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 À (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 Â 15 s at speed 4.0 and Gelfand et al., 2003) that was stocked with gilthead seabream 1 Â 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 LÀ1 (pool volume 2.3 m3)atarateof60LminÀ1 (Æ 20 L minÀ1). 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),10mgLÀ1 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(2Â 15satspeed5.0 extraction) or fixed in 4% paraformaldehyde (for FISH; and 1 Â 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 substra- tum 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 À1 NH4