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Applied and Environmental Microbiology, September 2002, p. 4629-4636, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4629-4636.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Abundance and Phylogenetic Affiliation of Iron Reducers in Activated Sludge as Assessed by Fluorescence In Situ Hybridization and Microautoradiography

Jeppe Lund Nielsen,1 Stefan Juretschko,2 Michael Wagner,2 and Per Halkjær Nielsen1*

Department of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark,1 Lehrstuhl für Mikrobiologie der Technischen Universität München, D-85350 Freising, Germany2

Received 8 March 2002/ Accepted 25 June 2002


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ABSTRACT
 
Microautoradiography (MAR) was used to enumerate acetate-consuming bacteria under Fe(III)-reducing conditions in activated sludge. This population is believed to consist of dissimilatory iron-reducing bacteria, because the applied incubation conditions and the use of specific inhibitors excluded consumption of radiolabeled acetate by other physiological groups such as sulfate reducers. By use of this approach, dissimilatory iron reducers were found in a concentration of 1.1 x 108 cells per ml, corresponding to approximately 3% of the total cell count as determined by DAPI (4',6'-diamino-2-phenylindoledihydrochloride-dilactate) staining. The MAR enumeration method was compared to the traditional most-probable-number (MPN) method (FeOOH-MPN) and a modified MPN method, which contains Ferrozine directly within the MPN dilutions to determine the production of small amounts of ferrous iron (Ferrozine-MPN). The Ferrozine-MPN method yielded values 6 to 10 times higher than those obtained by the FeOOH-MPN method. Nevertheless, the MAR approach yielded counts that were 100 to 1,000 times higher than those obtained by the Ferrozine-MPN method. Specific in situ Fe(III) reduction rates per cell (enumerated by the MAR method) were calculated and found to be comparable to the respective rates for pure cultures of dissimilatory iron-reducing bacteria, suggesting that the new MAR method is most reliable. A combination of MAR and fluorescence in situ hybridization was used for phylogenetic characterization of the putative iron-reducing bacteria. All activated-sludge cells able to consume acetate under iron-reducing conditions were targeted by the bacterial oligonucleotide probe EUB338. Around 20% were identified as gamma Proteobacteria, and 10% were assigned to the delta subclass of Proteobacteria.


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INTRODUCTION
 
Iron is universally present in most ecosystems, and the microbial reduction of Fe(III) oxyhydroxides plays a significant role in the biogeochemistry of these systems. Many microorganisms are capable of reducing iron, e.g., fermentative bacteria and some sulfate reducers, but in contrast to dissimilatory iron-reducing bacteria (DIRB), they do not effectively couple the oxidation of organic matter to the Fe(III) reduction (21). DIRB are usually divided into groups depending on their substrate requirements and on whether they can completely oxidize organic matter to CO2. It is thought that Fe(III) reducers capable of oxidizing important fermentation products such as acetate are responsible for the main reduction of ferric iron in many ecosystems (20).

At present, little is known about the identity and abundance of DIRB in different ecosystems. Several DIRB have been isolated from various environments, and it has been shown that they constitute a phylogenetically diverse group of organisms (for a review, see reference 19). Most of the DIRB that have been described are members of the delta subclass of the Proteobacteria that includes the genera Geobacter, Desulfuromonas, and Pelobacter. Other DIRB include Shewanella spp. and Geovibrio ferrireducens (8), which are affiliated with the gamma subclass of the Proteobacteria and the Flexistipes lineage, respectively. Furthermore, several gram-positive DIRB have been recognized (36).

Fe reduction has been demonstrated to occur in activated sludge and might, for example, influence floc structure and the precipitation of phosphorus (27). However, with the exception of a single isolate of the obligate iron reducer Geobacter sulfurreducens, no information is available about the identity and diversity of iron-reducing bacteria in this ecosystem (28). Furthermore, due to the lack of alternative techniques, the number of iron-reducing bacteria in activated sludge has been determined only by using a cultivation-dependent approach (28).

Different techniques for enumeration and isolation of Fe(III)-reducing bacteria have been developed (20). The most commonly applied method for enumeration of DIRB in various environments is the most-probable-number (MPN) technique (e.g., see references 10 and 28). Application of this technique must compensate for the insoluble nature of Fe(III); nevertheless, cultivation-based approaches such as this are assumed to greatly underestimate the actual numbers of DIRB occurring within natural systems, since not all DIRB might be culturable using standard MPN methods. Clumping and attachment to particulate matter might also cause problems for the enumeration of DIRB in complex environments, as has been previously shown for sulfate-reducing bacteria (SRB) (17).

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been used to detect and quantify bacterial species which are capable of catalyzing dissimilatory iron reduction (13). However, due to the phenotypic versatility of many bacteria, the in situ identification of a bacterial species capable of reducing iron in pure-culture experiments is not enough to conclude that an organism actually performed iron reduction at the time of sampling. Furthermore, it is impossible to design one or a few rRNA-targeted oligonucleotide probes for specific detection of all recognized DIRB as this physiological group comprises a phylogenetically diverse collection of species.

Microautoradiography (MAR) has previously proved valuable for the enumeration of actively metabolizing bacteria in many ecosystems (16, 23, 26). The challenge of the MAR technique is to offer the right radiolabeled substrate(s) and appropriate incubation conditions so that only the target organisms are enumerated. In this study, we were interested in the use of MAR and FISH for the enumeration and phylogenetic characterization of DIRB in activated sludge. As acetate is the most common substrate in most activated-sludge systems, we focused on bacteria that are capable of consuming radioactively labeled acetate under Fe(III)-reducing conditions. The specific labeling of DIRB by acetate uptake under iron-reducing conditions is, however, complicated by other concurrent processes, such as phosphate accumulation/release, sulfate reduction, and methanogenic activity. Therefore, the incubation conditions were carefully controlled, for example, by the inhibition of sulfate reduction by molybdate (30) and of methane production by bromoethanesulfonic acid (BES) (29). The impacts from other heterotrophic organisms (e.g., phosphate-accumulating organisms [PAOs]) were also evaluated. The MAR method used was evaluated and compared to traditional as well as newly modified MPN techniques for the enumeration of DIRB. The phylogenetic affiliation of the MAR-defined acetate-consuming Fe(III)-reducing population was subsequently examined by combining MAR with the use of fluorescently labeled rRNA-targeted group-specific oligonucleotide probes (MAR-FISH) (18).


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MATERIALS AND METHODS
 
Activated-sludge samples.
Activated sludge was collected from the Aalborg West wastewater treatment plant (WTP). This WTP has a presettling basin and performs biological nitrogen and phosphorus removal by the alternating BioDenipho process (14). The plant is designed for a population equivalent of 300,000 and has a sludge age of 25 to 30 days. The plant performs simultaneous chemical P removal by the addition of ferrous sulfate. The total amount of Fe in the activated sludge is approximately 0.4 g · liter-1. Activated-sludge samples were collected in May and June, when the mean temperature was around 17°C. All samples were taken from the aerobic process tank, and all incubation experiments were carried out on the day of sampling.

Cultivation and radiolabeling of Shewanella alga.
Shewanella alga strain BrY was obtained from F. Caccavo, Jr. (University of New Hampshire). The strain was cultivated by using the media described by Caccavo and coworkers (7). However, filtered effluent water from the Aalborg West WTP was used instead of distilled water. Radioactively labeled cells were prepared by the addition of 30 µCi of tritium-labeled acetate (100 mCi mmol-1; Amersham-Pharmacia Biotech, Buckinghamshire, England) per 50 ml of culture and 2 mM unlabeled acetate under aerobic conditions. S. alga strain BrY cannot utilize acetate under anaerobic conditions with Fe(III) as the electron acceptor, but it is able to take up acetate under aerobic conditions. After entering the stationary-growth phase, the cells were harvested by centrifugation (10,000 x g for 10 min) and washed twice with filtered effluent water from the Aalborg West WTP before use.

Enumeration of DIRB by the HFO-MPN and Ferrozine-MPN methods.
MPN enumerations were performed by serial dilution (to 10-9) of 1 ml of homogenized activated sludge. Homogenization was achieved by using a glass tissue grinder (Thomas Scientific, Swedesboro, N.J.). From this sample, serial dilution series were prepared in 5 ml of MPN medium. The medium for the Ferrozine-MPN assay contained 20 mM HEPES, 350 µM Ferrozine, 300 µM acetate, and 100 µM Fe(III) pyrophosphate. The medium for the hydrous ferric oxide (HFO)-MPN assay contained NaHCO3 (2.5 g · liter-1), NH4Cl (1.5 g · liter-1), KH2PO4 (0.6 g · liter-1), KCl (0.1 g · liter-1), vitamin solution (10 ml · liter-1), mineral solution (10 ml · liter-1), amorphous hydrous ferric oxide (30 mM), and acetate (2 mM). The media were made by using sterile filtered effluent water from the WTP to provide the conditions for growth, and the pH was adjusted to 7.5 by the addition of 1 N HCl or 1 N NaOH for both types of media. No change in pH was measured during any of the incubations. The amorphous Fe(III) oxyhydroxide was synthesized by repeated steps of neutralizing and washing of FeCl3 until the pH was constant. The composition of the vitamin and mineral solutions was as previously described by Balch et al. (6). All bottles were evacuated, and the headspaces were replaced with ultrapure N2. The bottles were autoclaved, except for the one containing Fe(III) pyrophosphate, which was added from a sterile filtered stock solution. Using strict anaerobic techniques, a 0.15-ml aliquot from each dilution series was transferred to each bottle before incubation at 30°C. The content of reduced iron was measured spectrophotometrically after 3 to 5 days and after 90 days. MPN values were calculated from standard MPN tables. The results from both methods were scored as positive if the absorbancy at 562 nm, measured after reaction with Ferrozine for the HFO-MPN assay or determined directly for the modified Ferrozine-MPN assay, was more than twice the value of a negative control, or equivalent to an increase of more than 2 µM Fe(II). All MPN investigations were performed in triplicate with a standard deviation of less than 30%. Controls with the addition of pasteurized activated sludge (10 min at 80°C) or without the addition of acetate showed no reduction of Fe(III). The addition of 2 mM sodium molybdate for inhibition of sulfate reduction did not cause any change in the reduction of Fe(III).

Incubation with radioactive and nonradioactive substrates.
Two milliliters of activated sludge was diluted with nitrate-free effluent water from the WTP to a final dry-matter concentration of approximately 1 g of suspended solids (SS) per liter and was incubated in a 9-ml glass vial closed with a butyl rubber stopper. The vials were evacuated with ultrapure nitrogen and were shaken gently for 1 h before addition of the substrate in order to ensure complete oxygen removal. Strict anaerobic techniques were used for all anaerobic incubations. Acetate was added to a final concentration of 2 mM together with 10 µCi of 3H-labeled acetate (100 mCi mmol-1) or 1-14C-labeled acetate (57 mCi mmol-1). All incubations were conducted at 21°C on a rotary table at 200 rpm. Inhibition of sulfate reduction was performed by the addition of 5 mM sodium molybdate (30), while methanogenic growth was inhibited by the addition of 1 mM BES (29). The addition of sodium molybdate and BES to these concentrations completely inhibited the sulfate reduction and methanogenesis in the activated sludge (data not shown). The iron reduction rates in activated sludge were measured as previously described by Nielsen et al. (28). Triplicate parallel incubations were performed with and without the addition of acetate (2 mM), with a standard error of the mean (SEM) of less than 5%. Preincubations were performed by adding 2 mM unlabeled acetate under anaerobic conditions prior to the addition of labeled acetate.

In order to determine the optimal preincubation time with unlabeled acetate, time course experiments of the consumption and incorporation of radioactive acetate into the biomass were conducted. The 14C content was measured directly in the sludge and in centrifuged (10,000 x g for 10 min) sludge bulk water samples. [14C]CO2 was stripped from the samples by lowering the pH to below 3 and placing the vials on ice and under a continuous stream of nitrogen for 30 min. One hundred to 200 µl of the stripped samples was allowed to react with 2 ml of scintillation liquid (Ultima Gold XR; Packard Instruments Co.) overnight in the dark before counting with a Packard model 1600 TR liquid scintillation analyzer.

Fixation and hybridization.
Incubation of the activated-sludge samples with [3H]acetate was terminated by the addition of paraformaldehyde to a final concentration of 4% at 4°C for 2 h. The samples were washed three times with filtered sludge water to remove excess labeled substrate. DAPI (4',6'-diamino-2-phenylindoledihydrochloride-dilactate) staining was achieved by applying DAPI to a final concentration of 1 mg/ml for 30 min in the last washing step. Subsequently, the sludge samples were homogenized in a glass tissue grinder (Thomas Scientific). All dilutions were performed in filtered tap water. Homogenized samples for enumeration were filtered onto 0.22-µm-pore-size white polycarbonate membrane filters (Millipore, Bedford, Mass.) and fixed on microscopic slides. Total counts were determined by counting more than 400 cells per microscopic slide and no fewer than 15 microscopic fields. The total DAPI count (DAPI-stained cells) was found to be 6.6 x 109 cells per ml. For in situ hybridization, paraformaldehyde-fixed samples were directly spotted on gelatin-coated cover glass. Dehydration and further application of the oligonucleotide probes were performed according to the procedure previously described by Amann et al. (1).

MAR.
Slides with fixed samples were carefully dipped in a prewarmed (43°C) liquid film emulsion (LM1; Amersham-Pharmacia Biotech). The slides were exposed in the dark at 4°C. For each incubation experiment, several slides were incubated in parallel. In order to test for the optimal exposure time, a slide was developed every day, and the increase in the number of cells covered with silver grains was determined microscopically. After an exposure period of 7 days, the number of positive cells did not increase further and all slides had been developed by this time. Chemography and other possible artifacts were never observed in controls without the addition of tracer or with pasteurized samples (10 min at 80°C). Cryosectioning was performed as previously described by Lee et al. (18).

Bright-field microscopy combined with confocal laser scanning microscopy was used for the identification of active (radiolabeled) cells by FISH with rRNA-targeted oligonucleotide probes as previously described by Lee et al. (18). A Zeiss LSM 510 scanning confocal microscope equipped with an argon ion laser (450 and 488 nm), two helium-neon lasers (543 and 633 nm), and a UV laser (351 and 364 nm) and standard Zeiss software (LSM 510, version 2.01) were used for the recording of images.

Enumeration by MAR.
Enumeration of MAR-positive bacteria was carried out on 0.22-µm-pore-size polycarbonate filters (Millipore), and the results were compared with the total number of DAPI-stained bacteria. In order to verify that all assemblages of silver grains actually did represent radiolabeled bacteria, radiolabeled homogenized sludge was stained with DAPI, immobilized on cover glass, and examined by inverse microscopy. This revealed that all assemblages of silver grains were located on top of DAPI-positive cells with a typical bacterial morphology.

The recovery of MAR-positive cells was determined by the addition of different amounts of radiolabeled S. alga BrY cells (see above) to 10 ml of activated sludge. Various amounts of S. alga BrY, equivalent to between 0.1 to 100% of the total number of bacteria in the activated sludge, were added to the activated sludge. After being mixed on a magnetic stirrer for 3 min, the sludge was fixed with paraformaldehyde as described above. The number of S. alga BrY cells was enumerated by using MAR and FISH with an S. alga BrY-specific probe, Sal216 (34).

Oligonucleotide probes.
The following previously described oligonucleotide probes were used for in situ hybridization: EUB338, Gam42a, Bet42a, SRB385, SRB687, SRB804, and Sal216. The probe specificities, probe sequences, and hybridization conditions are listed in Table 1. Probe SRB385 also hits several gram-positive bacteria (e.g., Clostridium spp.) (31), but the in situ hybridizations performed were carried out exclusively by using paraformaldehyde-fixed cells, which renders most gram-positive bacteria impermeable for fluorescent oligonucleotide probes, and consequently, most organisms detected by the SRB385 probe belong to the delta subclass of Proteobacteria. Oligonucleotides were labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS) or with the sulfoindocyanine dyes Cy3 and Cy5. All probes were purified by high-performance liquid chromatography (Interactiva Biotechnologie GmbH, Ulm, Germany).


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  		TABLE 1. 16S and 23S rRNA-targeted oligonucleotide probes used in this study

Analytical techniques.
Measurements of Fe(III) and Fe(II) were performed after HCl extraction by the Ferrozine-HEPES method as described by Rasmussen and Nielsen (32). Total SS and volatile SS (VSS) were determined by use of standard methods (3) to be 4.1 g · liter-1 and 2.6 g · liter-1, respectively. Acetate was measured with a Dionex ion chromatograph equipped with an IonPac AS11 high-capacity column and a suppressed conductivity detector using 0.2 mM NaOH as an eluent buffer. Phosphate was measured spectrophotometrically by standard methods (3).


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RESULTS
 
Fe(III) reduction and anaerobic acetate removal rates in activated sludge.
Fe(III) reduction took place in activated sludge from the Aalborg West WTP as soon as anaerobic conditions prevailed. The Fe(III) reduction rate was determined to be 31 µmol of Fe(III) g of VSS-1 h-1, corresponding to 95 µmol of Fe(III) liter-1 h-1, and it remained constant for at least 16 h. The addition of 2 mM acetate did not increase the Fe(III) reduction rate, indicating that the substrate did not limit the Fe(III) reduction.

Anaerobic acetate removal was followed by the tracking of the removal of [14C]acetate from the bulk water of the sludge and of the uptake into the biomass (Fig. 1). As the Aalborg West WTP is a treatment plant with biological phosphorus removal, the acetate was initially consumed very quickly, with rates up to 0.7 mmol g of VSS-1 h-1 (1.4 mmol liter-1 h-1), which was most likely due to the activity of PAOs (24). The storage capacity of the PAOs was exhausted after 2 to 3 h, and the acetate removal rate stabilized at a constant rate of approximately 0.007 mmol g of VSS-1 h-1 (SEM did not exceed 40%; n = 5), which remained constant for at least 16 h. The activity of PAOs during the first 2 to 3 h under anaerobic conditions was also supported by the observed release of orthophosphate (Fig. 2). After 3 h, no further release of orthophosphate was measured.



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  		FIG. 1. Time course of acetate removal (solid symbols) and uptake of acetate into activated-sludge biomass (open symbols) with (circles) or without (triangles) the addition of 2 mM molybdate. The initial concentration of acetate was 1 mM, the concentration of VSS of the sludge was adjusted to 2 g/liter, and the isotope amount was 0.25 µCi ml-1.



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  		FIG. 2. The release of orthophosphate in activated sludge under anaerobic incubation in the presence of acetate.

The rate of [14C]acetate incorporation was highest immediately after addition of acetate to the sludge (0.3 mmol g of VSS-1 h-1) and later declined until a constant value of 0.005 mmol g of VSS-1 h-1 was reached after 3 h. The addition of 2 mM sodium molybdate as an inhibitor of sulfate reduction resulted in reductions of both the acetate removal rate (9 to 12% reduction in three replicate experiments) and the incorporation of [14C]acetate (14 to 16% reduction in three replicate experiments). Interestingly, and in accordance with the work of Nollet et al. (29), the removal rate of acetate in activated sludge increased by 10 to 15% upon the addition of BES as an inhibitor of methane production compared to that of a control without the addition of BES.

The molar ratio between the amounts of Fe(II) produced and acetate consumed during the anaerobic incubation period from 4 to 16 h was calculated in three independent experiments to be 3.6 to 6.2. These values are in the same range as but slightly below the stoichiometric value of 8 that can be expected from a complete oxidation of acetate coupled to Fe(III) reduction.

Enumeration of DIRB by MPN studies.
By use of the modified MPN assay containing Ferrozine, the number of iron-reducing bacteria in activated sludge was determined to be 2.1 x 105 cells per ml, which is approximately 6 times more than the count obtained by the traditional HFO-MPN method (Table 2). The reduction of less than 1 µM of ferric iron could be determined by the modified method, which enabled us to detect positive samples after only 3 to 5 days of incubation. Further incubation for 90 days increased the number of positive samples by only 17%. Compared to the traditional HFO-MPN method, where amorphous iron oxides must be reduced in measurable amounts, the modified Ferrozine-MPN method was significantly faster and did not require filtration before determination of the Fe(II) content. The two methods were compared several times for enumeration of DIRB in activated sludge, and the modified Ferrozine-MPN method always yielded counts that were 6 to 10 times higher than those obtained by the HFO-MPN method. The SEMs for both MPN methods used did not exceed 30%. The addition of 2 mM molybdate did not result in a lower yield for either of the MPN methods.


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  		TABLE 2. Number of Fe(III)-reducing bacteria in activated sludge estimated by MPN techniques and by MAR

The average cell-specific Fe(III) reduction rates were calculated to be 1.9 x 10-12 mol cell-1 h-1 and 3.3 x 10-13 mol cell-1 h-1 by using the numbers obtained by the HFO-MPN and Ferrozine-MPN methods, respectively (Table 2, column 5).

Enumeration of DIRB by MAR.
Initially, we evaluated the accuracy of the MAR method by determining its recovery efficiency for different amounts of radioactively labeled S. alga BrY cells added to the activated sludge (Fig. 3). There was a linear relationship between the number of added S. alga BrY cells and the recovery of MAR-positive cells. A similar recovery of S. alga BrY cells was found when the cells were enumerated after FISH with the species-specific oligonucleotide probe (Sal216) (results not shown).



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  		FIG. 3. Recovery of radiolabeled S. alga BrY cells added to activated sludge.

MAR was used to enumerate acetate-consuming bacteria under iron-reducing conditions (AC-IRC). In order to exclude the uptake of radioactive acetate by PAOs, a period of preincubation with unlabeled acetate was included to saturate their uptake capacity. Preincubations for 0 to 6 h with unlabeled acetate were carried out before [3H]acetate was added to the activated sludge (Fig. 4). The number of MAR-positive AC-IRC was found to decrease from approximately 2.2 x 108 cells ml-1 without preincubation to 1.1 x 108 cells ml-1, corresponding to 4.3 x 1010 cells g of VSS-1, after 3 h of preincubation with unlabeled acetate (Table 2). Longer preincubation times did not reduce further the number of MAR-positive bacteria, indicating that the PAOs were active only within the first 3 h, which is consistent with the release of orthophosphate only in this period (Fig. 2). The number of labeled cells observed after 3 h of preincubation (1.1 x 108 cells ml-1) thus represents the number of AC-IRC in the sludge, as the inhibition of sulfate reduction and methanogenic activity by the addition of 2 mM molybdate and/or 1 mM BES lowered the number of MAR-positive cells to only 1.04 x 108 cells ml-1. AC-IRC accounted for 3.0% of the total DAPI count, and the number determined in situ is significantly higher than the viable counts obtained by both MPN methods. Increasing the time of incubation with radiolabeled acetate from 3 to 6 h (after a preincubation time of 3 h with unlabeled acetate) did not yield a higher number of MAR-positive cells, indicating that all active cells had taken up enough acetate during the incubation period of 3 h to be detectable.



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  		FIG. 4. Percentage of MAR-positive cells after incubation with [3H]acetate under Fe(III)-reducing conditions and after various periods of anaerobic preincubation with the presence of unlabeled acetate only.

Based on the MAR enumeration results, an average cell-specific Fe(III) reduction rate of 1.8 x 10-15 mol cell-1 h-1 was calculated, which is around 200 to 1,000 times lower than the rates inferred by the MPN methods.

Distribution of iron-reducing bacteria in activated sludge.
The spatial localization of AC-IRC in activated sludge was visualized by MAR on cryosections of unhomogenized samples of sludge flocs. The MAR-positive cells were present, either as single cells or in small assemblages of a few cells throughout the sludge flocs, at the surface as well as in the center. The typical number of cells within a cluster was less than 10, and these assemblages were distributed homogeneously throughout the different sludge flocs.

Phylogenetic affiliation of AC-IRC.
Between 50 to 60% of all EUB338-positive cells in the activated sludge hybridized with the Bet42a probe, 8 to 15% hybridized with the Gam42a probe, and ca. 5% hybridized with the Alf1b probe. A small number of bacteria within the delta group (estimated to be 0.5 to 1% of the total EUB338 count), encompassing important potentially sulfate- or iron-reducing bacteria, was identified by using the probe SRB385. These cells were often found in small cell clusters of 10 to 30 cells, although single cells were found as well. Sulfate reducers hybridizing with probe SRB804 could be detected in significantly lower numbers. No target cells were found which hybridized with the SRB687 probe or the probe for S. alga BrY.

All MAR-positive cells (AC-IRC) hybridized with the Bacteria-specific probe (EUB338). A considerable fraction of the AC-IRC (approximately 20%) was identified to be affiliated with the gamma subclass of Proteobacteria. Figure 5 shows an example of a small cluster of cells belonging to the beta subclass of Proteobacteria with a few cells of the gamma subclass flanking the cluster. On the corresponding MAR image, it can be seen that the beta Proteobacteria did not take up acetate under iron-reducing conditions but that the gamma Proteobacteria were covered with silver grains, indicating uptake of labeled acetate. Interestingly, some MAR-positive AC-IRC (less than 1%) could be identified as members of the beta subclass of Proteobacteria, which occurred as single cells. Furthermore, 5 to 10% of the AC-IRC hybridized with probe SRB385, indicating an affiliation with the delta Proteobacteria. Among the SRB385-positive cells, only some of the individually occurring cells and none of the clusters were found to be MAR positive. None of the cells hybridizing with the Alf1b, SRB687, or the SRB804 probe were MAR positive.



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  		FIG. 5. Incorporation of [3H]acetate under Fe(III)-reducing conditions, as detected by MAR and FISH. Shown are the results of hybridization with the Bet42a probe (red) (A) and the Gam42a probe (green) (B) and of MAR (C). Superimposing the images in panels A to C shows the combination of MAR and FISH (D). Bar = 5 µm.


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DISCUSSION
 
Enumeration.
In the activated-sludge sample examined, the number of AC-IRC determined by the MAR method was 350 to 2,500 times higher than the number of DIRB counted by the MPN methods. The corresponding cell-specific Fe(III) reduction rate calculated from the numbers obtained by the MAR method was 1.8 x 10-15 mol/cell, which is very similar to those reported for Desulfuromonas acetoxidans (33), Shewanella putrefaciens (4, 5), and S. alga (7) in pure-culture studies of iron-reducing bacteria. This indicates that the counts of iron-reducing bacteria in activated sludge were significantly underestimated by both MPN methods, while more realistic numbers were obtained by the applied MAR technique.

Incorporation of the labeled substrate into the target cells is a crucial step in the MAR enumeration procedure. In the activated-sludge system we investigated, we excluded the uptake of acetate by PAOs, sulfate reducers, and methanogenic bacteria in order to detect only AC-IRC. The PAOs are not believed to be able to grow under anaerobic conditions (9), so the inclusion of a preincubation step with unlabeled acetate in order to saturate their storage capacity prevented the uptake of labeled acetate by these microorganisms. The time course of acetate uptake and orthophosphate release showed that a 3-h preincubation time was sufficient to saturate the storage capacity, which is in agreement with the results of other studies of activated sludge from treatment plants with biological phosphorus removal (24). Thus, the constant rate of acetate consumption observed after 3 h of incubation was most likely not due to the activity of PAOs. This is also supported by the results from the FISH probing of the MAR-positive bacteria. Less than 1% of all MAR-positive bacteria belonged to the beta subclass of the Proteobacteria, where members of the most common PAOs belong (Rhodocyclus [15]). This result remained constant with preincubation times of 3, 6, and 9 h with unlabeled acetate (data not shown).

Several SRB can consume acetate (16), and any sulfide produced can chemically reduce ferric iron, which will affect the measured iron reduction rate. The activity of SRB was effectively inhibited by molybdate, as demonstrated also in other studies (e.g., see reference 16). Upon inhibition of SRB activity, the number of MAR-positive cells remained almost unchanged, indicating that the number of SRB was small compared to the population of acetate-utilizing iron reducers in the activated sludge investigated. With the probe SRB385, we consistently detected low in situ numbers of cells belonging to the delta subclass of Proteobacteria (<1% of total DAPI count), which encompasses many dissimilatory SRB (12). This is in accordance with the results obtained by Schramm et al. (35), who found that 1 to 2% of the total population hybridized with the SRB385 probe in activated sludge from another Danish WTP. Methanogens were effectively inhibited by the addition of BES, but as the number of MAR-positive bacteria did not significantly decrease, the number of methanogens was below 0.5% of the total DAPI count. The observed increase in the acetate removal rate after addition of BES indicates that BES increases the activity of the active cells rather than the number of active cells. This phenomenon is probably due to a stimulation of reductive acetogenic bacteria, which has also been described by Nollet et al. (29).

Other functional groups that could possibly grow on acetate under the incubation conditions applied include bacteria capable of using other electron acceptors such as manganese and elemental sulfur. However, as these compounds are hardly present in activated sludge in significant amounts, these bacteria are most likely not present in numbers comparable to the DIRB. Furthermore, the observed ratio between the amounts of Fe(III) reduced and acetate oxidized (3.6 to 6.2) also suggests that most MAR-positive bacteria are in fact DIRB. The molar ratio was below the stoichiometric value of 8 that can be expected from a complete oxidation of acetate coupled to Fe(III) reduction. A slightly lower value can be explained by the incorporation of some acetate into cellular material, uptake by other non-iron reducers, or inadequate iron extraction. However, although the MAR-based enumeration may represent a nearly twofold overestimation, this should be compared with the second-best method, the MPN technique, which underestimated the number of DIRB by at least a factor of 350. On the basis of these results and the phylogenetic analysis of the MAR-positive cells (see below), we find it very likely that the major fraction of AC-IRC were actually DIRB.

The cell-specific iron reduction rates determined on the basis of the two MPN methods were 100- to 1,000-fold higher than any value determined in pure-culture studies. It is known from studies of sulfate reducers that the specific activities in activated sludge and in pure culture are very similar (37). Assuming that this is also true for DIRB, both MPN methods significantly underestimated the actual number of iron-reducing bacteria. This is most likely caused primarily by the inability of certain iron-reducing bacteria to grow under the applied cultivation conditions and is not due to the clumping of the cells, because microscopic observation showed that the MAR-positive cells were scattered throughout the sludge flocs and that DIRB cell clusters contained only a few cells. The presence of the complexing agent, citrate, in the Ferrozine-MPN assay may potentially lead to an overestimation of the population size of iron-reducing bacteria due to the reduction of citrate by fermentative bacteria that can also indirectly reduce ferric iron. However, this was not considered a problem in our experiments since no iron was reduced in the controls without the addition of acetate.

The number of iron-reducing bacteria able to consume acetate was approximately 3% of the total population as determined by DAPI staining. However, the total number of iron-reducing bacteria may be higher because several known DIRB are not able to consume acetate (20), and it has been shown that substrates such as lactate can almost double the iron reduction rate in the activated sludge investigated (28). This number is significantly lower than the number of nitrate-respiring acetate-utilizing bacteria in the same treatment plant. These have been determined by MAR enumeration to be 71% of the total number of DAPI-stained bacteria (26), so if all enumerated DIRB were able to use nitrate as an alternative electron acceptor, then it can be calculated that only 5 to 6% of the nitrate reducers in the sludge were also able to reduce Fe(III). This shows that although many facultative DIRB are believed to be able to use nitrate as an alternative electron acceptor (20), most denitrifiers in the investigated treatment plant were not able to use Fe(III) as an electron acceptor.

Phylogenetic affiliation.
All MAR-detected DIRB in the activated sludge were identified as Bacteria by use of FISH. Only about one-third of these hybridized with the probe tested, leaving the majority of the DIRB unidentified. Most known DIRB belong to the delta subclass of the Proteobacteria, and a few belong to the gamma subclass (19). This is in accordance with our results, although most of the DIRB identified belonged to the gamma subclass (approximately 20% of the MAR-positive bacteria), corresponding to approximately 10% of this phylogenetic group. The delta subclass of the Proteobacteria accounted for 10% of the MAR-positive bacteria, corresponding to approximately 50% of all bacteria detected in this subclass. Beta Proteobacteria were most numerous in the sludge, but very few of the MAR-positive bacteria belonged to this group (1%), corresponding to approximately 0.1% of all beta Proteobacteria. Known DIRB belonging to the gamma subclass of iron reducers oxidize only multicarbon electron donors such as lactate and pyruvate to acetate and are not able to use acetate (20). Thus, our study indicates the existence of novel bacteria belonging to this lineage that are able to oxidize acetate. The lack of MAR-positive cells from the alpha subclass of the Proteobacteria, cells positive with the SRB804 probe, and the presence of only a few cells belonging to the beta subclass support the general knowledge that DIRB are not found in these groups. The group of unknown AC-IRC (70% of all MAR-positive cells) may contain gram-positive bacteria, but more detailed phylogenetic analysis is required.

In the present study, we have shown that the combination of MAR and FISH is a method well-suited not only for enumeration but also for preliminary phylogenetic classification of functional bacterial groups within complex environmental samples. Almost all types of functional groups can be investigated by this approach, if only the right incubation conditions and the appropriate radiolabeled substrate(s) are provided.


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ACKNOWLEDGMENTS
 
M. Stevenson and M. Fredsgaard are thanked for valuable technical assistance. We also thank F. Caccavo, Jr., and the anonymous reviewers for their helpful comments.

The Danish Technical Research Council supported this study under the framework program "Activity and Diversity in Complex Microbial Systems." S. Juretschko was supported by Sonderforschungsbereich 411 from the Deutsche Forschungsgemeinschaft (Research Center for Fundamental Studies of Aerobic Biological Wastewater Treatment, project A2 of M. Wagner).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Environmental Engineering, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark. Phone: 45 96358505. Fax: 45 98142555. E-mail: phn{at}civil.auc.dk. Back


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REFERENCES
 
    1
  1. Amann, R., W. Ludwig, and K.-H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169.[Abstract/Free Full Text]
    2. 2
  2. Amann, R., J. Stromley, R. Devereux, R. Key, and D. A. Stahl. 1992. Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl. Environ. Microbiol. 172:762-770.
    3. 3
  3. American Public Health Association. 1995. Standard methods for the examination of water and wastewater, 18th ed. American Public Health Association, Washington, D.C.
    4. 4
  4. Arnold, R. G., T. J. DiChristina, and M. R. Hoffmann. 1990. Regulation of dissimilatory Fe(III) reduction in Shewanella putrefaciens. Appl. Environ. Microbiol. 56:2811-2817.[Abstract/Free Full Text]
    5. 5
  5. Balashova, V. V., and G. A. Zavarzin. 1980. Anaerobic reduction of ferric iron by hydrogen bacteria. Microbiology 48:635-639.
    6. 6
  6. Balch, W. E., G. E. Fox, L. J. Magrum, C. R. Woese, and R. S. Wolfe. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296.[Free Full Text]
    7. 7
  7. Caccavo, F., Jr., R. P. Blakemore, and D. R. Lovley. 1992. A hydrogen-oxidizing, Fe(III)-reducing microorganism from the Great Bay estuary, New Hampshire. Appl. Environ. Microbiol. 58:3211-3216.[Abstract/Free Full Text]
    8. 8
  8. Caccavo, F., Jr., J. D. Coates, R. A. Rosselló-Mora, W. Ludwig, K.-H. Schleifer, D. R. Lovley, and M. J. McInerney. 1996. Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium. Arch. Microbiol. 165:370-376.[CrossRef][Medline]
    9. 9
  9. Chuang, S.-H., C. F. Ouyang, and Y.-B. Wang. 1996. Kinetic competition between phosphorus release and denitrification on sludge under anoxic conditions. Water Res. 30:2961-2968.[CrossRef]
    10. 10
  10. Cummings, D. E., A. W. March, B. Bostick, S. Spring, F. Caccavo, Jr., S. Fendorf, and R. F. Rosenzweig. 2000. Evidence for microbial Fe(III) reduction in anoxic, mining-impacted lake sediments (Lake Coeur d'Alene, Idaho). Appl. Environ. Microbiol. 66:154-162.[Abstract/Free Full Text]
    11. 11
  11. Daims, H., A. Bruehl, R. Amann, K.-H. Schleifer, and M. Wagner. 1999. The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22:434-444.[Medline]
    12. 12
  12. Devereux, R., M. D. Kane, J. Winfrey, and D. A. Stahl. 1992. Genus- and group-specific hybridization probes for determinative and environmental studies of sulfate-reducing bacteria. Syst. Appl. Microbiol. 15:601-609.
    13. 13
  13. DiChristina, T. J., and E. F. DeLong. 1993. Design and application of rRNA-targeted oligonucleotide probes for the dissimilatory iron- and manganese-reducing bacterium Shewanella putrefaciens. Appl. Environ. Microbiol. 59:4152-4160.[Abstract/Free Full Text]
    14. 14
  14. Harremoës, P., E. Bundgaard, and M. Henze. 1991. Developments in wastewater treatment for nutrient removal. Eur. Water Pollut. Control 1:19-23.
    15. 15
  15. Hesselmann, R. P. X., C. Werlen, D. Hahn, J. R. van der Meer, and A. J. B. Zehnder. 1999. Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Syst. Appl. Microb., 22:454-465.[CrossRef]
    16. 16
  16. Ito, T., J. L. Nielsen, S. Okabe, Y. Watanabe, and P. H. Nielsen. 2002. 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. Appl. Environ. Microbiol. 68:356-364.[Abstract/Free Full Text]
    17. 17
  17. Jørgensen, B. B. 1978. A comparison of methods for the quantification of bacterial sulfate reduction in coastal marine sediments. III. Estimation from chemical and bacteriological field data. Geomicrobiol. J. 1:49-64.
    18. 18
  18. Lee, N., K. Andreasen, P. H. Nielsen, S. Juretschko, J. L. Nielsen, K.-H. Schleifer, and M. Wagner. 1999. Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analysis in microbial ecology. Appl. Environ. Microbiol. 65:1289-1297.[Abstract/Free Full Text]
    19. 19
  19. Lonergan, D. J., H. L. Jenter, J. D. Coates, E. J. P. Phillips, T. M. Schmidt, and D. R. Lovley. 1996. Phylogenetic analysis of dissimilatory Fe(III)-reducing bacteria. J. Bacteriol. 178:2402-2408.[Abstract/Free Full Text]
    20. 20
  20. Lovley, D. R. 1991. Dissimilatory Fe(III) and Mn(IV) reduction. Microbiol. Rev. 55:259-287.[Abstract/Free Full Text]
    21. 21
  21. Lovley, D. R. 1995. Microbial reduction of iron, manganese and other metals. Adv. Agron. 74:176-217.
    22. 22
  22. Manz, W., R. Amann, W. Ludwig, M. Wagner, and K.-H. Schleifer. 1992. Phylogenetic oligonucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst. Appl. Microbiol. 15:593-600.
    23. 23
  23. Meyer-Reil, L.-A. 1978. Autoradiography and epifluorescence microscopy combined for the determination of number and spectrum of actively metabolizing bacteria in natural waters. Appl. Environ. Microbiol. 36:506-512.[Abstract/Free Full Text]
    24. 24
  24. Mino, T., M. C. M. Van Loosdrecht, and J. J. Heijnen. 1998. Microbiology and biochemistry of the enhanced biological phosphate removal process. Water Res. 32:3193-3297.[CrossRef]
    25. 25
  25. Nealson, K. H., and D. Saffarini. 1994. Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation. Annu. Rev. Microbiol. 48:311-343.[CrossRef][Medline]
    26. 26
  26. Nielsen, J. L., and P. H. Nielsen. 2002. Enumeration of acetate-consuming bacteria by microautoradiography under oxygen and nitrate respiring conditions in activated sludge. Water Res. 36:421-428.[Medline]
    27. 27
  27. Nielsen, P. H. 1996. The significance of microbial Fe(III) reduction in the activated sludge process. Water Sci. Technol. 34:129-136.
    28. 28
  28. Nielsen, P. H., B. Frølund, and F. Caccavo, Jr. 1997. Microbial Fe(III) reduction in activated sludge. Syst. Appl. Microbiol. 20:645-651.
    29. 29
  29. Nollet, L., D. Demeyer, and W. Verstraete. 1997. Effect of 2-bromoethanesulfonic acid and Peptostreptococcus productus ATCC 35244 addition on stimulation of reductive acetogenesis in the ruminal ecosystem by selective inhibition of methanogenesis. Appl. Environ. Microbiol. 63:194-200.[Abstract]
    30. 30
  30. Oremland, R. S., and D. G. Capone. 1988. Use of specific inhibitors in biogeochemistry and microbial ecology. Adv. Microb. Ecol. 10:285-383.
    31. 31
  31. Ramsing, N. B., H. Fossing, T. G. Ferdelman, F. Andersen, and B. Thamdrup. 1996. Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column. Appl. Environ. Microbiol. 62:1391-1404.[Abstract]
    32. 32
  32. Rasmussen, H., and P. H. Nielsen. 1996. Iron reduction in activated sludge measured with different extraction techniques. Water Res. 30:551-558.[CrossRef]
    33. 33
  33. Roden, E. E., and D. R. Lovley. 1993. Dissimilatory Fe(III) reduction by the marine microorganism Desulfuromonas acetoxidans. Appl. Environ. Microbiol. 59:734-742.[Abstract/Free Full Text]
    34. 34
  34. Rosello-Mora, R. A., F. Caccavo, Jr., K. Osterlehner, N. Springer, S. Spring, D. Schüler, W. Ludwig, R. Amann, M. Vanncanneyt, and K.-H. Schleifer. 1994. Isolation and taxonomic characterization of a halotolerant, facultatively iron-reducing bacterium. Syst. Appl. Microbiol. 17:569-573.
    35. 35
  35. Schramm, A., C. M. Santegoeds, H. K. Nielsen, H. Ploug, M. Wagner, M. Pribyl, J. Wanner, R. Amann, and D. De Beer. 1999. On the occurrence of anoxic microniches, denitrification, and sulfate reduction in aerated activated sludge. Appl. Environ. Microbiol. 65:4189-4196.[Abstract/Free Full Text]
    36. 36
  36. Slobodkin, A. I., C. Jeanthon, S. L'Haridon, T. Nazina, M. Miroshnichenko, and E. Bonch-Osmolovskaya. 1997. Dissimilatory reduction of Fe(III) by thermophilic bacteria and Archaea in deep subsurface petroleum reservoirs of Western Siberia. Curr. Microbiol. 39:99-102.
    37. 37
  37. Vester, F., and K. Ingvorsen. 1998. Improved most-probable-number method to detect sulfate-reducing bacteria with natural media and a radiotracer. Appl. Environ. Microbiol. 64:1700-1707.[Abstract/Free Full Text]

Applied and Environmental Microbiology, September 2002, p. 4629-4636, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4629-4636.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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