INTRODUCTION

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Wastewater biofilms are very complex multispecies biofilms, displaying considerable heterogeneity with respect to both the microorganisms present and their physicochemical microenvironments. Moreover, multiple electron donors and electron acceptors are present in the wastewaters. Therefore, successive vertical zonations of predominant respiratory processes occurring simultaneously in close proximity have been found in aerobic wastewater biofilms with a typical thickness of only a few millimeters (10, 22, 40, 42). In these studies, sulfate reduction was found in the deeper anaerobic biofilm strata, even though the bulk liquid was oxygenated. Accordingly, reoxidation of the produced sulfide with oxygen and/or nitrate was found in a stratum close to the sulfate reduction zone, depending on the oxygen and nitrate penetration depths.

A major drawback of sulfate reduction in wastewater treatments is the production of toxic H₂S, which is also a possible precursor of odorants and significantly enhances microbially mediated corrosion of treatment facilities (23, 24, 31, 37). Furthermore, sulfate reduction accounts for up to 50% of the mineralization of organic matter in aerobic wastewater treatment systems (22). Once sulfate reduction occurs in biofilms, internal sulfide reoxidation is expected to account for a substantial part of oxygen consumption (approximately up to 70%) (22, 32, 42). Therefore, the in situ detection of populations of sulfate-reducing bacteria (SRB) and their activity in wastewater biofilms is of great practical and scientific relevance. However, such studies have been hindered due to lack of analytical tools and the complexity of the internal sulfur cycle in aerobic biofilms. Since mass balance of sulfide or sulfate flux across a biofilm-liquid interface cannot describe sulfur transformations within the biofilm, the sulfur cycle in wastewater biofilm systems is not well known presently.

Therefore, we must explore analytical tools to overcome this problem. Microelectrode measurements are the most reliable way of studying several metabolic processes with high spatial and temporal resolution and have been used for studying nitrogen cycles (11, 14, 36, 43, 44) and sulfur cycles (22, 40, 42) in various environmental samples. One advantage of the use of microelectrodes is their ability to detect in situ microbial activities with minimal disturbance. Furthermore, the recent development of the fluorescent in situ hybridization (FISH) technique with oligonucleotide probes has been widely used to study microbial community structures in microbial flocs (44, 47, 48) and biofilms (3, 36, 40, 43). FISH has been successfully combined with microelectrode measurements (36, 40, 43, 44). However, so far, studies relating in situ spatial distribution of SRB populations to their activity in wastewater biofilms are scarce.

In the present study, we combined three techniques to determine the vertical distribution of SRB populations, substrate profiles, and distributions of sulfur pools (i.e., S⁰, FeS, and FeS₂) within aerobic wastewater biofilms. Firstly, the vertical distributions of SRB populations were investigated by FISH with the previously published phylogenetic probes in combination with confocal scanning laser microscopy (CSLM). This was done by counting positively probe-stained cells in vertical transects across biofilm sections. Secondly, the spatial distributions of in situ activities of sulfate reduction and sulfide oxidation were measured by means of several microelectrodes. The resulting picture was cross-evaluated with reference to one-dimensional vertical distributions of most-probable-number (MPN) counts of SRB populations and potential sulfate reduction rates (SRRs) and sulfide oxidation rates (SORs) in the biofilm, which were measured by slicing the biofilm parallel to the substratum without any pretreatment by the Microslicer (model DTK-1000; Dosaka EM Co., Ltd., Kyoto, Japan). Finally, a complementary analysis of sulfur compound (i.e., S⁰, FeS, and FeS₂) distributions was performed to evaluate the importance and contribution of an internal iron-sulfur cycle in the overall sulfur cycle. The combination of these three techniques provides more comprehensive information on a complex sulfur cycle occurring in the aerobic wastewater biofilms.

	MATERIALS AND METHODS

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Biofilm samples. Aerobic mixed-population biofilms were grown in fully submerged rotating disk reactors (RDR) consisting of 10 polymethyl-methacrylate disks. Eight removable slides (1 by 6 cm) were installed in each disk for sampling biofilms. The reactor volume was 5,600 cm³, and the total biofilm area was 4,020 cm². Disk rotational speed for 16-cm-diameter disks was fixed at 14 rpm (which gives a peripheral speed of ca. 14 cm s¹). The dilution rate in the reactors was kept at 0.2 h¹. The average dissolved organic carbon (DOC) loading rate was 11.2 g of DOC (m² of biofilm)¹ h¹. The primary settling tank effluent from the Soseigawa municipal wastewater treatment plant in Sapporo, Japan, consisting of stage 1 (215,000 population equivalents [PE] [1 PE = 60 g of biological oxygen demand day¹]) and stage 2 (210,000 PE) was fed into the reactor. To facilitate sulfide denitrification, the influent was supplemented with KNO₃ solution to give a final concentration of 350 µM NO₃. Since the bulk water was not aerated, the average dissolved oxygen (DO) concentration in the bulk water was 40 ± 30 µM during the experiment.

Vertical distributions of MPN counts and activity profiles. The biofilms collected from the RDR were sliced into 50- to 100-µm-thick sections parallel to the substratum without any pretreatment by means of the Microslicer as described previously (34) and then apportioned into samples representing four to six layers. The apportioned samples were homogenized and subjected to the enumeration of MPN counts and the measurement of potential SRRs and SORs.

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Measurement of reduced sulfur compounds. Elemental sulfur (S⁰), AVS (H₂S and FeS), and chromium-reducible sulfide (CRS; FeS₂) in biofilms were determined by the method described originally by Fossing and Jorgensen (18) and modified by Nielsen et al. (31). The biofilm samples were immediately fixed in 1% ZnAC solution, sliced into 50- to 100-µm-thick sections parallel to the substratum in 1% ZnAC solution without any prefixation by means of the Microslicer, and then apportioned into samples representing four to six layers. The apportioned biofilm samples were sequentially analyzed for elemental sulfur (S⁰), AVS (H₂S and FeS), and CRS (FeS₂). Elemental sulfur was extracted with 96% ethanol for 24 h at room temperature prior to AVS and CRS measurements. Samples extracted in ethanol were analyzed by a high-performance liquid chromatograph (HPLC) with a UV detector at 254 nm. A reversed-phase column (Partisil 5; octyldecyl silane 3; Whatman) was used with 100% high-performance liquid chromatography-grade methanol as eluent and a flow rate of 0.4 ml min¹. The sample injection volume was 100 µl. The remaining samples from ethanol extraction were suspended in 10 ml of 1% ZnAC solution, and AVS (H₂S and FeS) was volatilized by addition of 10 ml of 2 N HCl. The volatilized H₂S was trapped in 1% ZnAC solution (variable volumes) and measured colorimetrically by the methylene blue method (7). After the AVS distillation, 2.5 ml of 1 M Cr²⁺ in 0.5 N HCl solution was added directly to the remaining sample suspension from S⁰ and AVS analyses. CRS was volatilized, and H₂S was measured as described above. Recovery of FeS and FeS₂ was determined to be (87 ± 7)% (n = 3) and (73 ± 24)% (n = 3), respectively.

Measurements of total Fe and total Mn contents in the biofilm. Biofilm samples were embedded in Tissue-Tek OCT compound (Miles, Elkhart, Ind.) and rapidly frozen at 20°C. Horizontal sections (40 µm thick) of the frozen biofilm (about 1 by 1 cm) were obtained parallel to the substratum by use of a cryostat (Reichert-Jung Cryocut 1800; Leica) and then apportioned into samples representing four to six layers. Each of the apportioned samples was transferred into an acid-cleaned test tube containing 6.5 N HNO₃ (5 ml) and ultrasonicated for 10 min. Total Fe and Mn concentrations were determined by the polarized Zeeman atomic adsorption spectrophotometer (model Z-5700; Hitachi, Tokyo, Japan) after appropriate dilutions of the samples were made.

Microelectrodes. Concentration profiles of O₂, NO₂, NO₃, pH, and H₂S in the biofilms were measured by microelectrodes manufactured in our laboratory. Cathode-type oxygen microelectrodes with a tip diameter of about 15 µm were prepared and calibrated as described previously by Revsbech and Jorgensen (41). Liquid ion-exchanging membrane microsensors for NH₄⁺, NO₂, and NO₃ were prepared as described before (10, 11, 13) and calibrated in a dilution series (10³ to 10⁶ M) of NH₄⁺, NO₂, and NO₃ in the medium used for the measurements. pH electrodes with tip diameters of about 5 to 10 µm were constructed according to the procedure of deBeer and van den Heuvel (12). The pH microelectrode was calibrated in the medium with an adjusted pH in the range of 4 to 9. The sulfide electrodes were manufactured as described by Revsbech and Jorgensen (41) and were calibrated as described by Kuhl and Jorgensen (22). The total sulfide concentration (H₂S, HS, and S²) in a dilution series was determined by the methylene blue method (7). The total amount of dissolved H₂S, HS, and S² is designated H₂S or total sulfide in the rest of the paper. The 90% response times were approximately 1 to 5 min, depending on sulfide concentration. Since the pH profiles showed a significant variation (>0.1 pH unit) throughout the biofilm, pH correction of the measured sulfide profiles was necessary. The total sulfide concentration in an aqueous solution can be calculated as described by Kuhl and Jorgensen (22). We used the following dissociation constants for sulfide, pK₁ = 7.05 and pK₂ = 17.1 (29).

Microelectrode measurements. Biofilms were taken from the reactors and incubated in the synthetic medium for about 3 h before measurements were made, which ensured steady-state concentration profiles. The medium contained the following (in micromolar concentrations): NaNO₃ (270), NaNO₂ (100), MgSO₄ · 7H₂O (300), sodium propionate (600), NH₄Cl (600), Na₂HPO₄ (570), MgCl₂ · 6H₂O (84), CaCl₂ (200), and EDTA · 2Na (270). All measurements were performed in a water chamber containing 1.8 liters of the synthetic medium at 20°C. Each microelectrode was separately mounted on a motor-driven micromanipulator (model ACV-104-HP; Chuo Precision Industrial Co., Ltd., Tokyo, Japan). The electrode assembly was placed inside a Faraday cage to reduce electrical noise. Each measurement was performed three to five times by advancing the electrodes at depth steps of 50 to 100 µm through the biofilm. Average liquid flow velocity (2 to 3 cm s¹) above the biofilm was provided by a Pasteur pipette blowing a mixture of air and N₂ gas onto the water surface. The bulk DO concentration was kept the same as the one in the RDR. The biofilm-liquid interface was determined by using a dissection microscope (model Stemi 2000; Carl Zeiss).

Estimations of specific reaction rates and substrate flux. Net specific consumption and production rates (R; micromoles centimeter³ hour¹) were estimated from the measured microprofiles by using Fick's second law of diffusion. The details of this method have been described previously by Lorenzen et al. (26). Furthermore, the total diffusion fluxes (J; micromoles centimeter² hour¹) through the biofilm-liquid interface were calculated by using Fick's first law, J = D_s (dS/dz), where D_s is the molecular diffusion coefficient of compound S in the biofilm and dS/dz is the concentration gradient in the boundary layer at the biofilm-liquid interface, which is determined from microprofiles. We used the molecular diffusion coefficients of 2.09 × 10⁵ cm² s¹ for oxygen (4), 1.23 × 10⁵ cm² s¹ for NO₃ (4), and 1.39 × 10⁵ cm² s¹ for sulfide (22) at 20°C.

Oligonucleotide probes. In situ hybridization of biofilm sections was performed with the following 16S rRNA-targeted oligonucleotide probes: (i) EUB338 (1), (ii) SRB385 (3, 39), (iii) SRB385Db (38), and (iv) four group-specific probes (SRB687, SRB660, SRB129, and SRB221) (15). All probe sequences, their specificities, hybridization conditions, and references are given in Table 1. All probes were synthesized and labeled with tetramethylrhodamine-5- isothiocyanate (TRITC) at the 5' end by TaKaRa Shuzou Co., Ltd. (Shiga, Japan) (2).

FISH. Biofilm samples were fixed in 4% paraformaldehyde solution (2) immediately after the microelectrode measurements and embedded in Tissue-Tek OCT compound. Horizontal and vertical (cross section) thin sections (20 µm thick) of the fixed biofilm were prepared as described by Ramsing et al. (40). The previously published optimal hybridization conditions were used for each probe. All in situ hybridizations were performed according to the procedure described by Amann (1) in 8 µl of hybridization buffer (0.9 M NaCl, 20 mM Tris hydrochloride [pH 7.2], 0.01% sodium dodecyl sulfate; formamide concentrations are shown in Table 1) with 1 µl of probe solution at 46°C for 2 to 3 h in an equilibrated sealed moisture chamber. The final probe concentration was approximately 5 ng µl¹. Subsequently, a stringent washing step was performed at 48°C for 20 min in 50 ml of prewarmed washing solution (NaCl concentration is shown in Table 1; 20 mM Tris hydrochloride [pH 7.2], 0.01% sodium dodecyl sulfate). The stringency of the washing step was adjusted by lowering the sodium chloride concentration to achieve the appropriate specificity. The slides were then rinsed briefly with ddH₂O, allowed to air dry, and mounted in antifading solution (Slow Fade Light; Molecular Probes, Eugene, Oreg.).

Microscopy. An LSM 510 CSLM (Carl Zeiss) equipped with an argon laser (488 nm) and a HeNe laser (543 nm) was used to examine the biofilm specimens. Zeiss filter sets 09 and 15 and ×20, ×40, and ×63 oil-immersion lenses were used. Wastewater biofilm samples generally contain detrital matters and mineral grains, which generate problems with autofluorescence and unspecific staining. These mineral grains and detrital matters generally exhibit a wide range of emission spectra. Thus, although biofilm samples were hybridized with only TRITC-labeled probes, images were recorded by using simultaneous excitation of 488- and 543-nm lasers. By doing this, only TRITC-labeled probe-stained cells appeared red, and other debris and mineral grains appeared yellowish due to dual excitation. In this way, the probe-stained cells could easily be distinguished from other materials. 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 Adobe Photoshop 3.0J (Adobe Systems Incorporated, Mountain View, Calif.).

Vertical distribution of SRB. To quantify the vertical distributions of SRB populations from FISH images, we directly counted positively probe-stained cells along vertical transects through the biofilm (40). In parallel, surface fractions of probe-stained cell area to total biomass area (differential interference contrast [DIC] image) were measured for the horizontal biofilm sections taken from representative depths of the biofilm. Three randomly chosen slices were stained with TRITC-labeled SRB385 probe, three were stained with TRITC-labeled SRB660 probe, and three were used as controls without staining. The nine slides were randomly mixed, and the positive cells along three vertical transects on each slide were counted without knowledge of the probe that had been used to stain the particular slide. The transects were made by counting the cells within a scan frame. The counts were recalculated to absolute cell density from the scan frame area and the scan depth.

	RESULTS

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General biofilm reactor performance. Typical water quality in influent and effluent of the reactor after reaching the steady-state condition is shown in Table 2. The steady state was achieved after about 40 days. Relatively large standard deviations are attributed to fluctuations in the influent water quality. The average DO concentration in the bulk water was low (about 40 ± 30 µM) because of no aeration in the bulk water. Nitrification activity was not observed. Effluent SO₄² and NH₄⁺ concentrations were not statistically different from those of the influent, indicating that sulfur transformation in the reactor could not be seen from mass balance on SO₄². Consumption of nitrate indicated the occurrence of denitrification or reduction of nitrate to ammonium by SRB to a certain extent.

Biofilm architecture. The biofilm reactor had been at steady state for more than 1 month. Biofilm sloughing did not occur during this period. Figure 1 shows a composite cross-section (20-µm-thick) image of a 60-day-old wastewater biofilm (biofilm thickness, approximately 1,100 µm). It is clear that the wastewater biofilms studied have a complex heterogeneous structure consisting of discrete biomass (microbial aggregates) and interstitial voids, which connect the bulk water to the bottom part of the biofilm.

View larger version (126K): [in this window] [in a new window]   FIG. 1.   A cross section made with a cryomicrotome (20 µm thick) of an aerobic domestic wastewater biofilm. The biofilm thickness is approximately 1,000 µm.

In situ detection of SRB. Immediately after the microelectrode measurements (the results are shown below), a series of vertical sections of the biofilm were subjected to in situ hybridization. Firstly, four group-specific probes were used to specify possible numerically predominant species of SRB populations in the biofilm. Only a few positive cells were found when SRB129, SRB687, and SRB221 probes were used with any of the biofilm samples, and their fluorescence intensities were very low. An abundance of SRB660 probe-stained cells was found at all depths, and their fluorescence signals were strong. Based on these findings, Desulfobulbus spp. could be a numerically important member of SRB populations in this biofilm.

View larger version (76K): [in this window] [in a new window]   FIG. 2.   In situ hybridization of vertical sections (20 µm thick) of an aerobic wastewater biofilm with TRITC-labeled SRB660 probe (specific for Desulfobulbus spp.). (A) Composite DIC image of the entire biofilm vertical section (scale bar = 200 µm). The biofilm thickness is about 1,500 µm. (B and C) CSLM projection images of the microscopic fields enclosed by boxes. Images were obtained by using simultaneous excitation with 488- and 543-nm lasers. Weak yellow signals were autofluorescence. (D to G) Close-up views of SRB385 probe-stained cell clusters.

Vertical distribution of SRB population. Figure 3A presents the vertical distributions of SRB385 and SRB660 probe-stained cells in the biofilm. The SRB385 probe-stained cells were present in high numbers (approximately 10 × 10⁹ ± 1.6 × 10⁹ cells cm³) and evenly distributed throughout the biofilm, even in the oxic zone. The vertical distribution of the SRB660 probe-stained cells also revealed that there was no significant difference between the average cell counts in the oxic zones and those in the anoxic zones of the biofilms, approximately 10⁹ cells cm³. They accounted for about 6 to 23% of the SRB385 probe-stained cells. In addition, three slices of the samples at depths of approximately 200 and 900 µm hybridized with the SRB385Db probe. The numbers of SRB385Db probe-stained cells were approximately 0.7 × 10⁹ cells cm³ at depths of 200 and 900 µm, respectively, and were comparable with the numbers of SRB660 probe-stained cells.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Vertical distributions of SRB populations in the biofilm determined as SRB cell density (A) and surface fraction of SRB385 probe-stained cell area to the total biomass area (B). Average cell densities were determined from FISH direct cell counts along vertical transects through the biofilm after hybridization with TRITC-labeled SRB385 probe and SRB660 probe (specific for Desulfobulbus spp.). Error bars indicate the standard deviations of measurements. The biofilm surface is at a depth of 0 µm.

Vertical distributions of MPN counts and potential activity. To verify the high abundance of SRB in the upper part of the biofilm detected by in situ hybridization, the vertical distributions of potential SRR, MPN counts of cultivable SRB populations, ASOR, and ANSOR were simultaneously determined (Fig. 4). Figure 4A shows that the SRR in the oxic layer was approximately 1.1 µmol cm³ h¹ and was as high as the value at the lowest depth. However, the MPN counts decreased exponentially with depth, and the cell counts at the oxic surface layer (approximately 2 × 10⁷ MPN cm³) were 100 times higher than the cell counts found at the deeper part of the biofilm (2 × 10⁵ MPN cm³).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Vertical distributions of potential SRRs (A), MPN counts of SRB (B), and ASORs and ANSORs (C) in the biofilm. The biofilm surface is at a depth of 0 µm.

Distribution of reduced inorganic sulfur compounds in the biofilm. Figure 5A represents the spatial distribution of AVS, CRS, and S⁰ in a 1,200-µm-thick biofilm (a 65-day-old biofilm). Although AVS was not detectable in the surface and in the bottom of the biofilm, about 14 µmol of S (cm³ of AVS)¹ found at about 250 µm from the surface, at which the active H₂S production was detected by microelectrode measurements (see Fig. 6). Elemental sulfur (S⁰) seemed to be the most abundant sulfur pool at all depths of the biofilm. The concentration of S⁰ at about 150 µm from the surface was the highest (approximately 30 µmol of S cm³) and gradually decreased toward the bottom. The CRS concentration was below 10 µmol of S cm³ throughout the biofilm, constituting a relatively small fraction of the total sulfur pools. Since elemental sulfur was never detected in the influent, S⁰ accumulation was certainly due to internal reoxidation of the produced H₂S.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Concentration profiles of reduced inorganic sulfur compounds (S0, AVS, and CRS) (A) and total Fe and total Mn (B) in the biofilm. The biofilm surface is at a depth of 0 µm. Error bars indicate the standard deviations of measurements.

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Microelectrode measurements. Typical steady-state concentration profiles of O₂, H₂S, NO₃, NO₂, and pH in a biofilm incubated in the medium containing 70 µM DO, 270 µM NO₃, 100 µM NO₂, 300 µM SO₄², and 600 µM Na-propionate are shown in Fig. 6. The concentrations of O₂, NH₄⁺, SO₄², and NO₃ were in a range similar to those in the reactor bulk concentrations. Oxygen penetrated only about 100 µm from the surface in a biofilm approximately 1,000 µm thick, whereas NO₃ penetrated further down, to 300 µm. Sulfide was produced in a narrow zone 150 to 300 µm below the surface at a maximum specific rate of 21 µmol of H₂S cm³ h¹. Below the sulfate reduction zone, a constant H₂S concentration (approximately 60 µM) was observed, indicating no net sulfide production. A possible explanation could be a carbon limitation caused by an overall depletion of carbon source in the medium during the more-than-10-h measurement and a high level of competition for the carbon source with denitrifying bacteria in the presence of NO₃. This was indirectly supported by the fact that sulfide production increased with increasing propionate concentrations (data not shown). A narrow sulfide oxidation zone (50 to 150 µm from the surface) was found just above the sulfate reduction zone with a maximum specific H₂S oxidation rate of 20 µmol of H₂S cm³ h¹, giving a total H₂S oxidation rate of 0.20 µmol of H₂S cm² h¹ (specific reaction rates multiplied by the depth of the reaction zone). The specific O₂ respiration rate was 11 to 19 µmol of O₂ cm³ h¹, giving a total consumption rate of 0.24 µmol of O₂ cm² h¹. The specific NO₃ consumption rate was 3 to 19 µmol of NO₃ cm³ h¹, giving a total consumption rate of 0.59 µmol of NO₃ cm² h¹. Since the NO₂ profile indicates that NO₂ was produced in the anoxic biofilm stratum, the production of NO₂ at a rate of 0.16 µmol of NO₂ cm² h¹ was due to the result of nitrate reduction. The NO₂ profiles were measured only to a depth of 150 µm, because below that sulfide induced signal drift.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Steady-state concentration profiles of O2, NH4+, NO2, NO3, H2S, and pH in the aerobic wastewater biofilm incubated in DO-controlled (DO = approximately 70 µM) medium with 600 µM Na-propionate, 550 µM NH4+, 270 µM NO3, 300 µM SO42, and 100 µM NO2 (A) and the spatial distribution of the estimated specific consumption and production rates of O2, NO3, and H2S (B). The points are measured mean values of measurements in triplicate. The solid lines are the best fits from the model to calculate the specific consumption and production rates of O2, NO3, and H2S. The biofilm surface is at a depth of 0 µm. R, rate.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Vertical distributions of SRB determined by in situ hybridization. Aerobic wastewater biofilms displayed considerable structural heterogeneity (Fig. 1). In situ spatial organization of SRB within the biofilm was successfully visualized by FISH in combination with CSLM. We found that Desulfobulbus spp. could be numerically important species and were consistently present in high numbers (approximately 10⁸ to 10⁹ cells cm³) throughout the biofilm, even in the oxic surface. They accounted for about 6 to 23% of the number of SRB385 probe-stained cells (approximately 10⁹ to 10¹⁰ cells cm³). The relatively even distribution of SRB populations throughout the biofilm might indicate that the biofilm was grown under relatively dynamic conditions. The number of SRB obtained from the FISH analysis in this study was about 1 order of magnitude higher than the numbers of SRB in other wastewater biofilm systems (40, 42); accordingly, the SRR was higher with this factor. This is partly because the DO concentration in this study was lower than that in the other systems.

Vertical distributions of MPN counts of SRB and their activity. With FISH analyses, we found 10⁹ to 10¹⁰ SRB385 probe-stained cells per cm³ of biofilm (including pore [void] volumes), numbers which were about 3 to 4 orders of magnitude higher than the numbers of the MPN counts. The cultivation-based enumeration of SRB by MPN apparently used a medium with propionate as sole carbon source. Most sulfate reducers such as Desulfovibrio, Desulfobacter, and Desulfobacterium spp. and so on were not able to grow in this medium. It is thus most likely that the MPN counts reflect only propionate-utilizing SRB species (i.e., Desulfobulbus spp.), which may have led to a severe underestimation of the MPN counts. The results of the MPN counts were also several orders of magnitude (10² to 10⁴) lower than Desulfobulbus counts by FISH probing. Furthermore, the MPN counts decreased exponentially with depth, and the cell counts at the surface were 100 times higher than the cell counts at the base of the biofilm (Fig. 4B). This is quite different from the results of the FISH counts and the potential SRRs, which are relatively constant throughout the biofilm. This discrepancy could be explained by the fact that more Desulfobulbus bacteria were present in the form of densely packed clusters consisting of up to a few hundred cells in the deeper part of the biofilm than in the surface biofilm (Fig. 2), and thus, dispersion of clustered cells was not sufficiently done in the MPN counts.

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Oxygen consumption. An approximate budget of the oxygen consumption was estimated from the vertical distributions of the specific consumption rates of O₂, H₂S, and NO₃ calculated from the microprofiles (Fig. 6B). The H₂S profile overlapped with the O₂ and NO₃ profiles, indicating that the produced H₂S was aerobically and anaerobically oxidized in the biofilm. Sulfide denitrifiers, e.g., Thiobacillus denitrificans, preferentially utilize O₂ over NO₃ as electron acceptor in the presence of O₂ and NO₃. Therefore, we assumed that NO₃ was utilized by sulfide denitrifiers after O₂ was completely depleted in the zone where the H₂S profile overlaps with the O₂ and NO₃ profiles. We also assumed that the main product of both aerobic and anaerobic H₂S oxidation is SO₄². Taking into account the fact that oxidation of 1 mol of H₂S to SO₄² requires 2 mol of O₂ for aerobic oxidation and 4 mol of NO₃ for anaerobic oxidation (i.e., 4NO₃ + H₂S  4NO₂ + SO₄² + 2H⁺), the fraction of O₂ consumption for H₂S oxidation was determined within each measurement step and integrated throughout the reaction zone (Fig. 6B). As a result, a large fraction (up to 76%) of total O₂ consumption was due to the reoxidation of H₂S. Thus, sulfate reduction is as important as aerobic respiration in this biofilm. Based on the total H₂S consumption rate (ca. 0.20 µmol of H₂S cm² h¹) determined from the H₂S profile (at the point of the steepest gradient) and the integrated H₂S oxidation rate with NO₃ (ca. 0.11 µmol of H₂S cm² h¹) determined from the specific consumption rates of O₂, NO₃, and H₂S, approximately 55% of the sulfide produced was anaerobically reoxidized to SO₄². However, if more-reduced sulfur compounds such as S⁰ are formed as the product, the H₂S reoxidation becomes less important. Thus, it should be noted that the calculations indicate the upper limits of SRB contribution.

Sulfide oxidation. To investigate the potential sulfide oxidative pathways, average turnover times of O₂, NO₃, and H₂S in the H₂S-oxidizing zones were calculated as the ratio of the average concentration in the H₂S oxidation zone to the average reaction rate (both determined from microprofiles) as described by Kuhl and Jorgensen (22). These turnover times were extremely short (less than a minute) compared with possible spontaneous chemical reaction of O₂ and H₂S. The timescale of the O₂-H₂S reaction at wastewater temperature has been reported to be in the range of minutes to several hours (6, 17). Thus, the observed aerobic and anaerobic oxidation of H₂S was mediated mainly by microbial reactions and instantaneous reaction with metal ions. However, the latter reaction is less important (see below). Accordingly, SRRs in biofilms can be reliably measured in situ only by microelectrodes. It should be noted that the measured ASOR and ANSOR were about 1 to 2 orders of magnitude higher than the SRRs (Fig. 4C). Therefore, sulfate reduction was probably the rate-limiting step in the series of sulfur transformations in the biofilm.

Sulfur pools in biofilms. So far, measurements of inorganic reduced sulfur compounds (i.e., sulfur pools) in wastewater biofilm systems are scarce. Nielsen et al. (31) have reported that the maximum total sulfur pool in an alternating oxic and anoxic biofilm system attached on the metal coupon was 157 µmol of S cm³, which consisted mainly of AVS (FeS) and CRS (FeS₂). Compared with this figure, the total S pool in the biofilm in the present study was rather small (approximately 23 µmol of S cm³). However, it is important to note that elemental sulfur (S⁰) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms, which accounted for about 75% of the total S pool. S⁰ could be produced by both geochemical and biological H₂S oxidation processes. We speculate that the dominance of S⁰ at the surface biofilm (Fig. 5A) resulted from the high SRR followed by the intensive microbial sulfide reoxidation. The importance of the dominance of S⁰ in the biofilm is that S⁰ disproportionation is thermodynamically as favorable and important a process as sulfide production. High concentrations of S⁰ were found in the zone where the SRR was high according to the microprofiles (Fig. 6). This may suggest that a part of sulfide production is not due to sulfate reduction by SRB. S⁰ is also very corrosive to wastewater treatment facilities and could be reduced to H₂S and/or oxidized to SO₄² by some SRB and other species.

Contribution of an internal Fe-sulfur cycle in the overall sulfur cycle. The H₂S profile showed that H₂S diffused up to the very surface of the biofilm, indicating a relatively high in situ SRR (Fig. 6A). A comparison of the high SRRs and the slow accumulation of total reduced sulfur compounds in the biofilm indicated that intensive reoxidation of H₂S must have taken place. The average in situ SRR determined by the microelectrode measurement (Fig. 6) was approximately 13.0 ± 6.6 µmol of H₂S cm³ h¹. The mean accumulation rate of total reduced sulfur compounds in the biofilm was approximately 0.021 to 0.031 µmol of S cm³ h¹ (33). Thus, only up to 0.3% of the produced H₂S was retained as FeS, FeS₂, and S⁰, which must be regarded as an electron sink of aerobic biofilms. The remaining 99.7% was reoxidized to sulfate in the oxic and/or anoxic zone, indicating that the contribution of an internal Fe-sulfur cycle in the overall sulfur cycle is insignificant. Therefore, the role of AVS can be regarded as an important electron carrier from the deeper anoxic sulfate reduction zone to the oxic-anoxic interface. The degree of reoxidation of the H₂S produced in marine sediments was about 80 to 95% (20, 21), indicating that oxidized iron minerals (i.e., the Fe-S cycle) play a more important role than they do in wastewater biofilm systems. This can be explained by a shorter diffusion distance and a much higher SRR, which is due to the higher influx of organic matter and the higher abundance of SRB populations in wastewater biofilm systems.

Concluding remarks. The results of the combined study of in situ hybridization with the specific phylogenetic probes and microelectrode measurements provided a more detailed picture of the abundance, the spatial distribution, and the activity of SRB populations in the aerobic wastewater biofilm. In addition, a cross-evaluation of the FISH and microelectrode data was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance (approximately 10⁹ to 10¹⁰ cells per cm³) of SRB was present throughout the biofilms, even in the oxic surface layer. Probe SRB660-stained Desulfobulbus was found to be a numerically important member of SRB populations (approximately 10⁸ to 10⁹ cells per cm³). The biogeochemical measurements showed that elemental sulfur (S⁰) was an important intermediate of the sulfide reoxidation in thin wastewater biofilms, which accounted for 75% of the total S pool. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant.