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 thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures.

The effects of acetate, propionate, and butyrate on the anaerobic thermophilic conversion of propionate by methanogenic sludge and by enriched propionate-oxidizing bacteria in syntrophy with Methanobacterium thermoautotrophicum delta H were studied. The methanogenic sludge was cultivated in an upflow anaerobic sludge bed (UASB) reactor fed with propionate (35 mM) as the sole substrate for a period of 80 days. Propionate degradation was shown to be severely inhibited by the addition of 50 mM acetate to the influent of the UASB reactor. The inhibitory effect remained even when the acetate concentration in the effluent was below the level of detection. Recovery of propionate oxidation occurred only when acetate was omitted from the influent medium. Propionate degradation by the methanogenic sludge in the UASB reactor was not affected by the addition of an equimolar concentration (35 mM) of butyrate to the influent. However, butyrate had a strong inhibitory effect on the growth of the propionate-oxidizing enrichment culture. In that case, the conversion of propionate was almost completely inhibited at a butyrate concentration of 10 mM. However, addition of a butyrate-oxidizing enrichment culture abolished the inhibitory effect, and propionate oxidation was even stimulated. All experiments were conducted at pH 7.0 to 7.7. The thermophilic syntrophic culture showed a sensitivity to acetate and propionate similar to that of mesophilic cultures described in the literature. Additions of butyrate or acetate to the propionate medium had no effect on the hydrogen partial pressure in the biogas of an UASB reactor, nor was the hydrogen partial pressure in propionate-degrading cultures affected by the two acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment of thermophilic syntrophic anaerobic glutamate-degrading consortia using a dialysis membrane reactor

A dialysis cultivation system was used to enrich slow-growing moderately thermophilic anaerobic bacteria at high cell densities. Bicarbonate buffered mineral salts medium with 5 mM glutamate as the sole carbon and energy source was used and the incubation temperature was 55 degrees C. The reactor inoculum originated from anaerobic methanogenic granular sludge bed reactors. The microbial population was monitored over a period of 2 years using the most probable number (MPN) technique. In the reactor glutamate was readily degraded to ammonium, methane, and carbon dioxide. Cell numbers of glutamate-degrading organisms increased 400-fold over the first year. In medium supplemented with bromoethane sulfonic acid (BES, an inhibitor of methanogenesis), tenfold lower cell numbers were counted, indicating the syntrophic nature of glutamate degradation. After 2 years of reactor operation the predominant organisms were isolated and characterized. Methanobacterium thermoautotrophicum (strain R43) and a Methanosaeta thermophila strain (strain A) were the predominant hydrogenotrophic and acetoclastic methanogens, respectively. The numbers in which the organisms were present in the reactor after 24 months of incubation were 8.6 x 10(9) and 3.8 x 10(7) mL(-1) sludge, respectively. The most predominant glutamate-degrading organism (8.6 x 10(7) mL(-1) sludge), strain Z, was identified as a new species, Caloramator coolhaasii. It converted glutamate to hydrogen, acetate, some propionate, ammonium, and carbon dioxide. Growth of this syntrophic organism on glutamate was strongly enhanced by the presence of methanogens.     

Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules

16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35 degrees C) and thermophilic (55 degrees C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-, Methanobacterium-, Methanospirillum-, and Methanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genus Methanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655-2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related to Syntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.     

Effects of hydrogen and formate on the degradation of propionate and butyrate in thermophilic granules from an upflow anaerobic sludge blanket reactor.

Degradation of propionate and butyrate in whole and disintegrated granules from a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor fed with acetate, propionate, and butyrate as substrates was examined. The propionate and butyrate degradation rates in whole granules were 1.16 and 4.0 mumol/min/g of volatile solids, respectively, and the rates decreased 35 and 25%, respectively, after disintegration of the granules. The effect of adding different hydrogen-oxidizing bacteria (both sulfate reducers and methanogens), some of which used formate in addition to hydrogen, to disintegrated granules was tested. Addition of either Methanobacterium thermoautotrophicum delta H, a hydrogen-utilizing methanogen that does not use formate, or Methanobacterium sp. strain CB12, a hydrogen- and formate-utilizing methanogen, to disintegrated granules increased the degradation rate of both propionate and butyrate. Furthermore, addition of a thermophilic sulfate-reducing bacterium (a Desulfotomaculum sp. isolated in our laboratory) to disintegrated granules improved the degradation of both substrates even more than the addition of methanogens. By monitoring the hydrogen partial pressure in the cultures, a correlation between the hydrogen partial pressure and the degradation rate of propionate and butyrate was observed, showing a decrease in the degradation rate with increased hydrogen partial pressure. No significant differences in the stimulation of the degradation rates were observed when the disintegrated granules were supplied with methanogens that utilized hydrogen only or hydrogen and formate. This indicated that interspecies formate transfer was not important for stimulation of propionate and butyrate degradation.     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Enrichment of Thermophilic Propionate-Oxidizing Bacteria in Syntrophy with Methanobacterium thermoautotrophicum or Methanobacterium thermoformicicum

Thermophilic propionate-oxidizing, proton-reducing bacteria were enriched from the granular methanogenic sludge of a bench-scale upflow anaerobic sludge bed reactor operated at 55°C with a mixture of volatile fatty acids as feed. Thermophilic hydrogenotrophic methanogens had a high decay rate. Therefore, stable, thermophilic propionate-oxidizing cultures could not be obtained by using the usual enrichment procedures. Stable and reproducible cultivation was possible by enrichment in hydrogen-pregrown cultures of Methanobacterium thermoautotrophicum ΔH which were embedded in precipitates of FeS, achieved by addition of FeCl₂ to the media. The propionate-oxidizing bacteria formed spores which resisted pasteurization for 30 min at 90°C or 10 min at 100°C. Highly purified cultures were obtained with either M. thermoautotrophicum ΔH or Methanobacterium thermoformicicum Z245 as the syntrophic partner organism. The optimum temperature for the two cultures was 55°C. Maximum specific growth rates of cultures with M. thermoautotrophicum ΔH were somewhat lower than those of cultures with M. thermoformicicum Z245 (0.15 and 0.19 day^-1, respectively). Growth rates were even higher (0.32 day^-1) when aceticlastic methanogens were present as well. M. thermoautotrophicum ΔH is an obligately hydrogen-utilizing methanogen, showing that interspecies hydrogen transfer is the mechanism by which reducing equivalents are channelled from the acetogens to this methanogen. Boundaries of hydrogen partial pressures at which propionate oxidation occurred were between 6 and 34 Pa. Formate had a strong inhibitory effect on propionate oxidation in cultures with M. thermoautotrophicum. Inhibition by formate was neutralized by addition of the formate-utilizing methanogen or by addition of fumarate. Results indicate that formate inhibited succinate oxidation to fumarate, an intermediate step in the biochemical pathway of propionate oxidation.     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes.

In situ hybridization with fluorescent oligonucleotides was used to detect and localize microorganisms in the granules of two lab-scale upflow anaerobic sludge blanket reactors that had been fed for several months with either sucrose or a mixture of volatile fatty acids. Sections of the granules were hybridized with 16S rRNA-targeted oligonucleotide probes for Bacteria, Archaea, specific phylogenetic groups of methanogens, and two syntrophic propionate-oxidizing strains, MPOB and KOPROP1. Cells of the syntrophic strain KOPROP1 were not detected in either type of sludge granules. Hybridizations of the sucrose-fed granules showed an outer layer of mainly bacterial microcolonies with different morphologies. More inwards of these granules, a layer of different methanogenic microcolonies mixed with large colonies of the syntrophic strain MPOB could be detected. The MPOB colonies were intertwined with hydrogen- or formate-consuming methanogens, indicating their syntrophic growth. The granules fed with volatile fatty acids showed an outer layer of mainly bacteria and then a thick layer of Methanosaeta-like methanogens mixed with a few bacteria and a layer of methanogens mixed with syntrophic MPOB microcolonies. The centers of both sludge types consisted of large cavities and methanogenic microcolonies. These results indicate a juxtapositioning of syntrophic bacteria and methanogens and provide additional evidence for a layered microbial architecture of anaerobic granular sludge.     

Population dynamics of propionate-oxidizing bacteria under methanogenic and sulfidogenic conditions in anaerobic granular sludge.

Laboratory-scale upflow anaerobic sludge-bed reactors were inoculated with industrial granular sludge and fed with either propionate or propionate and sulfate. The population dynamics of the propionate-oxidizing bacteria Desulfobulbus sp. and the syntrophically growing strain SYN7 were studied in reactors by dot blot and in situ hybridization with 16S rRNA-based oligonucleotide probes.     

Start-up of a thermophilic upflow anaerobic sludge bed (UASB) reactor with mesophilic granular sludge

Summary Fast start-up of thermophilic upflow anaerobic sludge bed (UASB) reactors was achieved at process temperatures of 46, 55 and 64° C, using mesophilic granular sludge as inoculum and fatty acid mixtures as feed. The start-up was brought about by increasing the temperature of mesophilic UASB reactors in a single step, which initially led to a sharp drop in the methane production rate. Thereafter, stable thermophilic methanogenesis was achieved within a period of 1 or 2 weeks depending on the temperature of operation. Mesophilic granules functioned initially as effective carrier material for thermophilic organisms. However, long-term operation led to disintegration of the granules, resulting in wash-out of thermophilic biomass. The temperature optima for acetotrophic methanogenic activity of the sludges cultivated at 46, 55 and 64° C, were similar, but differed significantly from the temperature optimum of the mesophilic inoculum. All the sludges examined were dominated by Methanothrix-like rods. These could be distinguished by antigenic fingerprinting into two subpopulations, one predominant at 36° C and the other predominant at 46° C and above. Offprint requests to: J. B. van Lier     

In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I.

We previously showed that very thin filamentous bacteria affiliated with the division green non-sulfur bacteria were abundant in the outermost layer of thermophilic methanogenic sludge granules fed with sucrose and several low-molecular-weight fatty acids (Y. Sekiguchi, Y. Kamagata, K. Nakamura, A. Ohashi, H. Harada, Appl. Environ. Microbiol. 65:1280-1288, 1999). Further 16S ribosomal DNA (rDNA) cloning-based analysis revealed that the microbes were classified within a unique clade, green non-sulfur bacteria (GNSB) subdivision I, which contains a number of 16S rDNA clone sequences from various environmental samples but no cultured representatives. To investigate their function in the community and physiological traits, we attempted to isolate the yet-to-be-cultured microbes from the original granular sludge. The first attempt at isolation from the granules was, however, not successful. In the other thermophilic reactor that had been treating fried soybean curd-manufacturing wastewater, we found filamentous microorganisms to outgrow, resulting in the formation of projection-like structures on the surface of granules, making the granules look like sea urchins. 16S rDNA-cloning analysis combined with fluorescent in situ hybridization revealed that the projections were comprised of the uncultured filamentous cells affiliated with the GNSB subdivision I and Methanothermobacter-like cells and the very ends of the projections were comprised solely of the filamentous cells. By using the tip of the projection as the inoculum for primary enrichment, a thermophilic, strictly anaerobic, filamentous bacterium, designated strain UNI-1, was successfully isolated with a medium supplemented with sucrose and yeast extract. The strain was a very slow growing bacterium which is capable of utilizing only a limited range of carbohydrates in the presence of yeast extract and produced hydrogen from these substrates. The growth was found to be significantly stimulated when the strain was cocultured with a hydrogen-utilizing methanogen, Methanothermobacter thermautotrophicus, suggesting that the strain is a sugar-fermenting bacterium, the growth of which is dependent on hydrogen consumers in the granules.     

Identification and cultivation of anaerobic, syntrophic long-chain fatty acid-degrading microbes from mesophilic and thermophilic methanogenic sludges

We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55 degrees C or 37 degrees C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [(13)C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.     

Effect of ethylene glycol-bis(β-aminoethyl ether)-N,N-tetraacetic acid (EGTA) on stability and activity of methanogenic granular sludge

Summary The effect of the calcium-specific chelant ethylene glycol-bis(\-aminoethyl ether)-N,N-tetraacetic acid (EGTA) on methanogenic granular sludge from a laboratory-scale upflow anaerobic sludge-blanket (UASB) reactor fed propionate and from a full-scale reactor treating paper-mill waste-water was studied. Upon treatment with EGTA both sludge types showed a decrease in the calcium and phosphorus content and a release of protein and polysaccharides, leading to a decrease in strength of papermill granular sludge and a disintegration of propionate-grown granules. After treatment of propionate-grown granular sludge with high EGTA concentrations, the methanogenic activity with propionate and acetate as test substrates decreased by 88 and 33%, respectively. The marked reduction in propionate oxidation activity may be caused by a disruption of the special juxtapositioning of bacteria in the granules. Offsprint requests to: A. J. B. Zehnder     

Effect of medium composition and sludge removal on the production, composition, and architecture of thermophilic (55 degrees C) acetate-utilizing granules from an upflow anaerobic sludge blanket reactor.

A thermophilic upflow anaerobic sludge blanket (UASB) reactor degrading acetate was started by applying published methods (W. M. Wiegant and A. W. A. de Man, Biotechnol. Bioeng. 28:718-77, 1986) for production of granules dominated by Methanothrix spp. The reactor was inoculated with thermophilic digested sludge. No granules were observed during the first 7 months of start-up of the UASB reactor. However, after the concentrations of potassium, phosphate, ammonium, and magnesium in the medium were gradually increased, granules developed, indicating that there was a critical concentration of one or more of the ions required for production of granules from the starting material. After several years of stable operation, the effect of removing 60% of the granular sludge was investigated. Immunologic qualitative and quantitative studies showed that removal of the granular sludge resulted in an increase in the number of the predominant methanogens, antigenically related to Methanosarcina thermophila TM-1 and Methanosarcina mazeii S-6, and Methanobacterium thermoautotrophicum delta H and GC1. These changes were accompanied by modifications of the microanatomy of the granules, as demonstrated histochemically and immunohistochemically. The results indicated that different catabolic pathways dominated in different regions of the granules, i.e., acetate oxidation in the middle of the granules, where there is a low acetate concentration, and an aceticlastic reaction in the outer surfaces, with a high acetate concentration. The results also showed that removal of granules from a UASB reactor which has been under steady-state operation for a long period can improve the reactor's performance via formation of denser and larger granules with improved microbial activities.     

Microbial characteristics of methanogenic sludge consortia developed in thermophilic U ASB reactors

The effect of temperature on granulation and microbial interaction of anaerobic sludges grown in thermophilic upflow anaerobic sludge bed (UASB) reactors was investigated at two different temperatures, 55°C (Run 1) and 65°C (Run 2). Each run consisted of two phases. Phase 1 was conducted by feeding acetate for a period of 200 days. In Phase 2, both reactors were fed a mixture of acetate and sucrose for a further 100 days. During Phase 1, no granulation occurred in the sludge of either run. Microscopic observation revealed that the predominant methanogen was Methanothrix in Run 1, whereas Methanobacterium-like bacteria existed to a significant extent in Run 2. The acetate-utilizing methanogenic activity of both sludges increased with increasing test temperature in the range 55–65°C. Since the acetate-grown sludges exhibited far higher H₂-utilizing methanogenic activity than acetate-utilizing methanogenic activity, it is suggested that a syntrophic association of acetate-oxidizing bacteria with hydrogenotrophic methanogens was responsible for a considerable portion of the overall acetate elimination in thermophilic anaerobic sludge. During Phase 2, granules coated with either filamentous bacteria or cocci-type bacteria (both presumably acid-forming bacteria) were successfully established in Run 1 and Run 2, respectively. Since the acetate-utilizing methanogenic activities of the granular sludges were four to five times higher than those of the acetate-grown sludges (Phase 1), the co-existence of these coating bacteria appeared to contribute to the enclosing of acetate consumers inside granules. Correspondence to: S. Uemura     

Monitoring of specific methanogenic activity of granular sludge by confocal laser scanning microscopy during start-up of thermophilic upflow anaerobic sludge blanket reactor

Confocal, laser-scanning microscopy was applied to acquire coenzyme F₄₂₀-based autofluorescence images of middle sections of sludge granules during start-up of a thermophilic reactor that were seeded with mesophilically-grown microorganisms of granular sludge. Digital images were analyzed to calculate weighted averages of autofluorescence. The values were related (r ²=0.97) to specific methanogenic activities of granular sludge as the granules developed to steady state.     

Bacteriological composition and structure of granular sludge adapted to different substrates

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.     

Bacteriological composition and structure of granular sludge adapted to different substrates.

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.