final extension step at 72°C for 7 min. dates (“Brocadia anammoxidans”: AF375994, “B. fulgida”: Among the several primers shown in Table 2, we finally DQ459989, “Kuenenia stuttgartiensis”: AF375995, “Scalin- selected two primer sets, AMX368F-AMX820R and dua wagneri”: AY254882, “S. brodae”: AY254883, “S. AMX368F-BS820R, since they were most effective for a sorokinii”: AY257181, “Anammoxoglobus propionicus”: clone library analysis. The PCR products were recovered DQ317601) and other representative members of Plancto- ® and purified using a Wizard PCR and Gel purification sys- mycetales which are considered not to perform anammox tem (Promega, MA, USA) after 1.5% (w/v) agarose gel (non-anammox Planctomycetales) (Blastopirel- electrophoresis. Purified DNA fragments were ligated into lula marina: X62912, Gemmata obscuriglobus: AJ231191, the pGEM-T Easy vector (Promega), and 16S rRNA gene Isosphaera pallida: AJ231195, Pirellula staleyi: AJ231183, clone libraries were constructed by transforming E. coli Planctomyces maris: AJ231184), by using the CLUSTALX INVαF’ (Invitrogen, CA, USA) with the vectors. Nearly 1.83 program18). After constructing a Kimura’s two-parame- full-length 16S rRNA gene fragments were also amplified ter corrected distance matrix with the DNADIST program with the primer set Pla46F-AMX1480R. For this PCR, the from PHYLIP7), a group of sequences that had more than a elongation step was modified to 72°C for 1.5 min. certain level of similarity among them were combined into one OTU by using the “furthest neighbor assignment algo- Sequencing and phylogenetic analysis rithm” implemented in DOTUR. More than 30 clones from each library were sequenced with an ABI 3730xl (Applied Biosystems, CA, USA) by a Accession numbers sequencing service provided by Dragon Genomics Center Sequences of 16S rRNA gene clones described in this (Takara Bio Inc.). The NCBI-BLAST 2.2.12 program1) and study are available in the DDBJ/EMBL/GenBank databases

Table 2. Primers used for PCR amplification targeting anammox bacteria-related 16S rRNA genes

Primer Target Sequence (5'-3') Reference Pla46 Planctomycetales GGATTAGGCATGCAAGTC 17 AMX60F All anammox bacteria except for “Anammoxoglobus AGGGTGAGTAATGCATWGATWACCT this study propionicus” and “Scalindua wagneri” S-*-Amx-0368-a-A-18 All anammox bacteria except for “Anammoxoglobus TTCGCAATGCCCGAAAGG 25 (AMX368F) propionicus” S-*-Amx-0820-a-A-22 “Brocadia anammoxidans”, “Brocadia fulgida” and AAAACCCCTCTACTTAGTGCCC 24 (AMX820R) “Kuenenia stuttgartiensis” S-*-BS-820-a-A-22 “Scalindua wagneri” and “Scalindua sorokinii” TAATTCCCTCTACTTAGTGCCC 12 (BS820R) AMX1480R All anammox bacteria TACGACTTAGTCCTCCTCAC this study 236 AMANO et al.

29 under the accession numbers AB300468 to AB300486, and lated, which demonstrates that N2 production via nitrifica- AB303289 to AB303295. tion and subsequent denitrification was negligible. The activity was detected in another sample from the same site, Results YR-J, which was collected one month later. Considering the 15 + 15 dilution of NH4 (72.4% and 94.6% as the N labeling + Anammox activity measurement ratio of the NH4 pool in vials with sediment slurries of YR- Anammox activity was estimated based on production of M and YR-J, respectively), the potential anammox activities 29 29 N2 gas in strictly anoxic incubation of the marine sediment of the sediment samples YR-M and YR-J, expressed by N2 15 + − slurries with NH4 and NO2 . Among the nine sediment production, were determined as 0.29 and 0.69 nmol N2 pro- −1 samples collected from the Seto Inland Sea, Osaka Bay, Ise duction per 1 ml of wet sediment per hour (nmol-N2 ml 29 −1 Bay, and the Yodo River estuary, N2 gas was detected in h ), respectively (Table 3). Potential denitrification activi- the Yodo River sample YR-M collected on May 8, 2006 ties were also calculated in the same manner using the data 15 + 30 + (Fig. 2). When the slurry was incubated with NH4 in the sets of N2 production in vials amended with NH4 and 29 30 15 − absence of nitrite, neither N2 nor N2 gas was accumu- NO2 (Fig. 2). The level of denitrification was higher than −1 −1 that of anammox and reached 18 and 80 nmol-N2 ml h , respectively. No appreciable anammox activity was detected in other 30 samples (Table 3), whereas the production of N2 from 15 − NO2 added as an indicator of bacterial denitrification was observed (data not shown). From the sediment slurries of 29 the sample S3 collected from the Seto Inland Sea, N2 gas was detected, but neither reproducible nor robust evidence of anammox was obtained with this sample.

PCR amplification of anammox bacteria-related 16S rRNA genes and phylogenetic analysis In order to investigate the anammox bacterial commu- nity, we developed a selective PCR amplification protocol to detect anammox bacteria-related 16S rRNA gene frag- ments. Several PCR primers previously reported and newly designed with reference to the GenBank database were applied through several thermal cycle programs to the com- munity DNA of sample YR-J, in which the most anammox activity was observed. Two primer sets, AMX368F- AMX820R and AMX368F-BS820R, were most effective for amplification of the 16S rRNA gene fragments. The other combinations of primers provided unstable results in PCR-amplification and/or low recovery frequencies of anammox bacteria-related sequences after a clone library analysis. After constructing clone libraries containing PCR- amplified DNA fragments with the two primer sets, more than 40 clones in each were sequenced. Most sequences

29 30 from both libraries of AMX368F-AMX820R (39/40 clones) Fig. 2. Changes of N2 (a) and N2 abundance (b) in vials with sediment slurry of the sample YR-M. The slurries were incubated and AMX368F-BS820R (39/42 clones) were phylogeneti- 15 + − + 15 − 15 + with NH4 +NO2 (circles), NH4 + NO2 (triangles), or NH4 cally similar to some of the anammox bacterial 16S rRNA alone (squares). Open and closed symbols represent the experi- 29 + 15 − genes in the database, the similarity values being greater ment in duplicate. N2 production in slurries with NH4 + NO2 29 than 94%. The remaining clones were relatively similar to was not monitored because N2 can be produced even through 15 − denitrification unless the N labeling of the NO2 pool in vials is several environmentally derived bacterial 16S rRNA gene 100%. fragments (e.g. AB127625 and AB240495), and were obvi- Anammox in Coastal Marine Sediment 237

Table 3. Anammox activity and recovery of anammox bacteria-related 16S rRNA genes in clone libraries constructed with the primer set AMX368F-AMX820R from coastal sediment samples in Japan

Anammox activitya in anoxic incubation of sediment slurry amended with The number of The number of clones identified Sample clones analyzed as anammox bacteria-related (B)/(A) (%) 15 + − 15 + NH4 +NO2 NH4 (A) 16S rRNA genes (B) 29 −1 −1 29 −1 −1 (nmol- N2 ml h ) (nmol- N2 ml h ) S1 NDb —c 37 29 78 S2 ND — 33 29 88 S3 ND — 38 35 92 OB1 0.01 (0.01) −0.01 (0.03) 34 5 15 OB2 0.00 (0.01) 0.02 (0.01) 30 0 0 IB1 0.01 (0.00) 0.01 (0.01) 36 5 14 IB2 0.01 (0.02) 0.00 (0.01) 34 12 35 YR-M 0.29 (0.04) −0.01 (0.02) 42 30 71 YR-J 0.69 (0.03) 0.00 (0.01) 40 39 98 a Mean values of duplicate experiments. Standard deviations are shown in parentheses. b 29 ND, not determined. N2 gas production was monitored, but an anammox rate was not calculated. c —, not examined.

ously separate from the lineage of anammox bacterial can- high similarity (98%), and contained the identical sequence didates. region with dominant clones in phylotype 1 (Fig. 3). More- In the phylogenetic tree including the two libraries and over, the YR-6JSW clone has an identical region with reference sequences, two dominant groups (phylotypes 1 BS820R, which indicates the PCR conditions employed in and 2) were observed (Fig. 3). The topology of the tree this study allowed three base mismatches between demonstrated that the gene compositions of two libraries AMX820R and BS820R. The 16S rRNA gene clone library AMX368F-AMX820R and AMX368F-BS820R were not constructed with AMX368F-AMX820R from the sediment clearly distinguishable despite the difference in the reverse sample YR-M was also analyzed as on the sample YR-J. primer. The phylotype 1 was close to “Candidatus Scalin- Eighteen and 4 of 30 anammox bacteria-related clones were dua wagneri” (AY254882), which had been discovered in a affiliated to the phylotypes 1 and 2, respectively (Fig. 3). wastewater treatment plant25) and the sequence similarity A clone library analysis of the other sediment samples between the representative clones in phylotype 1 and “Ca. was also performed as on the sample of the Yodo River S. wagneri” was 98%. “Ca. S. wagneri” was also the closest estuary, since PCR amplicons were obtained with the relative of clones in phylotype 2 among the seven species of primer set AMX368-AMX820 even from the samples in known anammox bacteria, although the sequence similarity which anammox activity had not been observed. More than was only 95%. 30 clones in each of the libraries were randomly selected Nearly full-length 16S rRNA gene fragments of the and sequenced, and the determined sequences were anammox bacterial lineage were PCR-amplified from the assigned into OTUs after aligning them with the sequences same DNA sample of YR-J with the primer set Pla46- of known anammox bacterial candidates (seven species) and AMX1480R. Following the construction of clone libraries, non-anammox Planctomycetales in the GenBank database more than 50 clones were sequenced, but none of them were (see Materials and Methods). All of the known anammox considered anammox bacteria-related sequences. Hence, bacterial sequences in the corresponding region of the 16S dozens of transformants were screened by PCR amplifica- rRNA gene were grouped into one OTU clearly separated tion with SWA464F (5'-CCATTTATTTGACAAAAGCC- from the OTUs containing non-anammox Planctomycetales 3') and SWA614R (5'-TACACTCAAGATCTGCAG-3') sequences with a 19% cutoff value of the DOTUR program. targeting “Ca. S. wagneri”, and then sequenced. Only one Therefore, in this study, “anammox bacteria-related” 16S clone (YR-6JSW) was affiliated with known anammox bac- rRNA genes were tentatively defined as the sequences teria in databases. The nucleotide sequence (1460 bp) of the belonging to the “anammox bacterial OTU”, that is, the clone was related to that of “Ca. Scalindua wagneri” with sequences with at least 81% similarity to all of the seven 238 AMANO et al.

Fig. 3. A phylogenetic tree showing the relationships of anammox bacteria-related 16S rRNA gene fragments in the clone libraries of samples YR-M and YR-J (the Yodo River estuary) and known anammox bacteria. The tree was constructed based on partial 16S rRNA gene sequences (386–819; E. coli numbering) by the neighbor-joining (NJ) method. Branch points supported with bootstrap values of >95% are marked with solid circles, while those supported with values of >70% are marked with open circles. The representative clones in each opera- tional taxonomic unit (OTU) obtained in this study are indicated in bold. The clones are labeled with sample name, clone number, and the reverse primer used for constructing the library (AMX820R [A] or BS820R [B]); e.g., YR-6M24-A represents clone-24 recovered from sam- ple YR-M with the primer set AMX368F-AMX820R in 2006. The numbers of clones obtained as the same OTU are shown in parentheses. Each OTU comprises clones with greater than 99% sequence similarity among them. Two dominant groups in each library are specified as “phylotype 1” and “phylotype 2” and shown by vertical lines. Database accession numbers are shown after the names of species or clones. The sequence of Planctomyces maris (AJ231184) was used as an outgroup. species of published anammox bacterial candidates. With ities. The recovery frequencies of anammox bacteria-related this criterion, many clones of anammox bacteria-related 16S 16S rRNA genes were different among samples, and the fre- rRNA genes were recovered from each library, and the quencies from OB1, OB2, IB1, and IB2 were lower than remaining sequences were related to several environmen- those from samples YR-M and YR-J, and notably, no anam- tally derived clones in databases with low sequence similar- mox bacteria-related sequences were recovered from OB2 Anammox in Coastal Marine Sediment 239

+ − (Table 3). Most of the anammox bacteria-related sequences (400 µM and 1 mM of NH4 and NO2 , respectively) as well (183/184 clones) had 93% sequence similarity at least with as the disruption of natural oxygen and substrate gradients34) any of the anammox bacterial candidates. Some clones from or of local heterogeneous physical structures formed by OB1 were related to “Ca. Kuenenia stuttgartiensis” with macrobenthic bioturbation11), which influence on bacterial 93% and “Ca. Brocadia fulgida” with 96% sequence simi- metabolic activity, the results from vial experiments, such larity, respectively, whereas “Ca. Scalindua sorokinii” and as our activity assay system, may not completely reflect the “Ca. S. brodae” relatives with greater than 94% sequence intact activities of denitrification and anammox. Further similarity were recovered from samples IB1 and IB2. Inter- improvements of measuring methods including the optimi- estingly, in the case of samples from the Seto Inland Sea zation of substrate concentrations are needed to elucidate (S1, S2, and S3), anammox bacteria-related 16S rRNA gene correct estimates of these nitrogen conversion processes in fragments were recovered with relatively high frequency in coastal environments. the clone libraries. Among the anammox bacterial candi- dates, “Ca. Scalindua sorokinii” and “Ca. S. brodae” were Anammox bacteria-related 16S rRNA gene sequences most similar to the anammox bacteria-related sequences recovered from coastal marine sediment in Japan from the Seto Inland Sea. In this study, we attempted to obtain evidence of anam- mox through two independent methods, measuring the 15 Discussion activity with N tracers and detecting anammox bacteria by PCR amplification of anammox bacteria-related 16S rRNA Anammox activity in coastal marine sediment in Japan genes. Recently, there have been an increasing number In the present study, we detected potential anammox of reports showing anammox activity in marine sedi- + − 6,15,20,21,26,30,33) 4,12,13,27,31) activity (anaerobic NH4 oxidation with NO2 ) in two sam- ment and water column . However, ples collected from an intertidal sand bank in the Yodo very little information on the ecology of anammox-related River estuary by means of tracer experiments, although the bacteria has been available partly because the bacteria are anammox process was not detected ubiquitously in coastal difficult to cultivate. Even a culture-independent analysis marine sediment in Japan (Table 3). A few mechanisms targeting 16S rRNA gene homologues of known anammox + other than anammox such as the oxidation of NH4 by Mn bacteria could not provide sufficient information about the oxides under anoxic conditions14) and/or denitrification cou- or species composition of the anammox 16) 29 pled with anoxic nitrification could possibly produce N2 bacterial community probably due to the inadequacy of 15 + − gas from NH4 and NO2 . However, a contribution by primers, such as the low recovery frequency of target genes 29 these two other mechanisms to the production of N2 is not in the amplicons. Hence, we need a more effective, but spe- 29 30 likely, since there was no appreciable N2 or N2 produc- cific and comprehensive PCR-protocol for detecting anam- 15 + tion when nitrite was not added with NH4 amended (Fig. mox bacteria in natural environments as we have proposed 2 and Table 3). in the present study. The potential anammox activity in sediment samples YR- From the end of the 1990’s when the first candidate for −1 −1 28) M and YR-J was 0.29 and 0.69 nmol-N2 ml h , corre- an anammox bacterium was described , there have been sponding to 1.6% and 0.9% of the denitrification activity, seven strains of anammox bacterial candidates reported. respectively. The anammox activity observed in the sedi- Four strains (“Candidatus Brocadia anammoxidans”, “Ca. ment from the Yodo River estuary was comparable to that B. fulgida”, “Ca. Kuenenia stuttgartiensis”, and “Ca. in sediment from the mouth of the Thames River (Sites: Anammoxoglobus propionicus”) were enriched in Southend and Grain) in the UK5,33). Anammox activity pre- bioreactors9,10,24,28). Two strains (“Ca. Scalindua brodae” and viously reported for marine sediment based on vial experi- “Ca. S. wagneri”) were discovered in a wastewater treat- ments with homogenized samples varied greatly among ment plant (wwtp)25), “Ca. S. sorokinii” is the only species −1 −1 12) study sites and ranged up to 11 nmol-N2 ml h , and the discovered in marine environments . Although all genera relative anammox ratio was estimated at up to 79% of total of the anammox bacteria are similar to each other morpho- 5) N2 gas emission . The relatively minor contribution of logically and physiologically, their 16S rRNA gene anammox to total N2 emission determined in this study sequences are phylogenetically quite divergent. The (approximately 1–2%) might create the impression that sequence similarity between the two genera “Brocadia” and anammox is less significant than denitrification. However, “Scalindua” is only 85%, which corresponds to the degree considering the excess concentrations of added substrates of relatedness between two phenotypically distinct beta- 240 AMANO et al.

Fig. 4. NJ-tree based on partial 16S rRNA gene sequences (386–819; E. coli numbering) showing the relationships of seven anammox bacterial candidates and other non-anammox Planctomycetales. Branch points supported with bootstrap values are marked with solid and open circles as mentioned in Fig. 3. Database accession numbers are shown in parentheses. The scale bar represents 0.1 substitutions per nucleotide. proteobacteria25). Nevertheless, the anammox bacterial 16S primer. These results indicate that the reverse primer rRNA gene sequences were situated on one cluster in a phy- AMX820R was allowed to anneal with the target sequence logenetic tree, clearly divided from non-anammox Plancto- of BS820R well rid of three base mismatches at the 5'-end mycetales (Fig. 4). Therefore, we decided to use 16S rRNA under the PCR conditions used in this study. Given the orig- gene sequences as a landmark to explore the anammox bac- inal coverage of each reverse primer, most of the known teria. anammox bacterial lineage would be covered by the primer The two reverse primers AMX820R and BS820R used in set AMX368F-AMX820R in this PCR conditions. this study were originally used as FISH probes specific for Two dominant groups of clones affiliated with phylo- the genera “Brocadia” and “Kuenenia” and for the genus types 1 (18/30 clones) and 2 (4/30 clones) were recovered in “Scalindua” except for “S. brodae”, respectively5) (Table 2). the clone library constructed from the sample YR-M with Similarly, AMX368F is specific for all anammox bacteria the primer set AMX368F-AMX820R (Fig. 3). From the except for “Candidatus Anammoxoglobus propionicus”, results of measurements of anammox activity and of a phy- which was most recently reported10). First, we constructed logenetic analysis of 16S rRNA gene clone libraries, we two different clone libraries using the two primer sets concluded that the anammox process occurred undoubtedly AMX368F-AMX820R and AMX368F-BS820R from the in the intertidal sand bank at the Yodo River estuary and sediment sample YR-J from which the highest anammox that two dominant groups of Planctomycetales close to “Ca. activity was detected among samples (Table 3). Both clone S. wagneri” might be involved in this process. To our libraries were dominated by two groups (phylotypes 1 and knowledge, this is the first report demonstrating that the 2) despite the difference in reverse primer (Fig. 3). Both anammox process could contribute to N2 emissions in phylotypes were most closely related to “Candidatus Scal- coastal in Japan. The anammox bacterium “Ca. indua wagneri” among known anammox bacteria, and the S. wagneri” was discovered in a wwtp treating landfill sequence similarity was 98% (phylotype 1) and 95% (phy- leachate25) and has not been detected in marine environ- lotype 2), respectively. The sequence of the clone YR- ments yet. “Ca. S. wagneri” and its relatives possibly adapt 6JSW, which had been amplified with the primer set to moderately salty environments such as brackish habitats. Pla46F-AMX1480R, had completely identical region with Although each anammox bacterium has been considered to that of the clones of phylotype 1 (Fig. 3), and had no mis- have a distinct (but unknown) niche and rarely found matches with BS820R in the corresponding region of the together with other anammox bacteria10), the discernible and Anammox in Coastal Marine Sediment 241 fine clustering of anammox bacteria-related 16S rRNA gene References fragments shown in Fig. 3 also indicates an unexpected 1) Altschul, S.F., T.L. 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