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Molecular microbial diversity in a nitrifying reactor system without sludge retention Susanne Logemann, Julia Schantl, Saskia Bijvank, Mark van Loosdrecht, J. Gijs Kuenen, Mike Jetten * Kluyver Institute for Biotechnology, Research School for Biotechnological Sciences Delft-Leiden (BSDL), Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands Received 18 February 1998; received in revised form 26 June 1998; accepted 1 July 1998
Abstract Recently, the single reactor system for high activity ammonia removal over nitrite (SHARON) process was developed for the removal of ammonia from wastewater with high ammonia concentrations. In contrast to normal systems, this nitrifying reactor system is operated at relatively high temperatures (35³C) without sludge retention. Classical methods to describe the microbial community present in the reactor failed and, therefore, the microorganisms responsible for ammonia removal in this single reactor system were investigated using several complementary molecular biological techniques. The results obtained via these molecular methods were in good agreement with each other and demonstrated successful monitoring of microbial diversity. Denaturing gradient gel electrophoresis of 16S rRNA PCR products proved to be an effective technique to estimate rapidly the presence of at least four different types of bacteria in the SHARON reactor. In addition, analysis of a 16S rRNA gene library revealed that there was one dominant (69%) clone which was highly similar (98.8%) to Nitrosomonas eutropha. Of the other clones, 14% could be assigned to new members of the Cytophaga/Flexibacter group. These data were qualitatively and quantitatively confirmed by two independent microscopic methods. The presence of about 70% ammonia oxidizing bacteria was demonstrated using a fluorescent oligonucleotide probe (NEU) targeted against the 16S rRNA of the Nitrosomonas cluster. Electron microscopic pictures showed the typical morphology of ammonia oxidizers in the majority of the cells from the SHARON reactor. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Nitrosomonas; Cytophaga ; Molecular ecology ; Denaturing gradient electrophoresis ; 16S rRNA
1. Introduction The oxidation of ammonia to nitrite is of major importance in the global cycling of nitrogen in very diverse ecosystems and is widely used in wastewater * Corresponding author. Tel.: +31 (15) 2781193; Fax: +31 (15) 2782355; E-mail:
[email protected]
treatment as the ¢rst step to remove nitrogen from the water [1]. The conversion of ammonia to nitrite is assumed to be due to autotrophic bacteria, although heterotrophic nitri¢cation may contribute signi¢cantly in systems with abundant carbon supply [2]. The autotrophic bacteria responsible for the oxidation of ammonia are presumed to belong predominantly to the genera Nitrosomonas and Nitrosospira
0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 9 8 ) 0 0 0 7 0 - 1
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[3^6]. Investigation of autotrophic nitri¢ers has so far been limited by di¤culties in isolating and culturing ammonia oxidizers, especially elimination of heterotrophic contaminants [7]. To overcome these dif¢culties, the 16S rRNA approach has been used to analyze communities of ammonia oxidizers in natural and man-made ecosystems [8^16]. Recently, a single reactor system for high activity ammonia removal over nitrite (SHARON) was developed [17]. In this process ammonia is nearly completely converted to nitrite, with minor nitrate formation. This SHARON process is ideally suited to remove nitrogen from waste streams with a high ammonia concentration ( s 0.5 g N l31 ). In contrast to conventional systems, the SHARON process is performed in a single, stirred tank reactor without any sludge retention at a high dilution rate. In this way it is possible to e¡ectively outcompete the nitrite oxidizers at temperatures above 25³C [1,2]. This results in stable nitri¢cation with nitrite as the major end product. The SHARON process has been extensively tested on laboratory scale for the treatment of sludge digester e¥uents and is currently under construction at two Dutch wastewater treatment plants [17]. Although the engineering aspects of the system were well understood, little was known about the composition of the microbial community responsible for the SHARON process. The application of high dilution rates (liquid residence time is about 1 day) in combination with high ammonia concentrations 31 (1 g NH 4 -N l ) results in a system with unusually high nitrite concentrations in which the biomass is not retained. This is contrary to conventional treatment systems which are operated at hydraulic and sludge residence times of 5^10 days and treat waters with low ammonia concentrations (50 mg NH 4 -N l31 ). Since the conditions in the SHARON reactor are so di¡erent from conventional systems, it might be expected that the composition of the microbial community might also be di¡erent. It was therefore the aim of this study to investigate the microbial community of the SHARON reactor with di¡erent complementary (molecular biological) techniques, to determine the phylogenetic identity of the dominant ammonia oxidizing bacterium in the SHARON system, and to monitor changes in the 16S rRNA composition of the community with electrophoretic [18] and microscopic techniques.
2. Materials and methods 2.1. Operation of the nitrifying reactor The SHARON reactor consisted of a 2-l stirred vessel equipped with pH (7.2), dissolved oxygen (50%) and temperature (35³C) control (ADI 1020, Applikon, Schiedam, The Netherlands). At the time of biomass sampling, the reactor had been fed for 2 years with sludge digestion e¥uent obtained from the Rotterdam wastewater treatment plant Dokhaven-Sluisjesdijk [1,17]. The sludge digestion e¥uent contained 1053 þ 231 mg N l31 , 232 þ 74 mg BOD l31 [1,17]. In addition, another reactor system, inoculated with reactor £uid from the original SHARON system fed with sludge digester e¥uent, was started on synthetic wastewater containing 3.7 g l31 (NH4 )2 SO4 , 3.1 g l31 K2 HPO4 , minerals and trace elements [1]. Once a week the vessel was cleaned with a brush to remove bio¢lms growing on the glass wall of the reactor. 2.2. Enrichment of autotrophic and heterotrophic bacteria from the SHARON reactor Reactor £uid was diluted in phosphate bu¡ered saline. The dilutions were incubated in both liquid and on solid (1.6% Noble agar) synthetic medium. For enrichments of autotrophic bacteria the medium was composed of 2.1 g l31 (NH4 )2 SO4 , 1 g l31 NaHCO3 , 0.38 g l31 MgSO4 W7H2 O, 0.2 g l31 CaCl2 W7H2 O, 0.02 g l31 MnCl2 , 0.087 g l31 KH2 PO4 ¢nal pH 7.6 and 0.2 ml l31 trace element solution (g l31 ) 0.2 Na2 MoO4 W2H2 O, 0.24 CoCl2 W6H2 O, ZnSO4 W7H2 O, 0.25 CuSO4 W5H2 O, 0.2 NiCl2 W6H2 O, 0.2 g NaSeO4 W10H2 O, 0.05 H3 BO3 , 0.05 NaWO4 W2H2 O). For isolation of heterotrophic bacteria the medium contained per liter 5 g tryptone, 2 g yeast extract and 0.5 g acetate [19,20]. Media were also made up using ¢lter sterilized reactor e¥uent instead of distilled water. Cell numbers, ammonium conversion, nitrite or nitrate production in liquid media and colony formation on solid media were monitored for 2 months and analyzed as described by Koch [21] and van de Graaf et al. [22].
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2.3. Extraction and puri¢cation of total genomic DNA from the SHARON reactor About 1.5 ml of reactor £uid was harvested by centrifugation, resuspended in 250 Wl bu¡er I (0.1 M Tris-HCl, pH 8.0, 25 Wg ml31 RNase, 1.5 mg ml31 lysozyme, 0.5 mg ml31 Qiagen 9942 protease) and incubated at 37³C for 10 min. Then 250 Wl bu¡er II (50 mM Tris-HCl, pH 8.0, 5 mM NaCl, 2.5 mM EDTA, 1% SDS) was added and the mixture was incubated at 37³C for another 10 min. This mixture was successively extracted three times with one volume of phenol (Tris-HCl bu¡ered, pH 7.4), phenol: chloroform:isoamyl alcohol (25:24:1) and ¢nally chloroform. The DNA was precipitated with 0.1 volume of 3 M sodium acetate (pH 5.0) and 2 volumes of chilled 100% ethanol. After centrifugation, the DNA pellet was washed in 70% ethanol, dried and resuspended in 10^100 Wl TE bu¡er (10 mM Tris pH 8.0, 1 mM EDTA). 2.4. Cloning and sequencing of SSU rRNA The bacterial DNA was ampli¢ed by the polymerase chain reaction using a Robocycler Gradient 40 Cycler (Stratagene, La Jolla, CA, USA). The 100 Wl reaction mixture contained the following compounds: 10^100 ng DNA template, 20 pmol each of the primers 27f-BamHI: 5P-CACGGATCCAGAGTTTGATMTGGCTTCAG-3P, and 1492r-HindIII: 5P-TGTAAGCTTACGGYTACCTTGTTA CGACT-3P), 20 nmol dNTPs, 10 Wl 10UPCR bu¡er, 2.5^5 mM MgCl2 , 2.5 U of Taq polymerase (Goldstar DNA Polymerase, Eurogentec, Belgium; or Taq DNA Polymerase, Gibco BRL, UK) [23]. In some cases primer 1492r was replaced by primer 907r (5PCCGTCAATTCATTTGAGTTT-3P) [23]. The 16S rRNA of the SHARON community was ampli¢ed by 30 cycles of PCR. The ampli¢cation program of the PCR consisted of the following steps: denaturing for 1.5 min at 94³C, annealing for 1^2 min, and elongation for 2 min at 72³C, followed by a ¢nal synthesis step of 5 min at 72³C. The PCR products were analyzed on 1% agarose gels, subsequently puri¢ed (Qiaex kit, Qiagen, Germany), cloned into the pGEM-T plasmid, and transformed into Escherichia coli K-12 JM 109 (Promega, Madison, WI, USA). Plasmid DNA was extracted from recombinant
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colonies and digested with several restriction enzymes. The initial screening was performed with the enzymes NcoI and SalI. After the integrity of the insert (1490 bp) was veri¢ed on 1% agarose gels, 56 plasmids were further analyzed with the following enzyme combinations: BamHI/HindIII, EcoRI/SphI SphI/PstI, and SphI/SacI. The digests were performed according to the instructions of the supplier (Promega, Madison, WI, USA, or Boehringer, Mannheim, Germany). For PCR reactions from plasmids, primers 27f and 907R were used [23]. The nucleotide sequence of plasmid inserts was determined by semi-automated sequencing on an ALFExpress using T7 polymerase (Pharmacia Biotech, Uppsala, Sweden). 2.5. Database searches Sequence alignment was carried out with the GCG Sequence Analysis Package version 8.1 on the multiuser multi process Digital AXP computer RULGCA. Similarity searches were performed with the program FastA and in the Ribosomal Database Project [24]. Phylogenetic trees were constructed by the Jukes-Cantor and neighbor-joining algorithms including bootstrap analysis using the Treecon package [25]. Nucleotide sequence data reported in this paper will appear in the EMBL database under the accession numbers AJ224410^AJ224417. 2.6. DGGE Analysis The mixed 16S rRNA PCR product of the reactor and PCR products from individual plasmids were analyzed by a modi¢ed denaturing gradient gel electrophoresis (DGGE) [18] using a Macrophore Electrophoresis Unit and a Thermostatic Plate (Pharmacia Biotech, Uppsala, Sweden). The PCR samples were concentrated by ethanol precipitation and separated on 5.6% polyacrylamide gels in 1UTris-borate-EDTA (TBE) bu¡er containing a linearly increasing gradient between 25% and 55% of denaturant (100% denaturant is 7 M urea and 40% (w/v) formamide). The gel was poured using a two chamber gradient mixer and electrophoresis was carried out in 1UTBE bu¡er at 50 mA for 135 (for 1500 bp) or 155 min (for 900 bp). The bands were visualized by staining in ethidium bromide (0.5 mg l31 )
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under UV. In this way PCR fragments without a GC-clamp could be analyzed within 3 h. 2.7. Fluorescent in situ hybridization (FISH) The reactor samples for FISH analysis were ¢xed in 3 volumes of freshly prepared paraformaldehyde solution (4% in Tris bu¡ered saline, TBS) for at least 30 min. Cells were washed in TBS and one volume of cold ethanol was added. The cells were spread on slides coated with Vektabond (Vector Laboratories, USA) and dried. Alternatively, cells were ¢xed with one volume of ice-cold ethanol for 30 min, spread on slides and dehydrated in three steps (50%, 80%, 100% ethanol). After a pre-hybridization in ExpressHyb bu¡er (Clontec, USA), the hybridization was carried out overnight at 42³C in half-strength ExpressHyb1 bu¡er containing the appropriate amount of formamide and 20 pM of the 5P £uorescein labeled oligonucleotides EUB338 (recognizing most eubacteria), CF319 (recognizing most Cytophaga/Flexibacter/Bacteroides members), and NEU (speci¢c for nitri¢ers of the Nitrosomonas cluster) [12,23,26]. The probes NEU (5P-CCCCTCTGCTGCACTCTA-3P, 40% formamide [12]), CF319 (5P-TGGTCCGTRTCTCAGTAC, 35% formamide [26]) and EUB338 (5P-CGTGCCTCCCGTAGGAGT-3P, 35% formamide [23]) were purchased from Eurogentec (Seraign, Belgium). Unbound oligonucleotides were removed by washing twice in warm (42³C) buffer (0.9 M NaCl, 10 mM Tris-HCl; 0.01% SDS), rinsed with water and mounted in Vektashield (Vector Laboratories, USA). Microscopy was performed under oil immersion with an Olympus BH2 microscope (Olympus Optical Co., Tokyo, Japan) equipped with blue and green ¢lter sets (BL 0892, Olympus).
2 min), washed once with 0.5% glutaraldehyde solution and resuspended in the same solution.
3. Results 3.1. Classical microbial analysis As a ¢rst estimation of the diversity in the microbial community of the SHARON reactor, MPN enrichments for autotrophic and heterotrophic bacteria were carried out in selective media. The use of either distilled water or ¢lter sterilized reactor e¥uent showed no signi¢cant di¡erence in numbers of autotrophic nitri¢ers (9.7 þ 4U106 ml31 ) or heterotrophic bacteria (5.7 þ 3U106 ml31 ) obtained after 2 months of incubation. In both cases the recovery (as a percentage of total cell counts) was very low (1^5%). It was relatively easy to isolate several heterotrophic species (mostly Gram-negative species with polar £agella), but attempts to obtain pure cultures of autotrophic ammonia oxidizers failed. In order to gain more insight into the community responsible for the
2.8. Electron microscopy The electron microscopic analysis of the SHARON samples was performed at the Department of Electron Microscopy at the University of Groningen. The samples were ¢xed with fresh, cold glutaraldehyde solution (3% in 10 mM K2 HPO4 pH 7.5). One volume of cell suspension was mixed with 3 volumes of ¢xative, and the mixture was incubated on ice for at least 2 h. The cells were centrifuged (13 000 rpm,
Fig. 1. DGGE analysis of a 900-bp 16S rRNA PCR product from the SHARON reactor fed with sludge digestion e¥uent (lane 1, C/F is Cytophaga/Flexibacter-like ; NE is N. eutrophalike) ; from clone 74 (lane 2; 98.8% similar to N. eutropha); and from N. europaea chromosomal DNA (lane 3).
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Fig. 2. Cartoon of the DGGE analysis of 900-bp 16S rRNA PCR products from several clones of the 16S rRNA library of the SHARON reactor. No original gel could be shown because several clones were analyzed on di¡erent gels. Furthermore the quality of some of the gels was too poor to be visualized via photography. The percentage (25^35%) denaturing agent (100% denaturant is 7 M urea and 40% (w/v) formamide) is indicated on the Y-axis. Lane 1, clone 76 (same migration distance (md) as C/F1) ; lane 2, clone 54 (same md as C/F3); lane 3, clone 24 (same md as C/F2); lane 4, clone 51 (N. eutropha-like); lane 5, clone 74 (N. eutropha-like); lane 6, PCR product from N. europaea chromosomal DNA; lane 7, PCR product SHARON reactor fed with synthetic wastewater (SWW); lane 8, PCR product SHARON reactor fed with sludge digestion e¥uent (SDE).
ammonia conversion in the SHARON reactor, a molecular approach was chosen. 3.2. DGGE analysis of SHARON reactor The complexity and composition of the microbial community in the SHARON reactor was ¢rst investigated by a modi¢ed DGGE method [18]. This method allowed the separation of a mixture of di¡erent PCR fragments originating from individual types of bacteria with sequence variations in the 16S rRNA genes. A high number of bands on a DGGE gel would represent high complexity in the community. DNA was extracted from the SHARON biomass of a reactor fed with sludge digestion e¥uent, ampli¢ed using bacterial 16S rRNA primers and subjected to DGGE analysis. In contrast to observa-
tions made in other reactor systems [27], the DGGE pattern of SHARON biomass showed only a small number of bands (Fig. 1, lane 1). The PCR products from biomass of the reactor fed with sludge digester e¥uent separated into four distinct bands using both primer pairs 27f/907r and 27f/1492r. All bands showed the same intensity after staining with ethidium bromide (Fig. 1, lane 1). This ¢nding suggested the presence of at least four di¡erent organisms. 3.3. Analysis of 16S rRNA gene bank In order to identify the individual members of the microbial community in the SHARON biomass fed with sludge digestion e¥uent, the 16S rRNA PCR products were cloned and analyzed. A nearly fulllength PCR product (27f/1492r) was used to con-
Table 1 Biodiversity in the SHARON 16S rRNA clone library A¤liation
Nitrosomonas sp. Cytophaga/Flexibacter class 1 Cytophaga/Flexibacter class 2/3 L-Proteobacteria Unknown
Number of clones
Position of restriction digest by
(percentage)
SphI
EcoRI
SphI
39 2 6 2 7
60 230 ^ ^ ^
680 ^ ^ 600 ^
1360 ^ ^ ^ ^
(69%) (4%) (10%) (4%) (13%)
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Fig. 3. Dendrogram based upon the distance method of Jukes-Cantor (calculated with the TREECON software [25]), showing the phylogenetic position of the major clones present in the 16S rRNA library from the SHARON reactor. A : Phylogenetic position of clone 74 in comparison to some Nitrosomonas strains [14,28], analysis based on 1125 nucleotides available for the various 16S rRNA gene sequences of ammonia oxidizers (E. coli positions 124^1340). B: Position of clones 23 (class C/F2), 24 (class C/F2), 54 (class C/F3), 76 (class C/F1), and 88 (class C/F3) in comparison to some Cytophaga strains, analysis based on 5P-392 nucleotides. The bars represent two or 10 nucleotide substitutions per 100 nucleotides, respectively. Bootstrap values (200 samples) are given at the nodes when they exceeded 50% of the replicates.
struct a gene library. After initial screening of the recombinant plasmids with NcoI and SalI, 56 clones containing a full-size 16S rRNA insert were characterized by restriction digests (BamHI/HindIII, EcoRI/SphI SphI/PstI, and SphI/SacI) and by DGGE. The clones could be divided into four distinct classes according to their restriction pattern
(Table 1). The majority of the clones (69%) showed an identical restriction pattern and had an identical migration distance (i.e. clone 51 and clone 74 in Fig. 2) on DGGE gels. A partial DNA sequence was determined for 11 clones, and the full-length 16S rRNA sequences of two representative clones were analyzed. Database comparisons of the sequences
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obtained showed that the dominant clones were nearly identical to the 16S rRNA of Nitrosomonas eutropha. The 16S rRNA homology was 98.8% with N. eutropha, and 97.5% with N. europaea in the ¢rst 1340 nucleotides. The last part of the gene is not yet present in the database for N. eutropha and N. europaea. Phylogenetic analysis of 1125 nucleotides available for the various 16S rRNA gene sequences of ammonia oxidizing bacteria (between Escherichia coli positions 124 and 1340, Fig. 3A) showed that this clone positioned within the cluster of Nitrosomonas-like ammonia oxidizers [14,28]. Representatives from the other classes of clones were subjected to partial DNA sequence analysis of 392 bp. An overview of the molecular microbial diversity in the SHARON reactor is presented in Table 1. In contrast to the major clone, one class of clones (4%) was not digested by SphI, but did have an EcoRI site in the 16S rRNA. After sequence analysis the two clones could tentatively be assigned to the L-proteobacteria. The closest relatives (less than 85% DNA homology) of clone 19 were Azoarcus evansii and Thauera aromatica. Clone 75 was weakly related (less than 80% DNA homology) to Rhodocyclus tenuis and Nitrosospira briensis. Of the other clones, 14% were a¤liated with the Cytophaga/Flexibacter phylum. Phylogenetic analysis of 392 nucleotides of these clones is presented in Fig. 3B. All sequences are new to the database, but clearly group within the Cytophaga/Flexibacter phylum. The remaining clones in the library turned out to be chimeras, oligopolymers or could not be identi¢ed from partial sequence analysis. The results obtained via the di¡erent molecular methods were in good agreement with each other. Nevertheless, it was observed that two clones showed the same restriction pattern but a di¡erent DGGE migration distance. It is therefore advisable to use at least two di¡erent screening techniques before clones can be excluded from sequence analysis. 3.4. Monitoring of population changes via DGGE The PCR product of the SHARON reactor fed with sludge digestion e¥uent separated into four distinct bands on DGGE. All four bands were identi¢ed by sequencing and co-migration of the PCR products from the corresponding sequenced clones
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on the same gel (Fig. 2). With such a characterized DGGE system closely related species can be discriminated or changes in the bacterial community due to changes in the reactor parameters can be monitored. Two examples are provided below. The two ammonia oxidizing bacteria N. europaea and N. eutropha show 98.4% homology in their 16S rRNA and could not be distinguished via molecular techniques so far. However, via DGGE we were able to show a di¡erence in the migration behavior of the PCR products from 16S rRNA from a pure culture of N. europaea and the clone representing N. eutropha (Fig. 1, lanes 2, 3). In this way we were able to show that our major clone was not identical to N. europaea. Together with the phylogenetic analysis this result indicates that the dominant organism in the SHARON biomass might be a new strain of N. eutropha. The second example is that changes in this well characterized community due to di¡erent growth conditions could be demonstrated by the DGGE technique. For this purpose a new SHARON reactor was started on synthetic wastewater and the 16S rRNA PCR product from the community that had developed after 100 days was compared to that of the SHARON reactor which had been fed with sludge digestion e¥uent for 2 years. The DGGE analysis of the biomass fed with synthetic wastewater showed one clear and one very faint band (Fig. 2, lane SHARON SWW). The migration distance of the dominant band was the same as the band from the reactor fed with sludge digester e¥uent (Fig. 2, lane SHARON SDW) which had been identi¢ed as N. eutropha. This indicates that presumably this organism is responsible for ammonia removal in this (strictly autotrophic) type of SHARON reactor and that this organism is present in both reactor systems. Although the intensity of the DGGE band was the same for samples of both reactors, the number of N. eutropha cells may have increased considerably by replacing the wastewater with synthetic medium. The medium only contained ammonia as energy source whereas the wastewater had some residual carbon. However, two of the Cytophaga/Flexibacter bands disappeared in the DGGE pattern of the SHARON biomass from the reactor fed with synthetic medium. The amount of Cytophaga cells (see Section 3.5) did not seem to change in this system
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indicating that only one was able to maintain itself in this system not supplemented with an organic carbon source. 3.5. Fluorescent and electron microscopic analysis Both PCR and cloning of PCR products could theoretically cause several biases in the community analysis. To con¢rm data obtained via DGGE and cloning, we carried out in situ hybridization with di¡erent £uorescently labeled 16S rRNA targeted oligonucleotide probes. When the SHARON reactor was hybridized with a probe that is complementary to the 16S rRNA of most eubacteria (EUB338), more than 90% of the cells were labeled, indicating that the biomass in the SHARON reactor was almost completely composed of Bacteria (data not shown). The presence and number of ammonia oxidizing bacteria in the SHARON reactor was investigated in situ by hybridization with a oligonucleotide probe (NEU) speci¢c for Nitrosomonas-like bacteria [12]. The hybridization signal observed under the microscope was used to estimate the number of Nitrosomonas-like bacteria by comparing the phase contrast picture with the £uorescent image (Fig. 4) in several slides. This comparison revealed that between 50% (579 of 1164) and 70% (868 of 1236) of the cells hybridized with probe NEU. Independent analysis in another laboratory (Bioclear, Groningen, The Netherlands) con¢rmed these values. As reported in other studies we sometimes observed densely packed clusters (£ocs) of Nitrosomonas cells [7].
Fig. 5. Electron micrograph of suspended cells from the SHARON reactor grown on sludge digestion e¥uent. Magni¢cation 35 000U.
In contrast to the probe NEU which is targeted against Nitrosomonas-like bacteria, the probe CF319 recognizes nearly the whole phylum of Cytophaga/ Flexibacter [26]. In situ hybridizations with CF319 showed that approximately 5^10% of the bacteria belonged to this phylogenetic group. The probe reacted with both rod-shaped and ¢lamentous bacteria which were unevenly distributed in the £ocs. Electron microscopic pictures of the SHARON reactor showed cells with intracytoplasmic stacked membrane structures typical of aerobically growing nitrifying Nitrosomonas. These types of cells were very abundant in the SHARON reactor. In some preparations, however, cells with areas of high electron density were visible (Fig. 5). These cells looked very similar to those of Nitrosomonas under oxygen limitation [29,30].
4. Discussion
Fig. 4. In situ hybridization of cells from the SHARON reactor. Epi£uorescence micrograph after hybridization with £uorescein labeled probe NEU [12]. Magni¢cation 625U.
With the advancement of molecular ecological methods it has become clear that selective classical enrichment and isolation techniques have serious limitations [31]. During our investigation of the bacterial community in the SHARON reactor, these limitations were also very obvious. Due to the slow
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growth rate and frequent occurrence of culture contamination by heterotrophic bacteria, it was not possible to isolate or identify the dominant ammonia oxidizing bacteria in this process with classical techniques. Even isolation of relevant dominant heterotrophic species was very problematic. In both cases the recovery (as percentage of total cell counts) was less then 5%. To overcome these limitations, we have successfully applied several molecular biological methods to unravel the diversity of the microbial community in the SHARON reactor. 4.1. Identi¢cation of the dominant bacterium in the nitrifying community The molecular techniques used in this study, except for DGGE analysis, showed independently that there is a highly dominant bacterium present in the SHARON reactor. This ammonia oxidizing bacterium is closely related to N. eutropha. This is not very surprising since the set-up of the SHARON system (high dilution rate, high temperature, high ammonia concentration, no sludge retention, and weekly removal of bio¢lms from glass walls) e¡ectively selects ammonia oxidizing bacteria which tolerate high salt and ammonia concentrations and can grow relatively fast. The properties of N. eutropha obtained in pure culture from other sources so far meet these criteria fairly well [32]. It was observed to tolerate 600 mM NH4 Cl and had a doubling time of approximately 12 h and a Ks (NH 4 ) of 0.75 mM. In addition, Nitrosomonas species were found to be capable of nitrite reduction to N2 O with hydrogen, ammonia or hydroxylamine as electron donor, especially in co-culture with heterotrophic species [29,30]. This is in good agreement with the electron microscopic detection of cells typical of `denitrifying' nitri¢ers. The sequence similarity of the dominant clone with N. eutropha was 98.8%. This is in the same range as the similarity (98.1%) between N. eutropha and N. europaea [5] indicating that the dominant cells in the SHARON reactor might be a previously undescribed Nitrosomonas. 4.2. Identi¢cation of other micro-organisms in the SHARON community In addition to N. eutropha, a smaller (4%) subpo-
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pulation of L-proteobacteria was represented in the 16S rRNA library from the SHARON reactor. The 16S rRNA homology of the two clones analyzed showed that they clearly grouped within L-proteobacteria, but that they were not closely related to known species (less than 85% to Azoarcus evansii and Nitrosospira briensis). One of the clones showed 75% homology to both Nitrosomonas and Nitrosospira. Although molecular techniques have established that Nitrosospira-like ammonia oxidizing bacteria are ubiquitous in the environment including activated sludge [9,14], no information is yet available on their nitrite and salt tolerance [6]. The third group of bacteria represented in the 16S rRNA library were related to the Cytophaga/Flexibacter phylum. Since the sludge digestion e¥uent contained some residual organic carbon, heterotrophic bacteria were expected in the reactor system fed with e¥uent. The amount of Cytophaga cells (5^ 10%) detected by FISH was the same in samples from the reactor fed with e¥uent or synthetic wastewater. Although the amount of cells did not change, there was a reduction in the diversity of Cytophaga in the system fed with synthetic wastewater. From the three DGGE bands identi¢ed as Cytophaga from the system fed with e¥uent only one was observed in the DGGE gel of biomass fed with synthetic wastewater. Also others studies have revealed the presence of Cytophaga/Flexibacter-like organisms in (activated) sludge samples. In those studies the amounts of Cytophaga/Flexibacter was somewhat higher (10^50%) [26]. The presence of Cytophagalike bacteria is thought to be associated with either £oc formation or degradation of polymeric substances [26]. 4.3. Comparison of the molecular techniques used in this study Although each molecular method used in this investigation may have a particular bias, the present study demonstrates that complementary molecular biological techniques can be used to determine the diversity of complex microbial system. The results obtained in the various experiments showed a good agreement among the di¡erent molecular techniques. The restriction digest and sequence analysis of the 16S rRNA library demonstrated the presence of
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four major classes of clones. Furthermore, the 16S rRNA PCR product of the SHARON reactor showed four bands in a DGGE gel and all bands could be a¤liated to clones. In particular, the diversity of the clones in the library re£ected the diversity of bacteria in vivo. FISH analysis with oligonucleotide probe NEU showed that 50^70% of the bacteria in the SHARON reactor were a¤liated with Nitrosomonas-like organisms. This amount is in good agreement with the 69% found for the dominant clone in the 16S rRNA library. Furthermore the percentage of clones (14%) in the library encoding members of the Cytophaga/Flexibacter group is similar to the 5^10% of bacteria detected by the CF319 probe in situ. Finally, the modi¢ed DGGE method proved to be an excellent tool for rapid investigations of microbial complexity and for identi¢cation of community members if characterized clones or pure cultures for comparison by co-migration are available. By using a thermostatted macrophore electrophoresis unit long DGGE gels could be made, and 900- or 1500-bp PCR fragments without GC-clamp [33,34] could be separated within 3 h. In this way the same PCR product can be used for both DGGE analysis and construction of a gene library. Since no GC-clamp is used, PCR products migrate continuously in the gel and become single stranded when they reach high denaturant concentrations. The single stranded DNA is not visualized by staining, and therefore the electrophoresis has to be stopped in time. Furthermore, the DGGE technique is not yet a quantitative method. The four bands originating from SHARON biomass showed equal intensity on the DGGE gel, although, as shown above, the relative amount of the di¡erent types of bacteria was clearly not identical.
Acknowledgments This research was supported by the Dutch Science Foundation (NWO), the Foundation for Applied Water Research (STOWA) and the Royal Netherlands Academy of Arts and Sciences. K.T. Schoonen and J.C. de Bruijn are acknowledged for helpful advice in microscopy and B. Geurkink from Bioclear (Groningen, The Netherlands) for in situ hybridiza-
tions and supply of the oligo NEU. The authors thank Klaas Sjollema, Ineke Keizer, and Maarten Veenhuis (RU Groningen) for electron microscopy, Quirien Lisman and Jaques Monod for determining many of the sequences, Henry van Veldhuizen and Svein Horn for running some of the reactor systems, and two anonymous referees for many constructive comments. Julia Schantl and Saskia Bijvank have contributed equally to the paper.
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