Nitrosomonas Nm143-Like Ammonia Oxidizers and Nitrospira Marina -Like Nitrite Oxidizers Dominate the Nitri¢Er Community in a Marine Aquaculture Bio¢Lm Barbel¨ U

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RESEARCH ARTICLE Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina -like nitrite oxidizers dominate the nitri¢er community in a marine aquaculture bio¢lm Barbel¨ U. Foesel1,2, Armin Gieseke3, Carsten Schwermer3, Peter Stief3, Liat Koch4, Eddie Cytryn4,5, Jose´ R. de la Torre´ 6, Jaap van Rijn5, Dror Minz4, Harold L. Drake2 & Andreas Schramm1,2

1Department of Biological Sciences, Microbiology, University of Aarhus, Aarhus, Denmark; 2Department of Ecological Microbiology, University of Bayreuth, Bayreuth, Germany; 3Microsensor Group, Max Planck Institute for Marine Microbiology, Bremen, Germany; 4The Volcani Center, Institute for Soil, Water and Environmental Sciences, ARO, Bet-Dagan, Israel; 5Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel; and 6Department of Civil and Environmental Engineering, University of Washington, Seattle, WA, USA

Correspondence: Andreas Schramm, Abstract Department of Biological Sciences, Microbiology, University of Aarhus, Ny Zero-discharge marine aquaculture systems are an environmentally friendly Munkegade, Building 1540, DK-8000, alternative to conventional aquaculture. In these systems, water is purified and Aarhus C, Denmark. Tel.: 145 8942 3248; recycled via microbial biofilters. Here, quantitative data on nitrifier community fax: 145 8942 2722; structure of a trickling filter biofilm associated with a recirculating marine e-mail: [email protected] aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH Present address: Barbel¨ U. Foesel, Bereich to obtain a reliable and comprehensive picture of the ammonia-oxidizing Mikrobiologie, Department Biologie I Ludwig- community. Analysis of the ammonia monooxygenase gene (amoA) confirmed Maximilians-Universitat¨ Munchen, ¨ Germany. Eddie Cytryn, Department of Soil Water and the results. The most abundant ammonia-oxidizing bacteria (AOB) were members Climate and Biotechnology Institute, of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), University of Minnesota, Saint Paul, MN, USA. followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina- Received 26 June 2007; revised 16 October lineage (15.7% of the bacterial biovolume). Although more than eight other 2007; accepted 21 October 2007. nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer ‘Nitrosopumilus maritimus’, their collective abundance was DOI:10.1111/j.1574-6941.2007.00418.x below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible. Editor: Michael Wagner

Keywords marine aquaculture; nitrifying bioﬁlm; DNA extraction; Nitrosomonas sp. Nm143; Nitrospira marina.

enough to keep ammonia concentrations below toxic levels Introduction (e.g. o 30 mM for juvenile gilthead seabream; Wajsbrot Declining marine fish stocks have induced a rapid growth et al., 1993), and yet dynamic enough to buffer fluctuations in marine aquaculture (FAO, 2004), an industry that can in ammonium production, e.g., caused by changing fish have deleterious effects on local environments via uncon- population size, age, and feeding. This challenge is quite trolled discharge of nutrients and organic matter (Wu, different from nitrification in activated sludge and waste- 1995; Christensen et al., 2000; Holmer et al., 2003). As an water biofilms, where ammonium concentrations are often environmentally friendly alternative, a zero-discharge mar- high and ammonia-oxidizing bacteria (AOB) with a low ine aquaculture system has been developed with integrated substrate affinity but high reaction rates dominate (Wagner microbial biofilters that couple nitrification with sludge et al., 2002; Koops et al., 2003). Marine nitrifying biofilters digestion and denitrification (Cytryn et al., 2003; Gelfand have not been evaluated in detail, and nitrifier diversity has et al., 2003). Nitrification in such a system has to be efficient sometimes been described on the basis of only a few clone

FEMS Microbiol Ecol ]] (2007) 1–13 c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 2 B.U. Foesel et al. sequences. Such diverse nitrifiers as Nitrosomonas europaea biosensors (Larsen et al., 1997), respectively. Depth-resolved (Hovanec & Delong, 1996), Nitrosomonas cryotolerans, and NOx production rates were computed from the profiles using the nitrite-oxidizing bacterium (NOB) Nitrospira marina the software PROFILE v.1.0 (Berg et al., 1998). (Tal et al., 2003), Nitrosomonas marina (Grommen et al., 2005), or Nitrosomonas aestuarii and Nitrosomonas sp. DNA extraction Nm143 (Itoi et al., 2006) have been detected in marine DNA was first extracted from 3 cm plastic strip fragments by biofilters. Recently, even a crenarchaeal ammonia oxidizer bead-beating (= DNA extraction method A), based on a (AOA), ‘Nitrosopumilus maritimus’, was isolated from a modified version of the FastDNAs SPIN Kit for soil (Qbio- marine aquarium (Konneke¨ et al., 2005), and subsequently gene Inc., Carlsbad, CA). Extraction tubes contained-acid archaeal ammonia monooxygenase genes (amoA) were washed and baked glass beads (0.36 g with a diameter of detected in marine samples and activated sludge (Francis 106 mm; Sigma-Aldrich, St Louis, MO, and six to eight beads et al., 2005; Park et al., 2006). However, quantitative data on with a diameter of 5 mm; Marienfeld, Lauda-Koenigshofen, the nitrifying community structure of marine biofilters are Germany). Wash buffer was 50 mM NaCl, 10 mM Tris-HCl still lacking, and the importance of the respective species (pH 7.5), 2.5 mM EDTA, and 50% (v/v) ethanol. As this remains unclear. Therefore, the goal of this study was to method proved insufficient for yielding DNA of AOB, the provide a comprehensive and quantitative analysis of the FastDNAs Kit (Qbiogene) was combined with an enzymatic AOB and NOB communities in a marine, nitrifying biofilter. digestion step (Juretschko et al., 1998), hereafter referred to as the DNA extraction method B: biofilm pellet (0.25 g) was Materials and methods resuspended in 300 mL DNA extraction buffer [100 mM Tris- HCl (pH 8.0), 100 mM sodium EDTA (pH 8.0), 100 mM System operation and biofilm sampling sodium phosphate (pH 8.0), 1.5 M NaCl, 1% cetyltrimethy- Biofilm samples originated from the trickling filter of a zero- lammonium bromide], transferred to a Lysis matrix E tube discharge marine aquaculture system (Cytryn et al., 2003; (Qbiogene), and bead-beaten (2 15 s at speed 4.0 and Gelfand et al., 2003) that was stocked with gilthead seabream 1 15 s at speed 4.5) using a FastPreps Instrument (Qbio- (Sparus aurata). The trickling filter consisted of 1 m3 polyvinyl gene). Then, 50 mL of enzyme mixture I [lysozyme (Sigma- chloride (PVC) cross-flow medium with a surface area of Aldrich, Steinheim, Germany), lipase typ7 (from Candida 240 m2 (Jerushalmi Ltd., Ashod, Israel). The top of the filter rugosa, Sigma), pectinase (from Aspergillus niger,Sigma),and was continuously sprinkled with water from the fish pool b-glucuronidase (from Helix pomatia, Sigma), 10 mg L1 (pool volume 2.3 m3)atarateof60Lmin1 ( 20 L min1). each] was added and the biofilm was incubated for 30 min at Normal inlet ammonia concentrations were c.20mMwith 37 1C, followed by addition of 50 mLofenzymemixtureII peaks of up to 100 mM after feeding; outlet concentrations [proteinase K (Roche Diagnostics GmbH, Mannheim, Ger- ranged from 6 to 42 mM(Gelfandet al., 2003), and salinity was many), protease typ9 (from Bacillus polymyxa,Sigma),and kept at c. 20 psu. The system was restarted in spring 2003; for pronase P (from Streptomyces griseus,Sigma),10mgL1 each] sampling of intact biofilm, plastic strips were inserted into the and incubation for 30 min at 37 1C. Finally, the sample was trickling filter. Samples were taken after several months of incubatedwith75mL of 20% dodecyl sulfate for 2 h at 65 1C, stable operation in October 2003 and April/May 2004 by then 800 mL cell-lysis solution (CLS)-TC (Qbiogene) was removing the strips that were either directly frozen (for DNA addedforasecondbead-beatinground(2 15satspeed5.0 extraction) or fixed in 4% paraformaldehyde (for FISH; and 1 15 s at speed 5.5), and DNA extraction was continued Amann et al., 1990b). Vertical sections (thickness, 14–30 mm) with the FastDNAs Kit following the manufacturer’s instruc- were prepared from fixed biofilms as described earlier tions. In addition, the DNA extraction protocol described by (Schramm et al., 1996). Additionally, biofilm was scraped off Burrell et al. (2001) was tested, which includes enzymatic the PVC substratum and fixed for qualitative FISH-compar- digestion with lysozyme and proteinase K. ison of the nitrifier community on the original PVC substratum and the inserted plastic strips. PCR amplification of 16S rRNA genes and amoA Primer sets GM3/GM4 (Muyzer et al., 1995), 616V (Jur- Microsensor measurements etschko et al., 1998)/NSO1225R (published as probe by Biofilm samples (1–2 cm2 on plastic strips) were incubated in a Mobarry et al., 1996), and NSMR76F (published as probe laminar flow chamber with artificial seawater (20 psu Red Sea by Burrell et al., 2001)/NSO1225R were used for amplifica- salt, Red Sea Fish Pharm, Eilat, Israel) amended with 25 mM tion of bacterial, b-AOB, and Nitrosomonas marina-like 16S 1 1 NH4 at a flow velocity of 1 cm s .Verticalmicroprofilesof rRNA genes, respectively. The presence of Nitrobacter spp. dissolved O2 and NOx (nitrate1nitrite1N2O) were measured was tested using primers 26F (Hicks et al., 1992)/NIT3R with amperometric microsensors (Revsbech, 1989) and micro- (published as a probe by Wagner et al., 1996), and the

c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol ]] (2007) 1–13 Published by Blackwell Publishing Ltd. All rights reserved Nitrifying community of a marine aquaculture biofilm 3 presence of g-AOB was tested by following the protocol of bias-corrected Chao1 estimator (Chao, 1984), calculated Ward et al. (2000). PCR mixtures contained 75 mM KCl, with the freeware program ESTIMATES (Colwell, 2005). RFLP 1 10 mM Tris-HCl pH 8.8, 1.5 mM MgCl2, 0.4 mg mL groups with highly similar sequences were merged. Thresh- bovine serum albumin, 125 mM of each dNTP, 10 pmol of olds for the definition of operational taxonomic units (OTU) each primer, 1 mL of template, and 1 U of Taq DNA- were 97% sequence similarity (Stackebrandt & Goebel, 1994) polymerase (Sigma). Thermal cycling included an initial and 80% (Koops et al., 2003) for 16S rRNA and amoA gene denaturation step of 94 1C for 2–5 min, followed by 35 cycles sequences, respectively. In addition, collector’s curves were of denaturation at 94 1C for 1 min, annealing for 1 min at fitted with a hyperbola [NOTU =A (NClones/B1NClones)] in 42 1C (GM3/GM4), 52 1C (616V/NSO1225R), 54 1C which the asymptote (fit-parameter A from the formula) (NSMR76F/NSO1225R), or 60 1C (26F/NIT3R), and exten- describes the expected number of OTUs (Hill et al., 2003) sion at 72 1C for 2 min; cycling was completed by a final (supplementary Fig. S1). elongation step at 72 1C for 5 min. Bacterial amoA fragments were amplified using primer pair Phylogenetic analyses amoA-1F/amoA-2R (Rotthauwe et al., 1997), PCR mixture as described above, and thermal cycling with initial denaturation Nucleotide sequences were added to prealigned 16S rRNA of 94 1C for 2 min, followed by 35 cycles of denaturation at and amoA gene databases using the ARB program package 94 1C for 30 s, annealing at 58 1C for 40 s, and extension at (www.arb-home.de; Ludwig et al., 2004). After automated 72 1Cfor45s(11scycle1); final elongation was 5 min at alignment and manual refinements, phylogenetic trees were 72 1C. Crenarchaeal amoA fragments were amplified using the calculated with distance-matrix, maximum-parsimony, and primer pair CrenamoA-F2 (50-TGG TCT GGY TWA GAC maximum-likelihood methods implemented in ARB; the GMT GTA-30)andCrenamoA-2R(50-CCC AYT TTG ACC results are displayed as neighbor-joining trees with branch- ARG CGG CCA-30), PCR mixture as described above but with ings that were not supported by the other treeing methods 100 pmol of primers, and thermal cycling with initial denatura- drawn as multifurcations (Ludwig et al., 1998). Only Escher- tion of 94 1C for 10 min, followed by 40 cycles of denaturation ichia coli positions 27-1225, i.e. between primers 616V and at 94 1C for 30 s, annealing at 58 1C for 30 s, and extension at NSO1225R, were used to calculate 16S rRNA gene-based 72 1Cfor45s(11scycle1); final elongation was 10 min at trees. AmoA trees were calculated based on nucleic acid 72 1C(J.R.delaTorre´ & D.A. Stahl, unpublished data). Primer sequences with a filter using only positions between primers and PCR conditions described by Francis et al. (2005) were also amoA-1F and amoA-2R and excluding the third codon tested but without positive results. position.

Cloning, clone screening, and sequencing Probe design and testing Nitrosomonas marina 16S rRNA gene-specific and crenarch- Probes specific for the Nitrospira marina group and the aeal amoA PCR products were run on a 1.5% Nusieve 3 : 1 different clone clusters of ammonia oxidizers (see Table 1) agarose gel (FMC Bioproducts, Rockland, ME). The desired were designed and evaluated in silico using the ARB probe bands (991 and 630 bp, respectively) were excised, purified tools (Ludwig et al., 2004). Probe specificity and hybridiza- (Wizards SV Gel and PCR Clean-Up System, Promega, tion conditions were optimized by Clone-FISH as described Madison, WI), and cloned using the pGEM-T Easy Vector previously (Schramm et al., 2002), using pGEM-T as the System (Promega) according to the manufacturer’s proto- vector and E. coli JM109(DE3) (Promega) as the host strain. col. All other PCR products were directly cloned. Clones Mismatch-clones (three mismatches) as well as mismatch- were screened by PCR using vector-specific primers, and probes (one central mismatch) served as negative controls; a grouped after restriction fragment length polymorphism list of clones and probes used for Clone-FISH can be found (RFLP) analysis with restriction enzymes RsaI (16S rRNA in the supplementary Table S1. gene library), AluI (AOB library), or HhaI and RsaI (amoA library). For each RFLP group, one to three clones were FISH selected for plasmid extraction (Wizard Plus Minipreps FISH of dehydrated biofilm sections followed standard Purification System, Promega) and automated sequencing protocols (Manz et al., 1992; Pernthaler et al., 2001) with (Macrogen Inc., Seoul, South Korea), followed by BLAST probes EUB338-I to III (Amann et al., 1990a; Daims et al., search (Altschul et al., 1997). 1999) and NON338 (Wallner et al., 1993) as positive and negative controls, respectively. AOB were identified in situ Extrapolation methods by a set of 15 probes, i.e., probes NSO1225, NSO190, The coverage and genotype richness of the clone libraries NSM156, and NSV443 (Mobarry et al., 1996); NEU23a were estimated using Good’s coverage (Good, 1958) and the (Wagner et al., 1995); Nse1472 and NmV (Juretschko et al.,

Table 1. Probes for FISH designed in this study with hybridization conditions Target siteÃ FAw NaClz Probe Sequence (50–30) (16S rRNA gene) (%) (mM) Speciﬁcity AOB Nmar831‰ AAC CTA AAA AGG CCC GAC 831–848 5 636 Nitrosomonas marina-like clones, cluster 1 Nm143-446-1 AGT CGC GGT CTT TTC TTC 446–463 25 159 Nitrosomonas sp. Nm143-like clones, cluster 1 Nm143-446-2 CGT CGT AGC CTT TTC TTC 446–463 20 225 Nitrosomonas sp. Nm143-like clones, cluster 2 pAOB630 CCA CAG TCC TCA ATG CAA 630–647 10 450 Potential AOB clones NOB Nspmar62 GCC CCG GAT TCT CGT TCG 62–79 40 56 Nitrospira marina-like clones

ÃEscherichia coli numbering (Brosius et al., 1978). wFormamide (FA) concentration (% v/v) in the hybridization buffer. zNaCl concentration (mM) in the washing buffer. ‰Use with equimolar amount of unlabelled competitor cNmar831 (AACCTAATAAGGCCCGAC).

1998); NmII and NmIV (Pommerening-Roser¨ et al., 1996); Microautoradiography (MAR)--FISH NSMR76 (Burrell et al., 2001); Nmo218 (Gieseke et al., CO uptake under nitrifying conditions was tested for 2001); and four newly designed probes (Table 1). For NOB, 2 the major nitrifying populations and a group of potential six probes were applied, i.e. Ntspa712 and Ntspa662 (Daims AOB by combining MAR with specific FISH analysis (Lee et al., 2001a), NSR826 and NSR1156 (Schramm et al., 1998), et al., 1999). Short-term, oxic incubations (8.25 h) of intact NIT3 (Wagner et al., 1996), and one newly designed biofilm on plastic strips received 1.5 MBq NaH14CO , lead- Nitrospira marina-specific probe (Table 1). All probe se- 3 ing to a final molar activity of 220 kBq (mmol CO2)1, and quences and hybridization conditions can be found at http:// 3 contained 2 mM ammonium; analysis was performed as www.microbial-ecology.net/probebase/(Loy et al., 2003). described previously (Andreasen & Nielsen, 1997; Lee et al., Crenarchaeota were detected by catalyzed reporter deposi- 1999). tion-FISH using probe CREN554 (Massana et al., 1997) according to published protocols (Teira et al., 2004; Fuchs Nucleotide sequence accession numbers et al., 2005). The 16S rRNA and amoA gene sequences obtained in this study were deposited at EMBL under accession Microscopy and image analysis numbers AM295508–AM295533 for 16S rRNA gene se- Microscopic images of hybridized samples were recorded on quences of AOB, accession numbers AM295534–AM295536 either a confocal laser scanning microscope (Zeiss LSM 510, for 16S rRNA gene sequences of ‘potential AOB’, accession Carl Zeiss, Jena, Germany) or, for quantification, on a Zeiss numbers AM295537–AM295545 for 16S rRNA gene se- ApoTome (Carl Zeiss, Jena, Germany) using an AxioCam quences of NOB, accession numbers AM295546–AM295575 MRm camera and the AXIOVISION software (version 4.4). for bacterial amoA sequences, and accession numbers Biovolume fractions of the major AOB (Nitrosomonas sp. AM295170–AM295174 for archaeal amoA sequences. Nm143- and Nitrosomonas marina-related species) and NOB (Nitrospira marina-like species) populations were Results quantified using the DAIME software package (Version 1.1, Daims et al., 2006). For each probe combination (specific Biofilm structure and microenvironment CY3-labelled probe/CY5-labelled probes EUB338-I to III), a Biofilm thickness was on average 150 mm(n = 100 sections), set of random optical sections (86, 120, and 40 image sets for spanning a range of 50–500 mm but rarely exceeding 300 mm. Nitrosomonas sp. Nm143, Nitrosomonas marina, and Nitros- Under normal in situ ammonium concentrations (c.20mM), pira marina, respectively, thickness 1 mm) was recorded. O penetrated the whole biofilm, and nitrification occurred Thresholds were set manually for each image series before 2 across the entire biofilm depth without apparent stratifica- image defragmentation and biovolume determination. The tion at an NO production rate of 0.596 nmol cm3 s1 final biovolume fractions were calculated after correction x (Fig. 1). with the negative control probe NON338. Where possible, images were recorded separately from three layers (top, Nitrifier diversity middle, and bottom, each c. 100 mm thick) along the vertical biofilm sections, and AOB biovolume fractions were calcu- In total, four 16S rRNA gene libraries using different DNA lated separately for each layer. extraction methods and PCR primers were necessary before

Fig. 1. Average microprofiles of O2 (filled circles) and NOx (open squares) at in situ ammonium concentrations, and corresponding NOx production rates (horizontal bar). Shaded area, biofilm. Error bars: SE (n =5).

Fig. 2. Comparison of 16S rRNA and amoA gene clone libraries from a satisfying congruency with the FISH results was obtained. different DNA extraction methods. (a) 16S rRNA gene, extraction In an initial bacterial library (primers GM3/GM4, 300 method A (bead-beating); (b) 16S rRNA gene, extraction method B (bead-beating1enzymatic lysis); (c) amoA, extraction method A; (d) clones screened) based on extraction method A (bead-beat- amoA, extraction method B. Numbers indicate the percentage of clones ing, no enzymes), about 20% of the analyzed sequences had from each group referring to all AOB and potential AOB clones; numbers 97% similarity to the NOB Nitrospira marina but not a in brackets indicate the percentage of AOB population detected in situ single AOB sequence was found. Therefore, a semi-specific by FISH (1, detected, but in very low numbers; , not detected). The b-AOB library (primers 616V/NSO1225R) was constructed second most abundant AOB group, Nitrosomonas marina cluster 2 (FISH: from the same DNA extract. Fifty-seven of the 61 clones 25% of all AOB), was absent in both 16S rRNA gene clone libraries and screened belonged to a group termed Nitrosomonas marina its 16S rRNA gene sequence could only be amplified by specific PCR. cluster 1 (Fig. 2a and 3a), while four sequences were affiliated to non-ammonia-oxidizing Betaproteobacteria.As not shown). Sequences for the second most abundant AOB, these findings were not consistent with the FISH results, the Nitrosomonas marina cluster 2 (see FISH results below), DNA extraction was modified to include enzymatic lysis could first be retrieved after cloning a more specific PCR (method B), and the semi-specific PCR amplification (pri- reaction (primers NSMR76F/NSO1225R) tailored for that mers 616V/NSO1225R) and cloning were repeated. This group (Fig. 3a). Neither g-AOB nor NOB of the genus new library was dominated by Nitrosomonas sp. Nm143-like Nitrobacter were detected by specific PCR. sequences (two clusters, 34 of 59 clones screened), followed Analysis of amoA largely confirmed the 16S rRNA gene- by a group of sequences (13 clones) with 90% sequence based AOB community; in an amoA library constructed similarity to the AOB Nitrosospira briensis. Because the latter from DNA extraction method A, exclusively Nitrosomonas lineage only included uncultured species and branched off marina-like sequences were detected, while method B before the deepest AOB branch point (Fig. 3a), it was yielded both Nitrosomonas marina and Nitrosomonas sp. provisionally termed ‘potential AOB’. Of the remaining Nm143-like sequences (Fig. 2c and d, Fig. 3b). In contrast to clones, two sequences affiliated with Nitrosomonas marina the 16S rRNA gene data, which suggested two groups of cluster 1, one with Nitrosospira sp. Nsp58, and the remain- Nitrosomonas marina-like AOB, only one Nitrosomonas ing nine with non-AOB sequences (Figs. 2b and 3a). A third marina-like amoA sequence type was found; it is unclear to DNA extraction with enzymatic lysis (Burrell et al., 2001) which of the two 16S rRNA gene-defined Nitrosomonas yielded similar results and was not analyzed further (data marina cluster it belongs. One could, however, speculate

Fig. 3. Phylogenetic trees (neighbor joining method) of partial 16S rRNA gene (1200 bp) and amoA gene sequences. Branchings not supported by maximum likelihood and parsimony methods are drawn as multifurcation. Sequences or sequence groups retrieved within this study are depicted in bold; (a) 16S rRNA gene-based tree of AOB and NOB; (b) tree of bacterial and archaeal amoA. Scale bar = 10% sequence difference. that it may be from cluster 1, because it was detected in the by AOB of the Nitrosomonas europaea/eutropha- (probe first DNA extract, in which, at the 16S rRNA gene level, NEU23a) and the Nitrosomonas marina-lineage (probe exclusively Nitrosomonas marina cluster 1 sequences were NSMR76). However, no 16S rRNA gene or amoA sequences obtained. No deep-branching amoA sequences correspond- affiliated with the Nitrosomonas europaea/eutropha-lineage ing to the ‘potential AOB’ 16S rRNA gene sequence group could be detected, and the Nitrosomonas marina cluster 1- were found. Instead, crenarchaeal amoA sequences with like sequences obtained were not targeted by probe 97% similarity to the recently described AOA ‘Nitrosopu- NSMR76; in addition, sequences of the Nitrosomonas sp. milus maritimus’ (Konneke¨ et al., 2005) were detected. Nm143-lineage had been retrieved, for which no FISH All clone libraries described above appear to be well probes were available. Therefore, (1) probe NEU23a was sampled as indicated by their high coverage (98–100%) and compared with the Nitrosomonas sp. Nm143-like sequences, consistent richness estimates (Table 2 and supplementary (2) new probes were designed based on the Nitrosomonas sp. Fig. S1). Therefore, the differences between the libraries Nm143- and Nitrosomonas marina-like sequences retrieved, cannot be ascribed to insufficient sampling/screening efforts. and (3) probe NSMR76 was used as a primer to obtain the missing sequence information for that particular Nitroso- FISH probe set adjustment monas marina group. Sequence analysis revealed that probe NEU23a has only one weak mismatch to the Nitrosomonas Initial FISH analysis using published probes indicated that sp. Nm143-like sequences, which is not covered by the the nitrifier community was dominated by Nitrospira-like NEU23a-competitor probe CTE (supplementary Fig. S2; NOB (as detected by probes Ntspa712 and Ntspa662), and Wagner et al., 1995). Probe NEU23a was subsequently

c 2007 Federation of European Microbiological Societies FEMS Microbiol Ecol ]] (2007) 1–13 Published by Blackwell Publishing Ltd. All rights reserved Nitrifying community of a marine aquaculture biofilm 7 proven to hybridize to Nitrosomonas sp. Nm143-like cell Potentially active nitrifiers clusters by double hybridization using clone-specific probes After oxic incubation of intact biofilm with 2 mM ammo- Nm143-446-112 (Table 1) in combination with probe nium, both types of numerically dominant AOB and the NEU23a. It appeared that these Nitrosomonas sp. Nm143- NOB were clearly MAR-positive (supplementary Fig. S4 like cell clusters were surrounded by a highly autofluores- a–d). This result indicates that all in situ dominating cent layer or envelope, putatively of extracellular polymeric nitrifiers can take up CO under nitrifying conditions and substances (EPS) that enclosed every cell tetrade (supple- 2 were therefore potentially active. In contrast, the ‘potential mentary Fig. S3). Probe Nmar831 was specifically designed AOB’ group was MAR-negative under the assay conditions for Nitrosomonas marina cluster 1 (Table 1). Although the applied (supplementary Fig. S4 e and f). probe worked well in the clone-FISH tests, no cells could be detected in situ, indicating that Nitrosomonas marina cluster Discussion 2 (as targeted by probe NSMR76) was the in situ dominant Nitrosomonas marina group. The cloning-FISH discrepancy New probes were also designed and optimized for the Nitrospira marina-like sequences and the group of ‘potential Difficulties arose when trying to match the initially retrieved AOB’ (Table 1). Probe Nspmar62 hybridized to almost all sequence information to the nitrifier populations detected Nitrospira-like cells (detected with probe Ntspa662). The by FISH. These discrepancies were due to insufficient DNA ‘potential AOB’ were identified in situ as small cell clusters extraction, PCR bias, and mis-hits of one FISH probe. This with a typical AOB morphology (Fig. 4g). combination and the absence of the most dominant AOB after screening 4 60 AOB-specific clones (Table 3; Fig. 2) might seem extreme; however, in many studies it would not Nitrifier community structure even surface because the ‘full cycle’ rRNA approach (Amann Initial FISH comparison between biofilm grown on the et al., 1995) is often not completed. Without verification of original PVC support and biofilm grown on the inserted sequence data in situ (e.g. by FISH), or without relating plastic strips revealed no visible differences in community quantitative FISH data to sequence-defined populations, composition, i.e., the plastic strips did not select for (or artifacts and biases may easily remain undetected. In our against) certain nitrifiers compared with the original biofilm case, the inability to detect Nitrosomonas sp. Nm143 in the (data not shown). Therefore, all further analyses were first 16S rRNA and amoA gene libraries could be clearly performed on the plastic strip-grown biofilm. Biovolume traced to the DNA extraction method A, because both genes fractions for Nitrosomonas sp. Nm143- and Nitrosomonas were detected when using the improved extraction method marina-like AOB were not significantly different along a B. DNA extraction methods are known to influence micro- vertical biofilm profile (supplementary Table S2), i.e. no bial diversity studies (e.g. Miller et al., 1999; Krsek & spatial stratification of nitrifiers was observed in this mostly Wellington, 1999; Martin-Laurent et al., 2001), and espe- rather thin biofilm; therefore, quantification was averaged cially cluster-forming AOB have been proven difficult to over the whole biofilm depth. Using the optimized probe set extract (Juretschko et al., 1998; Schramm, unpublished (Tables 1 and 2), Nitrosomonas sp. Nm143-like and Nitroso- data). It can only be speculated, based on microscopic monas marina cluster 2-like AOB were quantified to account observations (supplementary Fig. S3) and the success of for c. 75% and 25% of the total AOB community, respec- enzymatic lysis (method B, Burrell et al., 2001; Itoi et al., tively. Several other AOB were occasionally detected but 2006), that a thick layer of EPS prevented mechanical lysis only in very low numbers, accounting together for less than (method A) of the Nitrosomonas sp. Nm143 cells. 1% of the total AOB community (Table 3, Fig. 4). Compared Even after the improved DNA extraction, neither the 16S with the total biofilm community, an AOB biovolume rRNA gene nor the amoA library reflects the different AOB’s fraction of 8.9% was determined, corresponding to 6.7% in situ ratio. Because the two genes yield diametrically Nitrosomonas sp. Nm143-like AOB and 2.2% Nitrosomonas opposed percentages for Nitrosomonas sp. Nm143, the most marina-like AOB. The ‘potential AOB’ group accounted for likely explanation are the different degrees of PCR bias (e.g. less than 1% of all bacteria. Crenarchaeota were also detected von Wintzingerode et al., 1997; Polz & Cavanaugh, 1998; in situ (Fig. 4h) but only in low abundance, about 0.1% Mahmood et al., 2006) for the 16S rRNA gene and the amoA of all cells. gene PCR. In addition, different copy numbers of the 16S The NOB community encompassed 15.7% of the total rRNA and AmoA genes in the different AOB may contribute biofilm volume and consisted almost exclusively of to the observed skewed ratio in the clone library. Nitrospira marina with very few Nitrospira moscoviensis-like Finally, the hybridization of probe NEU23a to a nontarget cell clusters (Table 3, Fig. 4i); Nitrobacter spp. were not sequence with a weak mismatch (supplementary Fig. S2) detected. suggests that checking published probes in silico may not be

Fig. 4. FISH detection of nitrifiers in biofilm sections of the marine aquaculture biofilter. (a) dense clusters of AOB (probe NSO1225, red) and NOB (probe Ntspa712, green); (b) Nitrosomonas sp. Nm143-like AOB; (c) Nitrosomonas marina cluster 2-like AOB; (d) Nitrosomonas cryotolerans-like AOB; (e) Nitrosomonas communis/nitrosa-like AOB; (f) Nitrosospira sp.-like AOB; (g) ‘potential AOB’, (h) single crenarchaeotal cell; (i) coexistence of Nitrospira moscoviensis- (yellow) and Nitrospira marina- (green) like NOB. Scale bars = 10 mm. sufficient to ensure probe specificity. Empirical testing may lineage were detected in estuarine (Bernhard et al., 2005; be warranted as new sequences become available. Freitag et al., 2006) and coastal samples (Urakawa et al., 2006a, b), and in a marine aquaculture system (Itoi et al., The nitrifying community 2006). The closest cultured relative of the AOB detected in our system, isolate Nitrosomonas sp. NS20 (sequence simi- Nitrosomonas sp. Nm143-like bacteria were the most abun- larity, 98–99% for 16S rRNA gene, 97% for amoA), was dant AOB in the system. The Nitrosomonas sp. Nm143- isolated from a heavily polluted and eutrophicated area in lineage has been described only recently (Purkhold et al., Tokyo Bay, Japan (Urakawa et al., 2006a). Despite the 2003) based on a few coastal isolates and clone sequences. diverse habitats, AOB of the Nitrosomonas sp. Nm143-line- During the last two years, additional sequences of this age seem to share a moderate salt requirement (brackish to

Table 2. Observed and estimated numbers of OTU in the AOB-speciﬁc clone libraries

Ã w z Source Type NOTU (Nclones) Coverage (%) Chao1 Fit-parameter A Extraction method A 16S rRNA gene 3 (61) 98 3 3 Extraction method B 16S rRNA gene 7 (59) 98 7 8 Extraction method A amoA 1 (101) 100 1 NA‰ Extraction method B amoA 2 (112) 100 2 NA‰ Ã NOTU, number of OTU found in the various AOB-specific libraries; thresholds for OTU definition were 97% and 80% sequence similarity for 16S rRNA and amoA gene sequences, respectively; Nclones, number of clones screened. wNumber of OTU estimated by the bias-corrected estimator Chao1. z Number of OTU estimated by hyperbolic curve fitting [NOTU =A (NClones/B1NClones)]. ‰NA, not applicable (cannot be calculated with less than three data points).

Table 3. Contribution of AOB, AOA and NOB tested to overall community composition Species/lineage/clone group Probe Fraction (%)Ã AOB Nitrosomonas europaea Nse1472 1 Nitrosococcus mobilis NmV 1 Nitrosomonas communis/nitrosa lineage NmII 1 Nitrosomonas marina-like clones, cluster 1 Nmar831 Nitrosomonas marina-like clones, cluster 2 NSMR76 2.2 (25) Nitrosomonas oligotropha lineage Nmo218 Nitrosomonas cryotolerans NmIV 1 Nitrosomonas sp. Nm143-like clones, cluster 1 Nm143-466-2 6.7 (75) Nitrosomonas sp. Nm143-like clones, cluster 2 Nm143-466-1 Nitrosospira spp. NSV443 1 NOB Nitrospira marina-like clones Nspmar62 15.7 (100) Nitrospira moscoviensis rel. group NSR826, NSR1156 1 Nitrobacter spp. NIT3 AOA Crenarchaeota CREN554 o 0.1

ÃNumbers give the fraction of all bacteria, numbers in brackets give the fraction of AOB or NOB, respectively; 1, detected, but in very low numbers; , not detected.

coastal sites) and a preference for low to moderate in biofilms as a consequence of fluctuating substrate (or O2) (5–300 mM) ammonium concentrations. Therefore, the concentrations has been postulated previously (Daims et al., Nitrosomonas sp. Nm143-like AOB appear to be well 2001b; Gieseke et al., 2001, 2003; Koch, 2007). adapted to the marine biofilters. The other in situ relevant Besides the two dominant AOB groups, five bacterial and AOB, Nitrosomonas marina cluster 2, can be linked to several one archaeal ammonia-oxidizing populations were detected, clone sequences (Burrell et al., 2001; Grommen et al., 2005) albeit in very low numbers, i.e., o 0.1% of all cells in the and one isolate (Nitrosomonas sp. Is343; A. Bollmann et al., biofilm. This AOB community composition was stable over unpublished data), all originating from low ammonium several months before the first sampling (Koch, 2007), and environments with various salinities. Thus, it is likely that the community structure did not change during the the occurrence of these Nitrosomonas marina-like AOB is 6 months between the two sampling periods. Although these indicative of low ammonium concentrations rather than of a marginal populations may become functionally relevant certain salinity. Given this differing habitat range of the two under certain conditions, it appears more likely that they dominant AOB groups, one may speculate that both Nitro- are simply remnants of the start-up period of the reactor, somonas sp. Nm143- and Nitrosomonas marina-like AOB when conditions were most different (J. van Rijn, pers. thrive well under the normally low ammonium concentra- commun.). tions in the marine aquaculture system but that the former The maximum genetic distance within the known beta- have an advantage under occasional substrate peaks (e.g. proteobacterial AOB is about 89% (Koops et al., 2003). With after feeding). The coexistence of multiple AOB populations 90% 16S rRNA gene sequence similarity to Nitrosospira

FEMS Microbiol Ecol ]] (2007) 1–13 c 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 10 B.U. Foesel et al. briensis and the typical AOB aggregate morphology Acknowledgements (Fig. 4g), the ‘potential AOB’ were possible candidates for a The authors thank Britta Poulsen and Pernille Thykier for novel AOB group. However, extended phylogenetic analyses excellent technical assistance, Rikke L. Meyer (University of did not support a monophyletic origin of the ‘potential AOB’ Aarhus, Denmark) for performing the PROFILE analyses, Dirk with the betaproteobacterial AOB; instead, the ‘potential de Beer (Max Planck Institute for Marine Microbiology, AOB’ belong to one of several groups of uncultured bacteria Bremen, Germany) for helpful discussion and comments on from diverse habitats (e.g. soil, bioreactors, or activated the manuscript, and David A. Stahl (University of Washing- sludge), which are clearly placed outside the AOB-Gallionella ton, Seattle, WA) for pointing out the archaeal ammonia branchpoint (Fig. 3a). This tree topology, together with the oxidizers to us. This project was financially supported by absence of a deep-branching betaproteobacterial amoA and GIF Grant no. I-732–165.8/2001, and by the Universities of the lack of CO fixation under nitrifying conditions do not 2 Bayreuth, Germany, and Aarhus, Denmark. support the authors’ hypothesis of ‘potential AOB’. Even though one might speculate that the unnaturally high ammonium concentration in the MAR incubation (2 mM vs. 20 mM in situ) may inhibit a low ammonia-adapted AOB References population, the combined results render a nitrifying meta- Altmann D, Stief P, Amann R, de Beer D & Schramm A (2003) bolism of the ‘potential AOB’ very unlikely. In situ distribution and activity of nitrifying bacteria in Crenarchaeal ammonia oxidizers have been implicated in freshwater sediment. Environ Microbiol 5: 798–803. marine nitrification (Beman & Francis, 2006; Hallam et al., Altschul SF, Madden TL, Schaffer¨ AA, Zhang J, Zhang Z, Miller W 2006), and diverse archaeal amoAs can be readily detected & Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new in marine samples and activated sludge (Francis et al., 2005; generation of protein database search programs. Nucleic Acids Park et al., 2006). In the present system, archaeal amoA Res 25: 3389–3402. diversity was low, and even if one assumes that all CARD- Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R & FISH-detected Crenarchaeota (0.1% of the total biofilm Stahl DA (1990a) Combination of 16S rRNA-targeted volume) were ammonia oxidizers, their contribution to oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56: overall ammonia oxidation would be minor. 1919–1925. The dominant NOB were closely related to the marine Amann RI, Krumholz L & Stahl DA (1990b) Fluorescent- nitrite oxidizer Nitrospira marina. Sequences of this oligonucleotide probing of whole cells for determinative, so-called Nitrospira lineage IV (Daims et al., 2001a) have phylogenetic, and environmental studies in microbiology. been identified previously in saltwater aquaria (Hovanec J Bacteriol 172: 762–770. et al., 1998) and a marine biofilter (Tal et al., 2003). The high Amann RI, Ludwig W & Schleifer KH (1995) Phylogenetic abundance of Nitrospira marina-like NOB (nearly twice the identification and in situ detection of individual microbial cells biovolume of AOB, which, due to their smaller cell size, will without cultivation. 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