3.1Design and evaluation of oligonucleotide probes
Complete 16S rRNA sequences of 67 strains of the main subgroups of mesophilic Gram-negative SRB within the δ-subclass of Proteobacteria were aligned with representatives of other major lineages of Bacteria and scanned for conserved, diagnostic tracts within these genes. A difference alignment of the oligonucleotides with the complementary 16S rRNA regions of target and non-target species is given in Fig. 2.
Figure 2. Difference alignments of the 16S rRNA target regions for the SRB-specific oligonucleotides. Indicated are the sequences for the respective target (0 mismatch) and non-target species (1–3 mismatches). Positions that differ from the homologous sequence are underlined in bold type.
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Potential signature sequences suitable for in situ probes were compared to the ARB database using the Probe_Match tool of the ARB software package to check for other organisms that may have the identical nucleotide sequences. The computer aided specificity check was supplemented by manual analysis of further 16S rRNA sequences of SRB obtained from the National Center for Biotechnology Information/GenBank. The phylogenetic relationships of the SRB and the sequence homologies of the bacteria to the corresponding oligonucleotide probes are given in Fig. 3.
Figure 3. 16S rRNA based schematic phylogenetic tree reflecting the relationships of SRB within the Desulfovibrionaceae (upper branch) and Desulfobacteriaceae (lower branch) and the lineages targeted by the respective oligonucleotides. For each lineage one representative strain is indicated, numbers in brackets give the SRB encompassed by the respective probe. The scale bar corresponds to 0.1 substitutions per nucleotide position.
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To complement the computer aided probe design, the specificity of each probe was verified under in situ conditions by the reproducible and discriminative staining of target and non-target species. Non-target organisms comprised SRB and cells of other major lineages within the domain Bacteria with one, two and three mismatches to the target sequences as well as single representatives of the Archaea and Eucarya.
For the in situ application of fluorescent dyes with high extinction coefficient and quantum yield such as the indocarbocyanine fluorescent dye Cy3 , the hybridization conditions need to be readjusted and should get closer to the dissociation temperature (Td) of the respective oligonucleotide. In general, in situ hybridizations using Cy3-labelled probes required formamide concentrations at least 20% higher than the same hybridization performed with fluorescein- or tetramethylrhodamine-labelled oligonucleotides. After the optimization of stringency conditions, non-target species displaying one mismatch to the target sequence could be unambiguously separated from the target species. The results of the sequence analysis and whole cell hybridizations are summarized in Table 4. The schematic phylogenetic tree given in Fig. 3 indicates the phylogenetic relationships of SRB and the lineages targeted by the respective oligonucleotide probes.
Table 4. Target organisms, sources and results of sequence analysis and whole cell hybridization Abbreviations: DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; acc., GenBank 16S rRNA accession number; +, target organism with complete homology with the probe sequence; #, organisms used for whole cell hybridizations.
3.2Oligonucleotides specific for members of the family Desulfovibrionaceae
The largest phylogenetic entity within the δ-Proteobacteria sulfate-reducers is formed by various Desulfovibrio species, which define for their part separated lineages.
For the family Desulfovibrionaceae, the probes DSV698, DSV1292, DSD131, DSV407 and DSV214 were developed and evaluated.
Analysis of the target regions and whole cell hybridizations revealed that probe DSV698 was specific for Desulfovibrio desulfuricans (DSM 6949), Desulfomonas pigra (DSM 749), Desulfovibrio salexigens (DSM 2638), Desulfovibrio profundus (acc. U90726), three strains of Desulfovibrio longus (DSM 6739, acc. Z24450, acc. X63623), Desulfovibrio termitidis (DSM 5308), Desulfovibrio gracilis (acc. U53464), Desulfovibrio bastinii (acc. U53462), Desulfovibrio caledoniensis (acc. U53465), nine strains of the genus Desulfovibrio (acc. X93146, acc. X93147, acc. X93994, acc. M98496, acc. M80617, acc. U07570, acc. U07569, acc. U53463, acc. L42995), Lawsonia intracellularis (acc. U30147), Bilophila wadsworthia (acc. L35148), Desulfovibrio halophilus (DSM 5663) and five bacterial species not yet further characterized.
Probe DSV1292 yielded strong hybridization signals after hybridization with the species Desulfovibrio desulfuricans (DSM 6949), Desulfovibrio vulgaris ssp. vulgaris (DSM 644) and Desulfovibrio desulfuricans (DSM 642) and showed 100% sequence complementarity to the 16S rRNA target region of Desulfovibrio halophilus (DSM 5663), Desulfovibrio desulfuricans (acc. M37314), Desulfovibrio termitidis (DSM 5308), Desulfovibrio gracilis (acc. U53464), Desulfovibrio longus (acc. Z24450), Desulfovibrio multispirans (acc. M37318), Bilophila wadsworthia (acc. L35148), Desulfovibrio fairfieldensis (acc. U42221) and five additional Desulfovibrio species (acc. X93146, acc. X93147, acc. M98496, acc. X86689 and acc. L42995).
Desulfovibrio desulfuricans strain El Agheila Z (DSM 1926) is neither encompassed by the probe DSV1292 nor by the probe DSV698. Therefore, probe DSD131 was developed which showed 100% homology to the 16S rRNA sequence of this species.
Probe DSV407 was developed for the identification of Desulfovibrio africanus (acc. 37315) and Desulfovibrio gigas (DSM 1382), constituting two distinct branches within the family Desulfovibrionaceae. Nevertheless, both have an identical target site within the 16S rRNA between base position 407–424, used for construction of probe DSV407, which additionally comprised the species Desulfovibrio gabonensis (acc. U31080).
Probe DSV214 displayed complete homology to the target sequence of Desulfovibrio desulfuricans strain Norway 4 (DSM 1741, suggested as Desulfomicrobium norvegicum), Desulfomicrobium apsheronum (DSM 5918), Desulfomicrobium baculatus (DSM 1743) and Desulfomicrobium escambium (acc. U02469), which form the genus Desulfomicrobium within the family Desulfovibrionaceae.
The combined use of probes DSD131, DSV214, DSV407, DSV698 and DSV1292 enabled the in situ characterization of 84% of the family Desulfovibrionaceae, for which 16S rRNA data are currently available.
3.3Oligonucleotides specific for members of the family Desulfobacteriaceae
Five oligonucleotides (DSS658, DSB985, DSBO224, DSMA488 and DSR651) were developed and evaluated specific for the family Desulfobacteriaceae (Desulfosarcina and relatives, Desulfobacter and relatives, Desulforhopalus vacuolatus, Desulfobotulus sapovorans, Desulfoarculus-Desulfomonile).
Probe DSS658 comprised all strains forming the taxon Desulfosarcina-Desulfococcus, represented by the species Desulfococcus multivorans (DSM 2059) and Desulfosarcina variabilis (DSM 2060), which share 92% similarity in their 16S rRNA sequences . One further species, Desulfonema limicola (DSM 2076), displayed 88% sequence similarity within the 16S rRNA gene; for this species, sequence comparison revealed one mismatch within the target sequence.
The Desulfobacter species comprise a closely related lineage with members sharing more than 95% sequence similarity. Most members of this group could be successfully hybridized with probe DSB985, which displays 100% homology to the target sequence of Desulfobacter hydrogenophilus (DSM 3380), Desulfobacter curvatus (DSM 3379), Desulfobacter postgatei (DSM 2034), Desulfobacula toluolica (DSM 7467) and two further members of the genus Desulfobacter (DSM 2057, DSM 2035).
For probe 221 , specificity analysis revealed four species with complete sequence homology: two strains of Desulfobacterium niacini (DSM 2650, acc. U51845), Desulfobacterium vacuolatum (DSM 3385) and Desulfobacterium autotrophicum (DSM 3382). Together with the oligonucleotide 221, the newly developed probe DSB985 covers the whole Desulfobacter-Desulfobacterium lineage.
Desulfobotulus sapovorans has a distinct phylogenetic position within the family Desulfobacteriaceae and consequently did not completely match with the target region of probe 221 . Therefore, the oligonucleotide DSBO224 was developed, allowing the specific hybridization of Desulfobotulus sapovorans (DSM 2055). For the separate Desulfoarculus-Desulfomonile branch within the mesophilic Gram-negative SRB, probe DSMA488 could be designed and hybridized with both species within this lineage, Desulfoarculus baarsii (DSM 2075, formerly described as Desulfovibrio baarsii) and Desulfomonile tiedjei (DSM 6799). Additionally, this probe has 100% homology to the target sequence of two syntrophic δ-subclass Proteobacteria (Syntrophus gentianae, DSM 8423; Syntrophus buswellii, DSM ‘2612’) to which Desulfoarculus baarsii and Desulfomonile tiedjei are the next relatives .
A further subbranch formed by Desulforhopalus vacuolatus and three not definitely affiliated strains was comprised by the probe DSR651 hybridizing with all strains within this lineage characterized by the species Desulforhopalus vacuolatus (DSM 9700) and two species of the genus Spirochaeta (S. africana DSM 8902; S. asiatica DSM 8901). For two distantly related Desulfobulbus species, which share 92% sequence similarity and appear to be phylogenetically more diverse, the formerly published probe 660  could be successfully used for whole cell hybridizations. Sequence comparison revealed for probe 660 complete homology to Desulfobulbus propionicus (DSM 2032), Desulfobulbus elongatus (DSM 2908) and Desulfobulbus sp. (DSM 2058).
In combination with the probes 221 and 660, the five newly developed oligonucleotides comprise 72% of the 16S rRNA sequences currently affiliated to the family Desulfobacteriaceae.
3.4Evaluation of specificity and in situ suitability of formerly published SRB probes
The specificities of formerly published, group-specific oligonucleotides for different lineages of Gram-negative SRB were analysed by computer aided sequence comparison as described for the newly developed probes. Additionally, the oligonucleotides have been labelled with Cy3 and tested for their in situ suitability by whole cell hybridizations. First of all, six oligonucleotides published by Devereux and coworkers , namely probes 129, 221, 660, 687, 804 and 814 have been evaluated.
The Desulfobotulus-specific probe 660 and a probe specific for the genus Desulfobacterium (221), displayed high specificities and could be shown to be well suited for in situ hybridization. They were therefore used for our studies after adjustment of in situ stringency conditions. Probe 687 encompassed different members of the family Desulfovibrionaceae and several non-sulfate-reducing species within the δ-subclass of Proteobacteria and was therefore not used for further examinations. The fourth probe (129), specific for the genus Desulfobacter, showed clear in situ hybridization signals as well. The newly developed probe DSB985, however, additionally comprised the species Desulfobacter curvatus (DSM 3379) and Desulfobacula toluolica (DSM 7467) and was therefore used for further investigations. Probes 804 and 814 revealed very weak fluorescent in situ hybridization signals even after labelling with Cy3 and could not be successfully used for whole cell hybridizations, which is in accordance with results reported by Ramsing and coworkers  and Rabus et al.  for the fluorescein derivatives.
The probes SRB385  and SRB385Db  comprised a great number of Gram-negative, mesophilic SRB and showed strong in situ hybridization signals. However, sequence comparisons for both probes also revealed a broad range of non-sulfate-reducing bacteria displaying 100% homology within the target sequence [10, 45]. Consequently, both oligonucleotides should be applied in combination with more specific probes for subgroups of sulfate-reducing species in a nested hybridization approach [46, 47].
3.5Occurrence and quantification of SRB in activated sludge
The relative abundance and spatial distribution of SRB within activated sludge obtained from a municipal sewage treatment plant (Berlin-Ruhleben, Germany) was determined using Cy3-labelled derivatives of the oligonucleotides specific for the family Desulfobacteriaceae (probes DSR651, DSB985, DSS658, DSBO224, DSMA488) and the family Desulfovibrionacae (probes DSV214, DSV407, DSV698, DSV1292 and DSD131), combined with epifluorescence and confocal laser scanning microscopy.
Individual members of the families Desulfovibrionaceae and Desulfobacteriaceae could be visualized to different amounts within the activated sludge flocs. The distribution and relative abundance of members of the families Desulfovibrionaceae and Desulfobacteriaceae determined by in situ probing is shown in Fig. 4.
Figure 4. In situ distribution and abundance of members of the families Desulfobacteriaceae and Desulfovibrionaceae within the activated sludge process. In situ cell counts obtained with the specific probes were standardized over total cell counts, determined by DAPI-staining. DSB: total of specific counts obtained with probes DSBO224, DSMA488, DSR651, DSS658 and DSB985. DSV: total of specific counts obtained with probes DSD131, DSV214, DSV407, DSV698 and DSV1292.
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The number of cells which could be successfully hybridized with the individual Desulfovibrionaceae-specific probes ranged from 0.1% (probe DSD131) to 2.4% (probe DSV698) of total cell counts. In the sum, Desulfovibrionaceae could be monitored to percentages ranging from 2.8 to 5.2%, depending on the sample sites determined. For Desulfobacteriaceae, in situ counts of specific probes ranged from below 0.1% (probe 660) to 1.8% (probe DSB985) of total cell counts. Summarized, after hybridization of the activated sludge with probes specific for the family Desulfobacteriaceae, 5.1 to 9.5% of all DAPI-stained cells showed strong fluorescent signals. During the anaerobic and anoxic treatments of the activated sludge process, in situ counts obtained with the Desulfobacteriaceae-specific probes ranged from 2.4 to 4.3%. The number of hybridized cells declined in samples obtained from the end of the aerobic stage to 2.3%; within the final clarifier 2.6% of the total cell counts could be hybridized (sampling site 6). The specific cell counts determined with the Desulfovibrionaceae-specific probe were 4.3% (site 1) and 4.2% (site 2) in the anaerobic compartment and 4.7% (site 3) and 5.2% (site 4) in the anoxic zone. As could be shown for members of the family Desulfobacteriaceae, the specific cell counts dropped at the end of the aeration zone (2.8%, sampling site 5) and increased again to 4.3% in samples obtained from the final clarifier (sampling site 6).
For probe SRB385, in situ counts ranged from 10.4% (site 1), 8.7% (site 2) and over 10.0% (site 3) to 12.0% at site 4. At the end of the aeration zone (sampling site 5) the SRB385 counts decreased to 7.0% and raised again slightly to 7.9% in activated sludge flocs obtained from the final clarifier.
In view of the spatial distribution, members of the family Desulfobacteriaceae were mainly located as part of assemblies or densely packed microcolonies as well as arranged around solid cores within the sludge flocs. Examples typifying the three-dimensional distribution are given in Fig. 5A and B. Using in situ hybridizations with the Cy3-labelled probes DSR651 and DSS658, coccoid bacteria and sarcina-like arranged cells were visualized. These cell morphologies could not be seen after hybridization with the probe SRB385.
Figure 5. CLSM micrographs of activated sludge flocs after in situ hybridizations with CY3-labelled SRB probes. Confocal sections were scanned along optical XY axis with increments of 0.5 μm. A: Gallery of 16 optical sections scanned in distances of 0.5 μm representing the spatial distribution of Desulfobacteriaceae around the solid core of an activated sludge floc after hybridization with Cy3-labelled probe DSS658. B: Transparent projection of all 24 confocal sections scanned in distances of 0.5 μm resulting in an extended focus image of the microcolony of cells strongly hybridizing with probe DSS658 within the activated sludge floc. C: Transparent projection of confocal sections after hybridization with probes DSV698 and DSV1292 reflecting the disperse arrangement of Desulfovibrionaceae within the activated sludge floc. All photomicrographs were done at a magnification of ×1000, bar=10 μm.
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In contrast to the spatial arrangement of the Desulfobacteriaceae, individual cells which could be affiliated by in situ hybridization to the family Desulfovibrionaceae appeared as slightly curved, vibrio-like or rod-shaped cells. They were mostly arranged as single cells and were homogeneously distributed within the sludge flocs, or to a lesser extent, loosely grouped and attached to inorganic particles. The typical spatial distribution of individual members of the Desulfovibrionaceae hybridized with a mixture of Cy3-labelled probes DSV698 and DSV1292 is given in Fig. 5C.