Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

The following organisms were used in this study. Strain SI was enriched and isolated in this study as a thermophilic propionate-oxidizing syntroph. Clostridium acetobutyricum (DSM 792), Thermodesulfovibrio yellowstonii (DSM 11347), Syntrophobacter wolinii (DSM 2805), Desulfotomaculum thermobenzoicum (DSM 6193),Desulfotomaculum nigrificans (DSM 574),Desulfotomaculum thermosapovorans (DSM 6562),Desulfotomaculum thermocisternum (DSM 10259),Methanosaeta thermophila (DSM 6194), andMethanobacterium thermoautotrophicum strain ΔH (DSM 1053) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Braunschweig, Germany).Methanobacterium thermoformicicum type II was isolated in our laboratory.

Granular sludge was sampled from a lab-scale upflow anaerobic sludge blanket (UASB) reactor (13-liter capacity) operated at a thermophilic temperature (55°C) as described previously (29, 30). The reactor had been fed with a synthetic substrate containing sucrose, acetate, propionate, and yeast extract (4.5:2.25:2.25:1; chemical oxygen demand [COD] ratio, final concentration, 4,000 mg of COD per liter) over 2 years of operation.

The culture medium used for enrichment and isolation of thermophilic propionate-oxidizing syntrophs was prepared as described previously (31). All cultivations were carried out at 55°C in 50-ml serum vials containing 20 ml of medium (pH at 25°C, 7.2) under an atmosphere of N₂-CO₂ (80/20, vol/vol) without shaking. Neutralized substrates, normally propionate (final concentration, 20 mM), were added to the vials from stock solutions prior to inoculation. The purity of syntrophic propionate oxidizers was routinely checked by microscopy and incubation of the cultures with the medium containing 0.1% yeast extract and 20 mM sucrose at 35 or 55°C.

To test growth and substrate utilization, autoclaved or filter-sterilized substrates were added to the medium. Growth and substrate utilization were determined by monitoring increments of optical density at 600 nm (OD₆₀₀) and the production of acetate and hydrogen, respectively. In syntrophic growth and substrate utilization tests,M. thermoautotrophicum strain ΔH was added to the medium (2% inoculum) and growth and substrate utilization were checked by measuring the OD₆₀₀ and methane production.

DNA extraction and PCR amplification of bacterial 16S rRNA genes (rDNAs) from an enrichment culture of thermophilic propionate oxidizers were done as described previously (29). The following primer set was used for the amplification: the bacterial domain-specific primer 341F (5′-GGTTACCTTGTTACGACTT-3′, Escherichia colipositions 341 to 357) (22) and the universal primer 1490R (5′-GGTTACCTTGTTACGACTT-3′, E. coli positions 1491 to 1509) (45). The PCR products were purified with a MicroSpin column (Amersham Pharmacia Biotech), followed by cloning into plasmids using a TA cloning kit (Novagen). Twenty clonal rDNAs were randomly picked and subjected to restriction fragment length polymorphism (RFLP) analysis using HaeIII (Nippon Gene) as the restriction enzyme. Digested clonal rDNAs were electrophoresed in 2.0% agarose gels, and identical rDNA clones were distinguished based on electrophoresis pattern. Representative clones having different RFLP patterns were then sequenced.

DNA extraction from pure culture of a propionate oxidizer was performed according to the method of Hiraishi (10). 16S rDNA was amplified by PCR as described above, except that the forward PCR primer used in the amplification was the bacterial-domain-specific primer 8F (5′-AGAGTTTGATCCTGGCTCAG-3′, E. colipositions 8 to 27) (45). The PCR products were purified with a MicroSpin column (Amersham Pharmacia Biotech) and were subjected to further analysis.

Sequences of representative rDNA clones as well as the 16S rDNAs of pure cultures were determined with a Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) and an automated sequence analyzer (model DSQ-1000L; Shimadzu). The 16S rDNA sequences were aligned by using the CLUSTAL X package (38). Phylogenetic trees were constructed by the neighbor-joining method (27) with the MEGA package (14). Bootstrap analyses (6) for 1,000 replicates were performed to estimate the confidence of tree topologies.

Fixation of cells in the enrichment and pure cultures of propionate oxidizers was performed as described previously (30). Intact sludge granules were also fixed using 4% paraformaldehyde and ethanol as described previously (29). Whole-cell in situ hybridization was performed based on the method described elsewhere (30). We used the following 16S rRNA-targeted oligonucleotide probes in this study: TGP690 for strain SI, which was isolated as a thermophilic, syntrophic, propionate-oxidizing bacterium in this study (5′-CTCAAGTCCCTCAGTTTCAA-3′, E. coli positions 690 to 709), the EUB338 probe for the domain Bacteria(1), the ARC915 probe for the domain Archaea(33), and the MB1174 probe for the familyMethanobacteriaceae (25). The oligonucleotide probes were labeled with either rhodamine or Cy-5. Hybridization was carried out at 46°C for 3 h with 5 ng of fluorescence probe per μl of hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 7.2], 0.01% sodium dodecyl sulfate). The stringency of hybridization was adjusted by adding formamide to the hybridization buffer (15% [vol/vol] for TGP690, 20% for EUB338, and 35% for ARC915 and MB1174). For the determination of probe specificity, the following reference organisms were used: C. acetobutyricum (DSM 792),T. yellowstonii (DSM 11347), S. wolinii (DSM 2805), D. thermobenzoicum (DSM 6193), D. nigrificans (DSM 574), D. thermosapovorans (DSM 6562), and D. thermocisternum (DSM 10259). For in situ hybridization counting of targeted cells, the samples were dispersed with sonication and immobilized on glass slides coated with Vectabond (Vector Laboratories). In order to enumerate the total cell number in the samples, the cells were stained with 4′,6′-diamidino-2-phenylindole (DAPI) at a final concentration of 5 μg/ml. Duplicate counts of over 1,000 DAPI-stained cells were performed to determine the ratio of the probe-labeled cells to the total cells.

Slot blot hybridization was also performed to estimate the specificity of the designed probe as described elsewhere (30). Digoxigenin (DIG)-labeled TGP690 and universal probe 530R (15) were used for the hybridization and were detected using a DIG nucleic acid detection kit (Boehringer Mannheim) essentially according to the manufacturer's instructions. For the determination of the probe specificity, rDNA clones, which were recovered in this study, and rDNAs from the reference organisms mentioned above were used.

The Gram-staining reaction was performed by the method of Hucker as reported by Doetsch (4). Phase-contrast micrographs were taken by using wet mounts on agar-coated slides (24) for exponential-phase cultures. Cells immobilized and hybridized on glass slides were viewed with a fluorescence microscope (Olympus model BX50F), and the sections hybridized with the probes were examined under a confocal laser scanning microscope (Olympus Fluoview model BX50). Scanning electron micrographs were obtained as described previously (29). Short-chain fatty acids were determined with a gas chromatograph (Shimadzu GC-14A with flame ionization detection; packing material, FAL-M (GL Science); column temperature, 125°C). Determination of alcohols and other compounds was performed by high-performance liquid chromatography using a RSpak KC-811 column (Shodex; eluent, 3 mM HClO₄; column temperature, 50°C) and a UV detector (wavelength, 210 nm; Shimadzu SPD-10A). Methane, hydrogen, and carbon dioxide were determined by gas chromatography (GL Science model 370 with thermal conductivity detection; packing material, Unibeads C; column temperature, 145°C). Sulfate was determined by ion chromatography (Shimadzu Shim-pack IC-A1 column; carrier, 25 mM potassium biphthalate; detector type, electrical conductivity detector).

The 16S rDNA sequence of strain SI was deposited in the EMBL, GenBank, and DDBJ databases under the accession no. AB035723 .

Sludge granules were sampled from a thermophilic (55°C), methanogenic, UASB reactor which had been receiving an artificial wastewater containing sucrose, propionate, and acetate as the main carbon sources (29, 30). The artificial wastewater contained approximately 8 mM propionate, but only a small amount of propionate was detected in the effluent, suggesting that propionate in the wastewater had been effectively removed by certain populations in the thermophilic sludge. For enrichment of propionate-degrading populations, thermophilic granules were gently washed, homogenized anaerobically, and used for primary enrichment with 20 mM propionate as the sole substrate. Growth and methane formation were obtained after 1 month of incubation. The culture was successively transferred into the fresh medium every 2 months (1% inoculum). Over 10 successive transfers, the culture produced methane and degraded propionate. However, during the transfer, the enrichment sometimes became unstable: the growth and methane production stopped unexpectedly, as was previously observed by Stams et al. (34). The addition of FeCl₂ (1 mM) (34) was effective to some extent, although it did not completely prevent the unexpected stagnation. Relatively stable growth was eventually observed when the culture was left without any agitation, such as shaking of the vials manually for daily observation, during the exponential growth phase. By combining this technique with the addition of FeCl₂, we were able to obtain a stable propionate-degrading enrichment.

As shown in Fig. 1 the enrichment contained at least four morphologically distinct organisms: (i) F₄₂₀ autofluorescent rods morphologically resemblingMethanobacterium, (ii) thick rods, and (iii) oval and (iv) rod-shaped microbes, both of which seemed to form spores. We focused on the spore-forming oval and rod-shaped microorganisms and attempted to establish a defined coculture with M. thermoautotrophicum. The enrichment culture after 10 transfers was pasteurized at 90°C for 20 min and serially diluted into propionate medium containing M. thermoautotrophicum strain ΔH (5% inoculum). We verified that the pasteurized enrichment no longer showed growth and methane formation coupled with propionate degradation without M. thermoautotrophicum ΔH cells. The culture receiving the 10⁻¹ to 10⁻⁴ serial dilutions showed growth, propionate degradation, and methane production. By employing serial dilution in combination with pasteurization several times, we obtained a highly defined enrichment coculture in which both the oval and rod-shaped microbes were still present.

Several attempts were made to further purify the coculture, i.e., by coculture isolation by the roll tube method on propionate medium in the presence of M. thermoautotrophicumΔH, but all attempts were unsuccessful. We could not observe any colonies in roll tubes containing propionate and M. thermoautotrophicum cells. Therefore, we then tried to identify the propionate oxidizers by using an rRNA approach. From the highly purified coculture after serial dilutions with pasteurization, bacterial 16S rDNAs were amplified and cloned into E. coli, and RFLP analysis with HaeIII was performed using 20 randomly selected rDNA clones. The cloning and RFLP analyses revealed that two types of clones could be recovered from the coculture: 19 clones had the same electrophoresis pattern, and the remaining clone had a different pattern. Sequence analysis of the two unique types of clones (type I and type II) showed that both types of clones were affiliated with the genus Desulfotomaculum. In particular, the type I clones were closely related to the 16S rDNA clones of propionate-oxidizing bacterial spores A and B (Fig.2) (sequence similarity, 94 and 95%, respectively), which have recently been reported to be mesophilic, spore-forming, syntrophic, propionate-oxidizing bacteria enriched from a freeze-dried granular sludge (7). Through more detailed analysis of the clonal sequences, the type II clone was thought to be a chimeric artifact because the clone seemed to contain two types of 16S rDNA; a half of the sequence showed a high similarity with type I clones (approximately 93% similarity), and the remainder formed a clade with members of the Clostridium-Bacillus subclass, but there were no close relatives in the databases (data not shown).

To determine whether the type I clones were derived from the dominant populations in the highly purified coculture, a specific oligonucleotide probe was designed and applied to the coculture. The specificity of the designed probe (TGP690) was first evaluated using seven reference organisms (Materials and Methods) in whole-cell in situ hybridization. While all bacteria reacted with the universalBacteria probe EUB338 (1), none of the cells hybridized with the TGP690 probe at any formamide concentrations in hybridization and washing buffers (data not shown). On the other hand, both the oval and rod-shaped cells in the highly purified coculture hybridized with the TGP690 probe (data not shown). By changing the formamide concentration in the hybridization and washing buffers, 15% of the formamide in the buffers was found to give the highest stringency for the probe with the oval and rod-shaped bacteria. Moreover, slot blot analysis with the DIG-labeled TGP690 probe revealed that the type I clones hybridized with the TGP690 probe, although none of the rDNAs from the reference organisms hybridized with the TGP690 probe (data not shown). Throughout the enrichment experiments, the culture contained a number of the oval and rod-shaped cells, which reacted with the TGP690 probe. These observations indicated that the microbe possessing the 16S rDNA detected as the type I clone was the dominant propionate-oxidizing bacterium in the highly purified coculture.

Phylogenetic analysis (Fig. 2) and in situ hybridization experiments indicated that the predominant propionate-oxidizing bacterium was a member of theDesulfotomaculum group. Therefore, the isolation strategy was planned based on the physiological properties of the genusDesulfotomaculum. Attempts to isolate the propionate oxidizer were made using different substrates which are generally utilized by Desulfotomaculum species, such as propionate, butyrate, pyruvate, hydrogen, ethanol, and lactate as the electron donors in the presence of sulfate. In this approach, we screened the cells grown on different substrates by using in situ hybridization with the TGP690 probe. Through this approach, we found that 10 mM ethanol and 20 mM sulfate allowed the TGP690-positive rods and M. thermoautotrophicum ΔH-like autofluorescent rods to grow. Other substrates yielded no growth or sometimes supported the growth of microbes that did not react with the TGP690 probe; for example, pyruvate plus sulfate and lactate plus sulfate supported fortuitous growth of vibrios, which were later identified asThermodesulfovibrio sp. (data not shown). After several successive transfers of the coculture on ethanol plus sulfate medium, the culture still contained autofluorescent methanogens and produced methane and acetate in addition to degrading ethanol. This coculture had a much higher specific growth rate (2.4 day⁻¹ based on methane production) than that on propionate (0.19 day⁻¹). We then applied serial dilution (roll tube) using ethanol (no sulfate) as the substrate again with M. thermoautotrophicum ΔH cells. This procedure resulted in isolation of the pure coculture of rod-shaped cells and M. thermoautotrophicum ΔH (Fig. 3A and B). The ethanol conversion of the defined coculture is shown in Fig. 4A. The coculture grown on ethanol was then inoculated into the medium containing propionate, resulting in the degradation of propionate with methane production and growth (Fig. 4B).

To isolate the propionate-oxidizing bacterium in pure culture, the defined coculture on ethanol medium was used as the inoculum with various substrates such as pyruvate, fructose, fumarate, glucose, crotonate, and yeast extract. Of the substrates tested, only pyruvate (20 mM) plus yeast extract (0.01%) supported the growth of the rod-shaped cells after a week of incubation. After three successive transfers into the pyruvate plus yeast extract liquid medium, we conducted roll tube isolation using pyruvate and yeast extract as substrates. Small colonies that were yellowish, lens shaped, and 0.1 to 0.2 mm in diameter were formed after 1 week of incubation. This step was repeated several times, and the purified strain, designated strain SI, was obtained (Fig. 3C). 16S rDNA sequence and in situ hybridization analyses indicated that the strain was the same bacterium as that detected in the previous enrichment cultures on propionate and the defined coculture on ethanol medium. By recombining strain SI withM. thermoautotrophicum ΔH, we again confirmed that the strain syntrophically oxidized propionate.

Strain SI had nonmotile, rod-shaped cells. They were 1.7 to 2.8 μm long and 0.7 to 0.8 μm wide. Gram staining was negative. Spore formation was not observed in pure culture on pyruvate or in coculture on propionate or ethanol with M. thermoautotrophicum ΔH. However, the strain formed spores when they grew on propionate medium in tri-culture with M. thermoautotrophicum ΔH andMethanosaeta thermophila, in which the complete conversion of propionate to methane was observed (Fig. 4C). In the tri-culture, the cells were rod shaped or oval. Both in pure culture and coculture, the growth of strain SI was stimulated by the presence of 0.01% yeast extract. Although the strain was closely related to members of theDesulfotomaculum group on the basis of phylogeny, we eventually found that the strain could not utilize sulfate as an electron acceptor in the presence of ethanol, lactate, or propionate. This finding was also verified by using concentrated cultures with lactate or ethanol in the presence of sulfate (data not shown).

To elucidate the abundance and spatial distribution of strain SI in the sludge granules which were used as the inoculum for the enrichment study, the fluorescence in situ hybridization technique combined with confocal laser scanning microscopy was applied to thin sections of the granules as described previously (29). In situ hybridization was performed with thin (10- to 15-μm) sections using the rhodamine-labeled TGP690 probe and the Cy-5-labeled ARC915 probe. As described in the previous report, the granules used in this study showed a layered structure; the outer layer was dominated mainly by bacterial cells, whereas the inner layer was occupied mainly by archaeal cells (29). The application of the TGP690 probe to the thermophilic sludge granule sections showed that a number of rod-shaped cells, showing a morphology similar to that of strain SI, were detected inside the granules (Fig.5). They formed large colonies inside the granules and were found to be located in the middle and inner layers of the sludge granules. Double staining with the TGP690 probe and theMethanobacteriaceae-specific probe MB1174 (25) showed a number of aggregates, in which TGP690-positive cells and Methanobacterium-like cells were closely associated with each other (Fig. 5B to E). Direct in situ hybridization counting after dispersing the granules revealed that strain SI-type cells in the sludge accounted for approximately 1.1% ± 0.6% of the total cells.

As with other syntrophs, it took a long time to isolate the syntroph responsible for propionate oxidation from the enrichment culture. One of the primary reasons for the difficulty in isolation was that the growth of the propionate oxidizer was very slow on propionate medium. The specific growth rate of strain SI on propionate medium was estimated to be 0.19 day⁻¹ in both the coculture with M. thermoautotrophicum and the tri-culture with M. thermoautotrophicum and M. thermophila. The value was similar to those of the other known species of propionate-oxidizing syntrophs; Stams et al. reported that the specific growth rates of a thermophilic propionate-oxidizing enrichment cocultured with M. thermoautotrophicum and tri-cultured with M. thermoautotrophicum and M. thermophila were 0.15 and 0.25 to 0.32 day⁻¹, respectively (34).

Another difficulty in isolation was that the oxidation of propionate by our thermophilic enrichment and defined coculture was somewhat unstable, similar to a previous finding (34). Such unstable growth was observed only when they grew on propionate. In contrast, they grew stably at much higher growth rates when ethanol (coculture) or pyruvate (pure culture) was used as the growth substrate. The reason why such unstable growth occurred is not clear. However, particularly with the syntrophic propionate oxidation reaction, it is well known that syntrophs and hydrogen scavengers have to be in close association with each other to keep the hydrogen partial pressure very low. In syntrophic coculture with strain SI and M. thermoautotrophicum on propionate medium, the hydrogen partial pressure was determined to be 26 to 40 Pa. On the other hand, that of the coculture grown on ethanol was 65 to 81 Pa. These results suggest that the cocultured cells grown on propionate had to keep close together by forming aggregates. In the early stage of enrichment, we shook vials by hand for daily observation. This was probably the cause of such instability in cultivation.

Based on the comparative analysis of 16S rDNA sequences, strain SI was found to be closely related to members of theDesulfotomaculum group. The genusDesulfotomaculum is characterized to contain gram-positive, spore-forming, sulfate-reducing microorganisms (46). The most notable difference that separates our strain from all the other members of this genus is that strain SI does not reduce sulfate. Although the strain could oxidize lactate and ethanol as well as propionate in syntrophic association with hydrogenotrophic methanogens, the strain could not utilize these compounds with sulfate reduction in pure culture. These facts indicate that the strain lacks its dissimilatory sulfate-reducing ability, similar to the 3-hydroxybenzoate-degrading anaerobe Sporotomaculum hydroxybenzoicum, which is also closely affiliated with members of the genus Desulfotomaculum (3). Another difference that separates strain SI from the others is its ability to grow syntrophically in coculture with hydrogenotrophic microorganisms. Of the Desulfotomaculum species, only D. nigrificans is known to grow syntrophically on ethanol and lactate with hydrogenotrophic methanogens in the absence of sulfate (13). In addition, only one species, D. thermocisternum, is known to be a thermophilic propionate-oxidizing syntroph, although a detailed description is not available (23). We tested whether the type strain ofD. thermocisternum could be cocultured with hydrogenotrophic methanogens (M. thermoautotrophicumΔH or M. thermoformicicum type II) on propionate. However, it never grew or oxidized propionate.

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy and in situ direct enumeration revealed that strain SI was widely distributed in the granules and that it was one of the significant populations. Double staining with the TGP690 and MB1174 probes, specific for the propionate-oxidizing strain and the familyMethanobacteriaceae, respectively, could detect a number of aggregates in thin sections of granules, in which strain SI-type cells were always surrounded by Methanobacterium-like cells. Such proximity between the two types of microbes suggested that strain SI was performing a hydrogen-forming (syntrophic) reaction in situ. According to the calculation described previously, the physical distance between propionate oxidizers and hydrogenotrophic methanogens must be kept under 1 μm to make the oxidation reaction energetically possible (35). Our observation also supported this calculation since the propionate-oxidizing syntrophs andMethanobacterium-like cells were very closely associated with each other (Fig. 5).

Strain SI was found to syntrophically utilize ethanol and lactate as well as propionate. It is reported that a sucrose-feeding thermophilic sludge produced lactate and ethanol as major intermediates (37); hence, strain SI may contribute not only to propionate oxidation but also to oxidation of such kinds of intermediates in the granules.

Our research has several important points. First, this report is the first unequivocal description of a thermophilic propionate-oxidizing syntroph. Second, the organism found in this study is a non-sulfate-reducing microorganism phylogenetically close to theDesulfotomaculum group but may have multiple functions in the methanogenic ecosystems in terms of syntrophic and nonsyntrophic fermentation of intermediate organic substances, although its in situ substrate remains to be clarified. Third, we were able to visualize the spatial distribution of this type of organism in the methanogenic granules by using 16S-rRNA-targeted, fluorescently labeled oligonucleotide probes combined with confocal laser scanning microscopy and found that the organism is widely distributed in the granules and forms close associations with hydrogenotrophic methanogens. More detailed growth and physiological properties will be reported in the future.