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Applied and Environmental Microbiology, January 2002, p. 356-364, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.356-364.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

Tsukasa Ito,1 Jeppe L. Nielsen,2 Satoshi Okabe,1* Yoshimasa Watanabe,1 and Per H. Nielsen2

Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, 060-8628, Sapporo, Japan,1 Environmental Engineering Laboratory, Aalborg University, DK-9000 Aalborg, Denmark2

Received 23 July 2001/ Accepted 5 October 2001


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [14C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H2-utilizing and 14CO2-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H2-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H2 utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sulfate-reducing bacteria (SRB) are known to be ubiquitous and to play an important ecological role in diverse environments, such as freshwater lake sediments (34), marine sediments (13, 14), oil production facilities (27), and wastewater treatment plants (17, 24, 29, 33). In particular, it has been estimated that sulfate respiration accounts for up to 50% of organic-carbon mineralization in some marine sediments (13) and wastewater biofilms (17). Furthermore, sulfate reduction causes extensive biocorrosion and biodeterioration of oil production and wastewater treatment facilities (26, 38).

Although SRB are generally considered to be obligatory anaerobic bacteria, a widespread occurrence of SRB has been reported in oxic environments, such as the highly oxic chemocline of lake sediments (34), oxic zones of cyanobacterial mats (15, 30), and wastewater biofilms grown under oxic conditions (24, 29, 33). In particular, a high abundance of Desulfobulbus and Desulfovibrio was found in the oxic surface regions of wastewater biofilms by using fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes (24, 33). Although these SRB species are known to be oxygen tolerant and to have versatile metabolic abilities (8, 26, 38), it is not clear that they could remain or proliferate there via respiration with nitrate or oxygen as an electron acceptor.

While more detailed information on the microbial structure and diversity of SRB in complex microbial communities has been accumulated by using 16S rRNA-based (9, 20, 21, 30, 35) and dissimilatory sulfite reductase gene-based approaches (22, 37), information about their in situ functions (nutritional characteristics) in their habitats, especially under oxic and anoxic conditions, is scarce. There are a few studies relating SRB community structure to their sulfate-reducing activity in wastewater biofilms in which FISH was successfully combined with microelectrode measurements (24, 29, 33). However, the spatial resolution of microelectrode measurements is not high enough to directly link phylogenetic information about individual SRB cells obtained by FISH to information about their in situ physiology (substrate uptake characteristics) on a single-cell level. Microautoradiography (MAR) has previously proved valuable in investigating substrate uptake patterns of actively metabolizing bacteria in many complex ecosystems (4, 5), and recently it has been successfully combined with FISH as a tool to simultaneously determine phylogenetic identification and in situ nutrient uptake patterns of various cultivable or uncultivable bacteria (11, 18, 23, 25).

The main goal of the present study was to investigate which phylogenetic SRB groups are numerically and metabolically important in organic carbon mineralization within a complex sewer biofilm and their physiology under oxic and anoxic conditions. To achieve this goal, MAR and FISH were combined to simultaneously determine substrate uptake patterns under different electron acceptor conditions by individual SRB cells that were phylogenetically identified by FISH, many of which may be uncultivable. The MAR-FISH technique provides a direct link between 16S rRNA phylogeny and substrate uptake patterns of both cultivated and uncultivated SRB in the delta Proteobacteria, leading to further insight into the ecological importance of SRB in oxic-anoxic natural environments.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Biofilm samples were obtained from a gravity sewer line located at Frejlev in Aalborg, Denmark. The concentration of organic matter (chemical oxygen demand) was 300 to 600 mg liter–1. The dissolved oxygen concentration in the sewer wastewater varied between 0 and 5 mg liter–1. The biofilm sample was collected, homogenized with a glass tissue grinder (Thomas Scientific), and transferred to a serum bottle which was closed with a thick butyl rubber stopper. The biofilm sample in the serum bottle was flushed with oxygen-free N2 gas to remove the oxygen, stored at 4°C, and subjected to the incubation and analyses described below within 24 h.

Sulfate reduction rate.
The homogenized biofilm sample was transferred to 50 ml of the filtered effluent water from an activated-sludge plant containing 2 mM sodium acetate and 0.5 mM sulfate in a serum vial (60 ml). The vials were anaerobically incubated for 2 days at room temperature. At regular intervals, subsamples were withdrawn with a sterilized syringe, and the acid volatile sulfide (S2–, HS–, H2S, and FeS) was measured colorimetrically by the methylene blue method (6).

Incubation with radioactive and nonradioactive substrates under various electron acceptor conditions.
Biofilm samples were incubated with four different radioactively labeled organic and inorganic substrates and three different electron acceptors, such as oxygen, nitrate (3 mM), or sulfate (0.5 mM), at room temperature. Since Fe(III) could be present in the biofilm as a potential electron acceptor, sufficient amounts of sulfide were added to all anaerobic incubations to secure reduced conditions and to remove Fe(III). Prior to incubation, the biofilm samples were diluted to final concentrations of 0.2 g (for oxic conditions) and 0.5 g (for anoxic conditions with nitrate and sulfate as electron acceptors) of volatile suspended solids (VSS) per liter with nitrate-free filtered effluent water from the Aalborg West activated-sludge plant. The biomass was well homogenized to make sure that no aggregate was present. For each experiment, 3.0-ml portions of the diluted biofilm sample were immediately transferred into 9-ml serum bottles. The serum bottles were sealed with gas-tight butyl rubber stoppers. The aerobic cultures were incubated with 2 mM unlabeled substrates and 20 µCi of radioactively labeled substrates per 3-ml culture for 5 h on a rotary shaking platform at 200 rpm to maintain oxic conditions. The anaerobic cultures were flushed and evacuated with oxygen-free N2 gas to remove the oxygen and then preincubated for 3 h with the same nonradioactive substrates that would be used in the incubation to ensure that oxygen was completely depleted. Subsequently, the samples were supplemented with sterile radioactively labeled substrates (final radioactivity, 20 µCi per 3-ml culture) and 2 mM unlabeled substrates and were incubated for 5 h at room temperature. For anaerobic incubations, radioactive substrates were injected anaerobically with sterile syringes through the rubber stoppers into the serum bottles. The following radioactively labeled organic and inorganic substrates were used: (i) sodium [3H]acetate (specific activity, 100 mCi mmol–1), (ii) [14C]propionic acid (56 mCi mmol–1), (iii) [14C]formic acid (56 mCi mmol–1), and (iv) sodium [14C]bicarbonate (56 mCi mmol–1). These radioactive chemicals were obtained from Amersham (Little Chalfonut, United Kingdom), NEN Life Science Products (Boston, Mass.), and Sigma (Deisenhofen, Germany), respectively.

Control experiments.
Two controls were prepared from the homogenized biofilm samples by treatment with 2 mM sodium molybdate to inhibit sulfate reduction and by pasteurizing cells at 80°C for 10 min. Duplicate incubations were performed with [3H]acetate or [14C]propionate as a carbon source and sulfate as an electron acceptor.

Liquid scintillation counting.
The uptake of radioactively labeled substrates was monitored for all cultures, including two controls, before the MAR-FISH procedure. The 3H and 14C contents in the biomass were directly measured. A 0.2-ml aliquot was withdrawn from each culture, centrifuged at 14,000 x g for 8 min, and washed three times with tap water. The harvested biomass was transferred to 2 ml of scintillation liquid (Ultima Gold XR; Packard Instrument Co., Meriden, Conn.). After the biomass was thoroughly mixed and stored in the dark at room temperature for 24 h, the radioactivity was measured with a Packard model 1600 TR liquid scintillation analyzer as recommended by the manufacturer. We calculated the percentage of radioactive substrate incorporated into the biomass for each radioactively labeled substrate.

Sample fixation and washing.
The incubations were terminated by adding 3 ml of 8% paraformaldehyde to the vials, giving the final concentration of 4% paraformaldehyde. The samples were fixed for 3 h at 4°C and centrifuged at 14,000 x g for 8 min. Then, the pellets were washed three times with tap water to remove excess radioactive substrate, gently mixed with a vortex mixer, spotted on gelatin-coated cover glasses (24 by 60 mm), and air dried. Subsequently, the prepared samples were subjected to FISH.

In situ hybridization.
Dehydration and in situ hybridization were performed according to the procedure described by Amann (2). The following previously published oligonucleotide probes were used: EUB338 (3), SRB385 (3), SRB385Db (27), and eight genus-specific probes (129 [9], 221 [9], DNMA657 [10], DSS658 [20], 660 [9], DSV698 [20], SRB814 [9], and DSB985 [20]). The oligonucleotide probes were synthesized and labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) or with the sulfoindocyanine dye Cy3 (Interactiva, Ulm, Germany). In this study, the sum of SRB detected by the probes SRB385 and SRB385Db was regarded as the total SRB population. Since SRB385 and some other genus-specific probes were not as specific as originally described (28), the genus-specific probes 660 and DSV698 were used simultaneously with the family-specific SRB385, and the genus-specific probes 129, 221, DNMA657, DSS658, SRB814, and DSB985 were used simultaneously with the family-specific SRB385Db. Only dual-hybridized genus-specific SRB cells were counted as positive SRB cells. However, only very few positive bacterial cells were detected when the probes 129, 221, SRB814, DSS658, and DSB985 were used, and their fluorescence intensities were low. Therefore, the 129, 221, SRB814, DSS658, and DSB985 probes were not used for further investigations. The probe specificity and typical carbon utilization characteristics of the target groups of SRB are summarized in Table 1. After in situ hybridization, some samples were stained with 4',6'-diamidino-2-phenylindole (DAPI) to enumerate total cell numbers by the direct-counting method of Hobbie et al. (12).


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  		TABLE 1. Specificities of 16S rRNA-targeted oligonucleotide probes used in this study and typical characteristics of electron donor utilization by target groups of sulfate-reducing bacteria

 
Autoradiographic developing procedure.
After FISH, the autoradiographic procedure was performed directly on the cover glasses by using liquid film emulsion (LM1; Amersham Pharmacia Biotech) as previously described by Andreasen and Nielsen (4) and Lee et al. (18). The optimum exposure time was adjusted to 2 to 6 days, depending on the radioactive substrate.

Microscopy and enumeration by MAR-FISH.
A model LSM510 confocal laser scanning microscope Carl Zeiss, Oberkochen, Germany) equipped with a UV laser (351 and 364 nm), an Ar ion laser (450 to 514 nm), and two HeNe lasers (543 and 633 nm) was used. All image combining, processing, and analysis were performed with the standard software package provided by Zeiss. Processed images were printed out by using the software package Photoshop version 3.0J (Adobe Systems Inc., Mountain View, Calif.).

Three covers glasses were prepared for each radioactive substrate, each electron acceptor, and each oligonucleotide probe. For each cover glass, we directly counted probe-hybridized MAR-positive and MAR-negative cells within a scan frame by switching the microscopic mode between the fluorescence and transmission modes. Since the percentage of SRB populations among the total cells was below 5%, the counting of the target SRB cells was continued until more than 100 cells were counted for each cover glass. For enumeration of eubacteria hybridized with probe EUB338, 15 microscopic fields, in which about 100 cells were present, were randomly chosen, and direct cell counting was performed. Cells covered with two or more silver grains were defined as MAR positive in this study.

DNA extraction and PCR amplification.
An aliquot of the homogenized biofilm sample (0.2 ml) was subjected to DNA extraction. DNA was extracted using a Fast DNA spin kit (BIO101), as described in the manufacturer’s instructions. The 16S rRNA genes (ribosomal DNA [rDNA]) from mixed bacterial DNAs were amplified by PCR with the primer sets DSV230f and DSV838r for Desulfovibrio and DBB121f and DBB1237r for Desulfobulbus, as described by Daly et al. (7). To minimize nonspecific annealing of the primers to nontarget DNA, a hot-start PCR program was used for all amplifications. The PCR products were evaluated on a 1% (wt/vol) agarose gel.

Cloning and sequencing of 16S rDNA and phylogenetic analysis.
One microliter of the PCR-amplified bacterial 16S rDNA was directly ligated into the pGEM-T vector cloning system (Promega) and transformed into competent cells (high-efficiency Escherichia coli JM109 [Promega]) as described in the manufacturer’s instructions. Plasmids were extracted and purified from clones with the Wizard Plus Minipreps DNA purification system (Promega) in accordance with the manufacturer’s instructions. Partial sequencing (465 bases for the Desulfovibrio clone library and 1,117 bases for the Desulfobulbus clone library) of the 16S rDNA inserts was performed with an automatic sequencer (Prism 310 Genetic Analyzer; Applied Biosystems). All sequences were checked for chimeric artifacts by the CHECK_CHIMERA program in the Ribosomal Database Project (19) and compared with similar sequences of the reference organisms by BLAST search (1). Sequence data were aligned with the CLUSTAL W package (36). Phylogenetic trees were constructed by the neighbor-joining method (32). Bootstrap resampling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.

Nucleotide sequence accession numbers.
The GenBank-EMBL-DDBJ accession numbers for the 16S rDNA sequences of clones obtained in this study are AB062616, AB062617, and AB062618.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SRB community analysis by in situ hybridization.
The relative abundances of members of the domain Bacteria detected with probe EUB338 and of SRB detected with family- or genus-specific probes in the sewer biofilm were determined by FISH (Fig. 1). The total DAPI count was 6.7 x 1010 cells (cm of biofilm–3) or 3.0 x 1010 cells (g of VSS–1). The abundance of bacteria detected with probe EUB338 was 93.5% ± 3.4% of the total DAPI-stained cells. The number of SRB detected with probes SRB385 and SRB385Db (defined as total SRB) accounted for only 4.8% of the total DAPI-stained cells.



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  		FIG. 1. SRB community structure identified by family- and genus-specific 16S rRNA probes.

 
Control experiments.
Addition of 2 mM sodium molybdate almost completely inhibited uptake of all radioactively labeled substrates with sulfate as an electron acceptor by SRB cells detected with SRB385, SRB385Db, and other genus-specific probes (Fig. 2 and 3). Consequently, no silver grain-coated SRB cells were observed (Fig. 4A), whereas other bacteria detected with probe EUB338 were covered with silver grains. On the contrary, significant accumulation of silver grains was observed around SRB cells without the addition of molybdate (Fig. 4B). This result confirms that SRB hybridized with probe SRB385 did not take up radioactively labeled substrates by using other possible electron acceptors, such as Fe(III) and elemental sulfur, under anoxic conditions and that uptake of radioactively labeled substrates was truly due to sulfate reduction.



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  		FIG. 2. Substrate uptake patterns with oxygen, nitrate, and sulfate as electron acceptors by different phylogenetic SRB groups identified by family- or genus-specific probes. The fractions of probe-hybridized cells that simultaneously took up different radiolabeled substrates (MAR-positive cells) are shown as percentages of the total SRB counts (the sum of SRB detected with probes SRB385 and SRB385Db). Only [3H]acetate and [14C]propionate were used when oxygen and nitrate were the electron acceptors. The error bars indicate standard errors.

 


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  		FIG. 3. Substrate uptake patterns with oxygen, nitrate, and sulfate as electron acceptors by different phylogenetic SRB groups identified by family- or genus-specific probes. The fractions of probe-hybridized cells that simultaneously took up different radiolabeled substrates (MAR-positive cells) are shown as percentages of each family- and genus-specific probe-hybridized cell count. Only [3H]acetate and [14C]propionate were used when oxygen and nitrate were the electron acceptors. The error bars indicate standard errors. N.D., not determined.

 


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  		FIG. 4. Combined MAR-FISH images of homogenized sewer biofilm samples. (A) After 5-h incubation with [14C]propionate and sulfate as electron acceptors with 2 mM sodium molybdate and in situ hybridization with Cy3-labeled probe SRB385 (red) and FLUOS-labeled probe EUB338 (green); the yellow cells are SRB, and the green cells are other bacteria. (B) After 5-h incubation with [14C]propionate and sulfate without molybdate and in situ hybridization with the same probes as for panel A. (C) After 5-h incubation with [3H]acetate and nitrate and in situ hybridization with Cy3-labeled probe SRB385 (red) and FLUOS-labeled probe EUB338 (green). (D) After 5-h incubation with [3H]acetate and nitrate and in situ hybridization with Cy3-labeled probe 660 (red) and FLUOS-labeled probe SRB385 (green). (E) After 5-h incubation with [14C]propionate and oxygen and in situ hybridization with Cy3-labeled probe SRB385 (red) and FLUOS-labeled probe EUB338 (green). (F) After 5-h incubation with [14C]bicarbonate plus H2 with sulfate and in situ hybridization with Cy3-labeled probe SRB385 (red) and FLUOS-labeled probe EUB338 (green). Bars, 10 µm.

 
Furthermore, adsorption of radioactively labeled substrates onto bacterial cells was determined by liquid scintillation counting. Pasteurized cells contained only less than 0.04% of all added radioactively labeled substrates after 5 h of incubation under three different electron acceptor conditions, but it was not sufficient to be detected by MAR (data not shown). In experiments with nonpasteurized cells, significant uptakes (2.0 to 10.0% of the added radioactively labeled substrates) were detected for all organic substrates. This result confirms that radioactively labeled substrates were not adsorbed onto the cell surface. The uptake of [14C]bicarbonate into the biomass in the presence of H2 under three electron acceptor conditions was approximately 0.5%. This was partly due to a lower specific activity of [14C]bicarbonate utilization.

Substrate uptake patterns of different phylogenetic SRB groups under various electron acceptor conditions. (i) General overview.
Under anoxic conditions with sulfate as an electron acceptor, 18 to 34% of the total SRB population took up either [3H]acetate, [14C]propionate, [14C]formate, or [14C]bicarbonate plus H2 (Fig. 2 and Table 2). The acetate-utilizing SRB population was the most abundant (34%) in the SRB community. The SRB were numerically important members of the acetate- and H2-utilizing populations in this biofilm, as shown by the fact that SRB constituted 21 and 42% of bacteria that could utilize [3H]acetate and [14C]bicarbonate in the presence of H2, respectively (Table 2). Desulfobulbus hybridized with probe 660 showed a high activity for all organic substrates tested, especially [14C]propionate (Fig. 2).


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  		TABLE 2. Summary of uptake patterns of radioactively labeled substrates ([3H]acetate, [14C]propionate, [14C]formate, and [14C]bicarbonate plus H2) by SRB and bacteria within a sewer biofilm consortium

 
Under anoxic conditions with nitrate as an electron acceptor, 17 and 7% of the total SRB took up [3H]acetate and [14C]propionate, respectively (Table 2). Examples of MAR-FISH images taken under this condition are shown in Fig. 4C and D. Desulfobulbus hybridized with probe 660 was the most active and dominant phylogenetic SRB subgroup for the uptake of both [3H]acetate and [14C]propionate (Fig. 2).

Under oxic conditions, a small but significant fraction of the total SRB (mainly SRB cells hybridized with probe SRB385 but not with probe 660 and Desulfobulbus hybridized with probe 660) clearly showed a positive uptake of [3H]acetate and [14C]propionate (Fig. 2 and 4E).

(ii) Probe 660.
More than 90% of Desulfobulbus isolates hybridized with probe 660 took up [14C]propionate with sulfate as an electron acceptor (Fig. 3), demonstrating the high substrate specificity of Desulfobulbus for propionate. More than 20% of Desulfobulbus cells could also take up [14C]formate and [14C]bicarbonate plus H2. Interestingly, approximately 9 and 27% of Desulfobulbus cells clearly indicated a positive uptake of [14C]propionate with oxygen and nitrate, respectively, as an electron acceptor. Furthermore, it was noted that more than 40% of Desulfobulbus cells could incorporate [3H]acetate into cells under anoxic conditions (Fig. 3).

(iii) Probe DSV698.
About one-third of SRB cells (mainly Desulfovibrio) hybridized with probe DSV698 showed a positive uptake of [14C]bicarbonate in the presence of H2 with sulfate as an electron acceptor (Fig. 3). This group of SRB could also take up [14C]formate and [3H]acetate with sulfate as an electron acceptor but did not show a detectable uptake of [14C]propionate under any electron acceptor conditions tested.

(iv) Probe DNMA657.
Desulfonema hybridized with probe DNMA657 could utilize [3H]acetate with sulfate and nitrate as electron acceptors (Fig. 3). Desulfonema did not show significant positive uptake of the other substrates tested.

16S rDNA clone analysis.
Two 16S rDNA clone libraries were constructed from the sewer biofilm sample to further identify the Desulfovibrio spp. hybridized with probe DSV698 and the Desulfobulbus spp. hybridized with probe 660. Thirty-one and 15 clones were randomly selected from the Desulfovibrio and Desulfobulbus clone libraries, respectively. Phylogenetic-tree analysis was performed to affiliate the clone sequences to the previously identified SRB (Fig. 5). Among the clones analyzed, two phylogenetically distinct sequences were found in the Desulfovibrio library. Twenty-three clone sequences (represented by DSV08) were closely related to Desulfovibrio desulfuricans MB (96% similarity). Eight clone sequences (represented by DSV16) were affiliated with the Desulfomicrobium cluster. Desulfomicrobium hypogeium was the organism closest to the clone DSV16 (98% similarity). On the other hand, all 15 16S rDNA clone sequences from the Desulfobulbus library were closely related to the sulfate-reducing bacterium R-PropA1 (99% similarity). The clone DBB18 showed less than 95% similarity to the previously identified Desulfobulbus spp.



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  		FIG. 5. Phylogenetic-distance trees of members of the family Desulfovibrionaceae (A) and the family Desulfobacteriaceae (B) based on the comparative analysis of partial sequences of 16S rRNA genes retrieved from a sewer biofilm. The trees were calculated by the neighbor-joining algorithm and a 50% conservation filter. The numbers in parentheses indicate the frequency of appearance of completely identical sequences (clones) among the total clones. E. coli is used as the outgroup. The scale bar denotes 2% sequence divergence, and the numbers at the nodes represent bootstrap values (1,000 replicates).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MAR-FISH approach and incubation conditions.
The combined MAR-FISH technique applied in this study was very suitable for simultaneous and quantitative determination of the phylogenetic identification and substrate uptake patterns of SRB inhabiting a complex sewer biofilm consortium on a single-cell level. This was mainly due to the very clear inhibiting effect of the specific inhibitor (i.e., molybdate), which verified that only SRB were detected by most of the 16S rRNA probes applied (Fig. 2 and 4A). Similar specific inhibitors exist for only a few other distinct phylogenetic groups, e.g., nitrifiers.

In order to avoid counting SRB cells that accidentally coincided with background silver grains as MAR-positive cells, a minimum of two silver grains was considered necessary. The probability that SRB cells accidentally coincided with two background silver grains could be evaluated by counting SRB cells that coincided with two or more silver grains (MAR-positive cells) in the control experiments with the addition of molybdate. Only less than 1.0% of the total SRB were associated with two background silver grains for all radioactively labeled substrates, as shown in Fig. 2. Thus, a minimum of two silver grains was sufficient to distinguish between MAR-positive and MAR-negative SRB cells under incubation conditions with the concentration of radioactively labeled substrates applied in this study. The optimization of radiolabeled substrate concentrations and incubation time is very important, especially for detecting active SRB cells under oxic conditions, because aerobic heterotrophs were much more active and thus produced many silver grains. However, the aerobic incubation conditions used in this study (5 h without preincubation) with the applied tracer concentrations allowed MAR-positive SRB cells to produce enough silver grains to be distinguished from MAR-negative cells and the background, as shown in Fig. 4E.

A small but significant number of SRB showed uptake of [3H]acetate and [14C]propionate under oxic conditions. The oxygen concentrations during the aerobic incubation and the presence of microcolonies are critical points to discuss. We did not observe any visible microbial aggregates in the culture media or in the MAR-FISH images, as shown in Fig. 4. The medium was also rigorously shaken and was incubated for only 5 h. It is therefore unlikely that bulk oxygen was reduced or depleted or that anoxic zones developed in microbial aggregates during this period. Furthermore, since the nitrate concentrations in aerobic cultures were under the detection limit, it is unlikely that uptake of radioactively labeled substrates was coupled with nitrate or sulfate respiration.

Specific SRB activity.
SRB were present in high numbers (ca. 3 x 109 cells per cm3 of biofilm) in the sewer biofilm studied, and the relative abundance of total SRB detected with probes SRB385 and SRB385Db was about 4.8% of the total DAPI-stained cells. These values are in the range of the SRB densities in wastewater biofilms grown under oxic conditions (24, 29, 33). However, Desulfobulbus hybridized with probe 660 and Desulfovibrio hybridized with probe DSV698 together made up less than 50% of the SRB population (mainly the family Desulfovibrionaceae) hybridized with probe SRB 385. Thus, it is likely that many other unidentified species which could not be detected with the probes used in this study exist within the currently identified phylogenetic SRB groups.

Sulfate reduction took place in the sewer biofilm with a specific sulfide production rate of 1.3 x 10–6 mol of H2S g of VSS–1 h–1 when 2 mM acetate was used as the sole carbon and energy source. The total SRB cell density of this biofilm sample was approximately 3.0 x 1010 cells g of VSS–1. About 34% of the total SRB could utilize acetate with sulfate as an electron acceptor (Table 2). Based on these data, we can estimate the average sulfide production rate per cell to be 1.3 x 10–16 mol of H2S cell–1 h–1. This rate is in the lower range of the previously reported specific sulfate reduction rates of pure cultures on acetate: 4.2 x 10–16 to 4.8 x 10–15 mol of SO42– cell–1 h–1 (38). The relatively low rate observed could be due to a certain underestimation of the sulfide production rate because only acid volatile sulfide was measured.

Dominant phylogenetic SRB subgroups.
Desulfobulbus hybridized with probe 660 and Desulfovibrio hybridized with probe DSV698 were numerically important SRB subgroups in this sewer biofilm and may play an important role in carbon metabolism. The sewer biofilm studied was grown under mainly dynamic oxic conditions with high organic input, so the presence of oxygen-tolerant SRB species would be expected. In previous studies, Desulfobulbus and Desulfovibrio were known to be oxygen tolerant and were found to be the numerically important SRB members in wastewater biofilms grown under oxic conditions (24, 33), in the oxic layers of a stratified fjord (35), and in oxic regions of microbial mats (15). In this study, 9 and 27% of Desulfobulbus cells hybridized with probe 660 could utilize [14C]propionate with oxygen and nitrate, respectively, as an electron acceptor (Fig. 3). This result supports the previously described ability of the versatile metabolism of Desulfobulbus to utilize propionate with SO42–, NO3–, or even O2 as an electron acceptor (8, 39) and might explain the higher abundance or survival of Desulfobulbus in various oxic and anoxic environments.

More than 90% of Desulfobulbus cells could utilize [14C]propionate with sulfate as an electron acceptor, whereas only about 20 to 46% of them utilized [14C]formate, [14C]bicarbonate plus H2, and [14C]acetate in this study. For acetate utilization, Desulfobulbus might have grown on other substrates (e.g., propionate or H2) produced in the biofilm suspension and incorporated acetate as a carbon source. Different uptake and degradation kinetics for different substrates probably contributed to the difference in utilization patterns determined by MAR-FISH. A comparative 16S rDNA sequence analysis revealed that all clones retrieved from the sewer biofilm were closely related to the previously identified Desulfobulbus propionicus, with less than 95% similarity. D. propionicus cannot utilize formate (31, 39). The data from MAR-FISH (i.e., positive utilization of propionate, formate, and H2) and the comparative 16S rDNA sequence analysis suggest that Desulfobulbus detected with probe 660 in this study might be an as-yet-unrecognized Desulfobulbus species. Rooney-Varga et al. (31) have also suggested that the genus Desulfobulbus must be considered affiliated with a new family due to the phylogenetic width and physiological traits of this group and that as-yet-undescribed species are present in significant numbers.

An SRB group detected with probe SRB385Db (mainly the family Desulfobacteriaceae excluding Desulfobulbus) was primarily involved in acetate oxidation in this biofilm. Our MAR-FISH results revealed that some of the SRB in this group could utilize [14C]acetate with oxygen and nitrate as electron acceptors (Fig. 3), coinciding with the previous findings that some SRB species (e.g., Desulfobacter postgatei) belonging to the family Desulfobacteriaceae are able to oxidize H2 and organic compounds, including acetate, with O2 and NO3– as terminal electron acceptors (8). The ability of this SRB group to utilize acetate under oxic and anoxic conditions might explain its high abundance in oxic zones or near the chemocline in various natural environments (22, 30, 35).

H2-utilizing SRB populations.
SRB constituted 42% of bacteria that took up [14C]bicarbonate in the presence of H2 with sulfate as an electron acceptor (Table 2). Desulfobulbus was one of the main H2-utilizing SRB groups in this biofilm. There were at least two other H2-utilizing SRB groups. One group was Desulfovibrio detected with probes SRB385 and DSV698. The other group was the SRB population that hybridized with probe SRB385 but not with probes DSV698 and 660 (Fig. 2). Based on the comparative 16S rRNA gene analysis, we found two phylogenetically distinct SRB populations (DSV08 and DSV16) closely related to D. desulfuricans MB (96% similarity) and to D. hypogeium (98% similarity), respectively (Fig. 5). Both species are well-known H2-utilizing SRB (16). The sequences of clone DSV08 and clone DSV16 contained zero and one mismatch with probe DSV698, respectively. The SRB cells represented by the clone DSV16 sequence could not be detected with probe DSV698 under the hybridization conditions applied in this study. Based on these 16S rDNA analysis data, it is speculated that these SRB species, closely related to the D. hypogeium and the D. desulfuricans MB lineages, are potential candidates for important H2-utilizing SRB in this biofilm.


   ACKNOWLEDGMENTS
 
This research has been supported by CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST); by a Grant-in-Aid (no. 13650593) for Developmental Scientific Research from the Ministry of Education, Science and Culture of Japan; and by the Danish Technical Research Council within the framework program Activity and Diversity in Complex Microbial Systems.

Marianne Stevenson is acknowledged for technical assistance for MAR-FISH analysis.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, North-13, West-8, Kita-ku, Sapporo 060-8628, Japan. Phone: 81-(0) 11-706-6266. Fax: 81-(0) 11-707-6585. E-mail: sokabe{at}eng.hokudai.ac.jp. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, January 2002, p. 356-364, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.356-364.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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