INTRODUCTION

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Sulfate-reducing bacteria (SRB) are of great ecological importance in the mineralization of organic matter in anaerobic environments. For example, in marine sediments up to 50% of the organic matter may be oxidized by sulfate reduction (25). In some low-sulfate environments, such as freshwater lakes, bacterial sulfate reduction may still be important in the mineralization process (5, 14, 16, 20). SRB also have great economic importance in the oil industry, where they cause severe problems, including souring of oil and gas deposits and corrosion of production facilities (13, 21, 33).

Considerable efforts have been directed toward the development of rapid and dependable methods for detection and enumeration of SRB in natural and industrial environments. In general, the methods used to enumerate SRB can be divided into the following two categories: (i) direct detection methods and (ii) culture methods. The direct detection methods developed recently include the use of antibodies raised against SRB (7, 28), an immunoassay for the enzyme adenosine-5'-phosphosulfate (APS) reductase (34), and the use of 16S rRNA probes (2, 37). Although promising, these techniques are still in the developmental phase, and several problems are encountered when they are used in situ (1, 41). For example, rRNA fluorescent probes are difficult to use in sediments due to the high background autofluorescence of inorganic particles (32). Furthermore, not all known types of dissimilatory sulfate reducers in environmental samples can be unequivocally identified with RNA probes described previously (37, 42).

Culture methods for enumeration of SRB based on the most-probable-number (MPN) technique (3) have been used extensively for several decades. A variety of MPN media have been developed for specific environments, including activated sludge, marine sediments, and samples from the oil drilling industry (10, 22, 35, 36, 40). Most of these enumeration media contain lactate as the main carbon and energy source. In all cases, the presence of SRB in MPN tubes is evaluated by the formation of a black precipitate of ferrous sulfide (FeS).

Several studies have demonstrated that the numbers of viable SRB in marine sediments are underestimated by a factor of at least 1,000 when standard MPN techniques are used with synthetic growth media (12, 24, 37). In this paper, we describe the development and evaluation of a greatly improved MPN technique for enumeration of SRB in environmental samples.

	MATERIALS AND METHODS

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Sampling procedures. Sediment samples were collected by obtaining cores (23) from shallow permanently water-covered sediments in Kysing Fjord on the east coast of Jutland, Denmark. Material used to prepare sediment medium was collected from the top sediment layer (upper 2 cm), which had an organic content of 2 to 10% (dry weight) (18, 43). Activated sludge samples were obtained from an aeration basin at a municipal wastewater treatment plant (Marselisborg, Aarhus, Denmark). Sludge and sediment samples were stored in 100-ml serum flasks that were filled to capacity and sealed with gas-tight rubber stoppers while they were transported to the laboratory.

Strain. Desulfobulbus propionicus DSM 2032 was obtained from the Deutsche Sammlung von Microorganismen und Zellkulturen, Braunschweig, Germany.

Preparation of natural MPN media. Sediment medium for tracer MPN (T-MPN) enumerations performed with Kysing Fjord sediment was prepared as follows. Sediment was diluted 1:1 (vol/vol) with water from the sampling site and homogenized in a blender. The sediment suspension was successively passed through 1-, 0.5-, and 0.25-mm-mesh sieves (Endecotts Ltd., London, United Kingdom) and finally autoclaved for 20 min at 121°C. After autoclaving, the sediment suspension was cooled during vigorous magnetic stirring while it was purged with oxygen-free N₂. Aliquots (8.9 ml) of the sediment suspension were anaerobically dispensed into culture tubes (type 2047; Bellco), and the tubes were sealed under an N₂ atmosphere with butyl rubber stoppers. The tubes were incubated for 24 h at room temperature, autoclaved a second time, and stored at 5°C. The sediment medium was reduced immediately before inoculation by aseptically adding 0.1 ml of a freshly prepared sodium dithionite (Na₂S₂O₄) solution to a final concentration of 200 µM.

Preparation of synthetic MPN media. API-RST medium, an improved version of the API RP-38 medium used for enumeration of SRB, was prepared as described by Tanner (40), except that sodium dithionite (final concentration, 200 µM) was substituted for ascorbic acid and cysteine-HCl as the reducing agent. Baar's medium (culture medium 1249) was prepared as described previously (4), with the following modifications: the NaCl concentration was adjusted to 1% (wt/vol) (as in API-RST medium), and sodium dithionite was used as the reducing agent at a final concentration of 200 µM. Postgate's B medium was prepared as described by Postgate (36) by using autoclaved seawater from the sampling site. DSM medium 194, a bicarbonate-buffered, sulfide-reduced, defined medium having a low iron content (9), was used for pure-culture experiments performed with Desulfobulbus propionicus. All MPN media (except DSM medium 194) contained yeast extract and lactate as the main electron donor. Below, these media are referred to as synthetic media.

General anaerobic techniques. Strictly anaerobic and aseptic conditions were maintained throughout the experiments. The anaerobic techniques used were essentially the syringe methods of Macy et al. (30) performed with anoxic gases, N₂, or N₂-CO₂ (90:10).

Measurement of sulfate reduction rates in sediment slurries, activated-sludge enrichment cultures, and pure cultures. (i) Sediment slurries. Sulfate reduction rates and MPN values were determined as follows. Sediment slurries were prepared under constant N₂ gassing by diluting anaerobic surface sediment 1:1 with anoxic water from the sampling site in serum flasks containing glass beads. Each slurry was mixed vigorously by shaking and preincubated under an N₂ atmosphere for 2 h at 22°C. After preincubation, 20 ml of the sediment slurry was removed with a syringe, and 10-ml portions were placed into two sterile Bellco culture tubes containing an N₂-CO₂ (90:10) gas phase. One tube was immediately used to inoculate MPN tubes containing natural and synthetic media (see below). The other tube was used to measure the sulfate reduction rate with radiolabeled sulfate as follows. A 1-ml sample was removed for sulfate analysis before 0.1 ml of a sterile isotope solution (200 kBq of carrier-free ³⁵SO₄²; Isotope Laboratory, Risø, Denmark) was injected. The tube was incubated in the dark at 22°C, and 0.5-ml samples were removed at appropriate times and injected into 2 ml of a 20% (wt/vol) zinc acetate solution in order to stop the biological activity and preserve the ³⁵S-sulfides produced.

(ii) Activated-sludge enrichment cultures. Samples (100 ml) of activated sludge were amended with sulfate (1 ml of a 0.3 M Na₂SO₄ solution) and preincubated under an N₂ atmosphere for 6 days at 22°C to increase the indigenous population of SRB. Enrichment was used only during evaluation of the method in order to facilitate the statistical analysis because low numbers of SRB were sometimes observed in sludge samples. After preincubation, 10.0-ml aliquots of the enriched sludge were transferred with a 50-ml sterile syringe and an 18-gauge needle into three sterile Bellco culture tubes containing an N₂-CO₂ (90:10) gas phase. The same syringe was filled only once in order to reduce experimental error. One tube (tube 1) was immediately used to inoculate MPN tubes containing natural and synthetic media (see below). Tubes 2 and 3 were used for radiotracer measurements of the sulfate reduction rate as described above for sediment slurries. To one of these tubes (tube 2) chloramphenicol and streptomycin were added at final concentrations of 20 and 100 mg/liter, respectively.

(iii) Pure cultures. Sulfate reduction rates in pure cultures of Desulfobulbus propionicus were determined in the presence and absence of antibiotics. The experiments were carried out in culture tubes (type 2047; Bellco) containing 9 ml of defined growth medium (DSM medium 194). Three tubes were each inoculated with 1 ml of an exponential-phase culture, and the experiment was conducted as described above for the sludge sample experiment. The initial cell density in tube 1, which was used for T-MPN determination with DSM medium 194, was determined immediately after inoculation with a Bürker-Türk counting chamber. The tubes were incubated at 30°C for 25 h, and samples were removed at intervals as described above.

Enumeration of SRB by the T-MPN and N-MPN methods. T-MPN enumeration was performed as follows. Each 10-ml sample examined was transferred into a sterile Bellco culture tube containing glass beads and mixed vigorously by vortexing. The suspended sample was immediately used to prepare 10-fold MPN dilutions (in triplicate) in synthetic and natural media; each tube contained 200 kBq of ³⁵SO₄². In each T-MPN experiment, three uninoculated tubes were included as controls for medium sterility and isotope carryover during the distillation procedure. The tubes used for the MPN analysis and to determine sulfate reduction rates were incubated at the same temperature (22 or 30°C). During incubation, 1-ml subsamples were removed with a syringe from the T-MPN tubes and immediately injected into test tubes containing 2 ml of 20% (wt/vol) zinc acetate. Unless stated otherwise, the presence of SRB in MPN dilution tubes containing synthetic media was evaluated by using the normal MPN (N-MPN) method (formation of black FeS precipitate). In MPN dilution tubes containing natural media, the numbers of SRB were estimated only by the T-MPN method.

Recovery of reduced ³⁵S by distillation. The amounts of reduced ³⁵S-sulfur in subsamples from T-MPN tubes were determined by using the single-step chromium reduction method described by Fossing and Jørgensen (11), which allows simultaneous measurement of acid-volatile sulfur and chromium-reducible sulfur, yielding total reduced inorganic sulfur (TRIS). Sulfate reduction rates (SRR) were calculated with the following equation: SRR = [a × 1.06 × (SO₄²)]/[(a + A) × T], where (SO₄²) is the sulfate concentration, a is the total radioactivity of ZnS, A is the total radioactivity of sulfate after incubation, T is the incubation time, and 1.06 is the ³²S/³⁵S correction factor for the expected isotope fractionation (23). Sulfate reduction rates were expressed as number of moles per liter per day.

Sulfate analysis. The sulfate concentrations in activated sludge, sediment slurries, and DSM medium 194 were determined by suppressed ion chromatography as previously described (6).

MPN calculations and statistical analysis. A computer program (15) was used to calculate MPN values and the standard errors of the MPN estimates. The statistical method of Cochran (8), a Student t test, was used to determine whether MPN values obtained with different media were significantly different. Differences were considered significant at the 95% confidence level.

	RESULTS

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MPN enumeration of SRB in sediment slurries with natural and synthetic media. The MPN of SRB in a sediment slurry from Kysing Fjord determined by the T-MPN method with sediment medium (natural medium) and by the N-MPN method with API-RST medium (synthetic medium) differed by a factor of ~3,000 after 34 days of incubation (Table 1). Estimates obtained by the N-MPN method (API-RST medium) yielded significantly higher bacterial counts only on day 2. No significant differences in the MPN values between the two methods were observed from day 5 to day 11. However, from day 14 on, the bacterial numbers determined by the T-MPN method with sediment medium were significantly higher than the bacterial numbers determined by the N-MPN method with API-RST medium. A constant sulfate reduction rate of 0.98 mM SO₄² day¹ (0 to 9 h; r² = 0.996) was obtained for the Kysing Fjord slurry used for the enumeration experiments shown in Table 1. Based on this sulfate reduction rate, specific sulfate reduction rates (qSO₄²) of 75 × 10¹⁵ and 228 × 10¹² mol cell¹ day¹ were calculated by using the viable counts obtained by the T-MPN method with natural medium and the N-MPN method with synthetic medium, respectively (Table 1, day 34).

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View this table: [in this window] [in a new window]   TABLE 3.   Comparison of qSO42 values determined by different experimental methods

View larger version (49K): [in this window] [in a new window]   FIG. 1.   Time course of TRIS production during T-MPN enumeration of SRB in a sediment slurry from Kysing Fjord (sediment medium; initial sulfate concentration, 17 mM; 20 kBq of 35SO42/ml). TRIS values are the fractions of added 35SO42 converted to TRIS. Panels A, B, and C show the results obtained with three independent dilution series (series A, B, and C, respectively) prepared with the same sample. The detection limit for the presence of SRB in MPN tubes was 0.1% TRIS. After 34 days of incubation, the dilution series A, B, and C were positive with 107 ml of sample (1.30% TRIS), 106 ml of sample (2.56% TRIS), and 106 ml of sample (0.25% TRIS), respectively, yielding a viable count of 1.3 × 107 cells/ml. TRIS values for tubes considered negative for SRB on day 34 are shown by numbers. Note the enlarged scale for TRIS values of  1%.

Enumeration of SRB in activated-sludge enrichment cultures by T-MPN and N-MPN methods. SRB in an anaerobic sludge enrichment culture were enumerated by the T-MPN method (sludge medium and API-RST medium) and the N-MPN method (API-RST medium) at 30°C (Table 4). As shown in Table 4, the viable counts of SRB determined by the T-MPN method with sludge medium were significantly higher than the viable counts determined by both the N-MPN method and the T-MPN method with API-RST medium from day 8 on. After 34 days of incubation, the SRB counts determined by the T-MPN method (sludge medium) and the N-MPN method (API-RST medium) were very different (1.5 × 10⁷ and 4.3 × 10⁴ cells · ml¹, respectively). There was a general tendency for T-MPN values to be lower than N-MPN values with the same synthetic medium, which indicates that blackening of the medium may not always be due to dissimilatory sulfate reduction (Tables 2 and 4).

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View larger version (16K): [in this window] [in a new window]   FIG. 2.   Time course of sulfate reduction in an activated sludge enrichment culture used for MPN determination (Table 4). Sulfate reduction was measured in the presence () and in the absence () of antibiotics (chloramphenicol and streptomycin). The initial sulfate concentration was 4.2 mM. The isotope concentration was 20 kBq of 35SO42/ml. The inset shows a 28-h time course for the same experiment.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Sulfate reduction by Desulfobulbus propionicus after inoculation into defined growth medium (DSM 194 medium) in the presence () and in the absence () of antibiotics (chloramphenicol and streptomycin). The initial sulfate concentration was 23.1 mM. The isotope concentration was 20 kBq of 35SO42/ml. The inset shows a 24-h time course for the same experiment.

T-MPN enumeration of a pure culture of Desulfobulbus propionicus. The average cell density in the experimental culture was 8.3 × 10⁶ cells · ml¹ as determined by direct counting, whereas the T-MPN method yielded a slightly lower value, 1.4 × 10⁶ cells · ml¹, after 18 days of incubation (95% confidence limits, 0.4 × 10⁶ to 5.1 × 10⁶ cells · ml¹). A sulfate reduction rate of 0.34 mM day¹ (r² = 0.996) was obtained for the Desulfobulbus propionicus culture used for MPN enumeration (Fig. 3). From these data a qSO₄² value of 41 × 10¹⁵ mol of SO₄² cell¹ day¹ was calculated by using direct count cell numbers, and a qSO₄² value of 243 × 10¹⁵ mol of SO₄² cell¹ day¹ was calculated when the cell number estimated by the T-MPN method was used (Table 3).

	DISCUSSION

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The viable counts of SRB determined by the T-MPN method with natural media (slurries of marine sediment and anaerobic sludge) were typically 100- to 1,000-fold higher than the N-MPN and T-MPN estimates obtained when different synthetic media were used (Tables 1, 2, and 4) and yielded qSO₄² values similar to those reported in pure-culture studies of SRB. This indicates that viable counts of SRB were not seriously underestimated by the T-MPN method with natural media described here. The high counting efficiency of the T-MPN method with natural media was primarily due to the use of natural media prepared from sterilized sample material, since the MPN values obtained with the T-MPN and N-MPN methods with the same synthetic media were much lower (Tables 2 and 4).

Source material is often included in isolation and enumeration media. Thus, clarified rumen fluid has been used extensively as a constituent of media used for the isolation of rumen microorganisms (29, 31). Other examples include the use of soil extract (47) and bacterial extracts (44) in isolation media. Furthermore, many media contain extracts from blood, serum, animal tissues, fecal material, or digestor or sewage sludge supernatant mainly in order to promote growth of microorganisms with fastidious and/or unknown requirements. However, unsupplemented natural media have to our knowledge not been used previously for MPN enumerations of SRB.

In a comprehensive study of dissimilatory sulfate reduction in marine sediments, Jørgensen (24) concluded that sulfate reduction rates in coastal sediments were roughly proportional to colony counts of SRB, even though the actual numbers of bacteria appeared to be grossly underestimated. Jørgensen reported qSO₄² values for SRB in different marine sediments on the order of 10¹² to 10¹¹ mol of SO₄² cell¹ day¹, values which are much higher than the values determined in pure-culture studies (10¹⁵ to 10¹⁴ mol of SO₄² cell¹ day¹). This difference in metabolic rates (qSO₄²) led Jørgensen to the conclusion that viable counts of SRB in marine sediment obtained by the N-MPN method with synthetic media underestimate the true numbers by at least 2 orders of magnitude. He suggested that the low recovery of SRB from sediments could be due to clumping of cells and the inability of many naturally occurring SRB to grow in the enumeration media used. Many media used for enumeration of SRB contain lactate as the sole electron donor, and this compound is a substrate which cannot be utilized by several species of cultured SRB, including most Desulfobacter species, Desulfotomaculum acetoxidans, Desulfovibrio baarsii, some Desulfobacterium species, Desulfococcus niacini, and Desulfonema magnum (45). In order to improve the recovery of SRB, Gibson et al. (12) used MPN media containing surfactants and different growth substrates (acetate, lactate, propionate, butyrate, and H₂-CO₂) known to be important for sulfate reduction in situ. Although these modifications did increase the viable counts, the numbers of SRB were still underestimated, as shown by the resulting high qSO₄² values (mean qSO₄², 4.42 × 10¹² mol of SO₄² · cell¹ · day¹). Gibson et al. suggested that a large part of the natural population of SRB is physiologically different from laboratory isolates and is not able to grow in enumeration media containing high levels of organic substrates. Bak and Pfennig (5) also proposed that substrate inhibition was the cause of the low bacterial counts obtained during enrichment of SRB at butyrate concentrations of >7 mM. It is well-known that many bacteria from oligotrophic environments can be isolated only in media containing low levels of substrate (38). The concentration of organic substrates in enumeration media is typically 20 mM, which could be inhibitory to ecologically important types of SRB not yet isolated.

Addition of particles to growth media has been reported to stimulate bacterial metabolism and growth (26, 39, 46). The high particle content (approximately 20 to 30%, vol/vol) of the natural media could have contributed to the high MPN numbers observed in the present investigation. The natural media used for enumeration of SRB in the present study were not amended with electron donors and therefore contained only naturally occurring substrates at in situ concentrations. As shown in Fig. 1, natural media prepared from Kysing Fjord sediment contained sufficient electron donors to support complete reduction of approximately 17 mM sulfate in the tubes containing the lowest dilutions (natural media prepared from activated sludge supported reduction of at least 9 mM sulfate). Part of the electron donors used for sulfate reduction were probably produced during incubation by hydrolytic and fermentative bacteria coinoculated with the SRB. It should be noted that MPN enumerations carried out with sediment media having lower organic contents did not result in complete reduction of sulfate in the media; instead, TRIS values between 15 and 60% were typically obtained after 4 weeks of incubation.

The detection limit (scoring limit) for the presence of SRB in T-MPN enumeration tubes was set at 0.1% TRIS. This value was chosen on the basis of numerous distillations of sterile media (control tubes injected with 20 kBq of radioactive sulfate ml¹). Usually, TRIS values obtained with sterile media were much lower than 0.05% and not significantly higher than the radioactive background values, showing that the carryover of ³⁵SO₄²-containing aerosols into the zinc acetate traps was negligible.

Theoretical calculations (assuming worst-case scenarios), growth experiments performed with Escherichia coli, and MPN enumerations with sulfate-depleted freshwater sediments showed that interference from assimilatory sulfate reduction (i.e., false positives) could be neglected at sulfate concentrations greater than approximately 6 mM (data not shown).

Consequently, when the T-MPN method with natural media is used for enumeration of SRB in low-sulfate environments, such as freshwater sediments, false positives in the highest-dilution tubes can be avoided by adding nonradioactive sulfate at a final concentration of 6 mM to the natural media. Alternatively, the detection limit can be increased to 0.5% or a higher percentage of TRIS.

As shown in Fig. 1, a prolonged incubation period (34 days) was required before a TRIS value of 0.1% (scoring limit) was observed in the series C tube containing 10⁶ ml of sample. Hence, our method does not decrease the long incubation periods often used in MPN studies. However, compared to synthetic media, significantly higher estimated T-MPN values were obtained after 14 days of incubation in sediment medium (Tables 1 and 2) and after only 8 days of incubation in sludge medium (Table 4).

Table 3 summarizes qSO₄² values calculated from data obtained in this study. Cell densities obtained by the T-MPN method with natural media yielded qSO₄² values on the order of 10¹⁴ to 10¹³ mol of SO₄² cell¹ day¹ for both sediment slurries and activated sludge. Our qSO₄² values are thus at least 2 orders of magnitude lower than previously reported values obtained with marine sediments (10¹² to 10¹¹ mol of SO₄² cell¹ day¹) (24), as well as activated sludge and anaerobic sludge (10¹¹ and 10⁷ mol of SO₄² cell¹ day¹ (27), and similar to the values obtained in pure-culture studies (10¹⁵ to 10¹⁴ mol of SO₄² cell¹ day¹) (17, 19, 24).

All MPN techniques inherently tend to underestimate the actual numbers of SRB in environmental samples due to clumping of cells and attachment of cells to particulate matter. Gibson and coworkers (12) demonstrated that viable counts of SRB in marine sediment could be increased by 0 to 200% (average, 53%) by adding surfactants to enumeration media. The T-MPN method with natural media described in the present study always yielded 100- to 1,000-fold-higher viable counts of SRB than the T-MPN and N-MPN methods with synthetic media without any use of surfactants. The high MPN counting efficiencies obtained in this study thus suggest that cell clumping and attachment of SRB to particulate matter are not the main causes of the low levels of recovery of SRB observed with synthetic media.

The T-MPN method with natural media has several advantages over previously described methods for enumerating SRB: (i) it uses the natural environment of the SRB as the growth medium; (ii) it can easily be adapted to different environments; (iii) it yields significantly higher MPN counts than synthetic enumeration media; (iv) it selectively counts SRB performing dissimilatory sulfate reduction; and (v) it is insensitive to false positives due to assimilatory sulfate reduction by coexisting anaerobic bacteria and other H₂S-producing reactions.

The natural media used in the present study were prepared from sample material which had been sterilized twice by autoclaving and diluted to a suitable consistency. It is noteworthy that even though this treatment must have resulted in drastic changes in in situ conditions (chemically and physically), it produced growth media which were far superior to any of the synthetic enumeration media tested. Since the ecological niches of uncultured SRB in nature are not known, it may be very difficult to develop suitable cultivation techniques for these bacteria based on synthetic media. Attempts should be made to identify and isolate those SRB present in the highest MPN dilution tubes containing natural media since these bacteria are likely to be the bacteria which are predominant in the environment investigated.

The principle of using natural media should be widely applicable to MPN enumeration of microorganisms in diverse ecosystems, especially those physiotypes which can be detected by radiotracers or sensitive chemical methods (e.g., methanogens, methanotrophs, and denitrifiers).