Department of Environmental Engineering, National Cheng Kung University, Tainan, Taiwan1
Graduate Institute of Environmental Engineering, National Central University, Chungli 32054, Taiwan2
Department of Biology, National Cheng Kung University, Tainan, Taiwan3
Author for correspondence: Wen-Tso Liu. Tel: +886 3422 7151 ext. 4683. Fax: +886 3426 9401. e-mail: liuwt{at}cc.ncu.edu.tw
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ABSTRACT |
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Keywords: denaturing gradient gel electrophoresis, clone library, 16S rDNA, in situ hybridization, UASB
Abbreviations: DGGE, denaturing gradient gel electrophoresis; FISH, fluorescent in situ hybridization; GNS, green non-sulfur; OTU, operational taxonomic unit; TEM, transmission electron microscopy; UASB, upflow anaerobic sludge bed
The GenBank accession numbers for the sequences obtained in this work are AF229774AF229793.
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INTRODUCTION |
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![]() | (1) |
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Recently, the upflow anaerobic sludge bed (UASB) reactor was successfully demonstrated to treat terephthalate-containing wastewater (Cheng et al., 1997 ; Kleerebezem et al., 1997
). In this system, microbial populations aggregate as granules or as individual micro-ecosystems. Each micro-ecosystem is composed of acetogenic bacteria which degrade complex organic compounds to a mixture of acetate, hydrogen and formate, and methanogenic Archaea, which continuously mineralize the intermediates to methane and carbon dioxide. These populations interact syntrophically, because the fermentative step from terephthalate to acetate is energetically unfavourable (
G0'>0) (equation 1) and requires the methanogenesis step (equations 2 and/or 3) as a coupling reaction to proceed (Theile & Zeikus, 1988
). Kleerebezem et al. (1999a
, b
) proposed that in a methanogenic consortium, terephthalate was degraded via decarboxylation to the transient intermediate benzoyl-CoA, and then to acetate and hydrogen, which were further mineralized to methane and carbon dioxide. They suggested that the degradation of terephthalate to acetate and hydrogen was performed by a fermentative population, and the methanogenesis step was performed by acetoclastic and hydrogenotrophic methanogens. However, due to the difficulty of isolating syntrophic bacteria (Schink, 1997
), the proposed metabolic pathway remains to be verified. Furthermore, the diversity and phylogenetic position of the fermentative population responsible for the initial terephthalate degradation have not been identified.
The advent of molecular techniques has made it feasible to study the microbial community of environmental ecosystems without cultivation (Amann et al., 1995 ). 16S rDNA-based methods employing denaturing gradient gel electrophoresis (DGGE) (Muyzer & Smalla, 1998
; Nielsen et al., 1999
) and molecular cloning (Barns et al., 1994
; Godon et al., 1997
; Sekiguchi et al., 1998
) can provide a rapid estimate of the microbial diversity and composition of a community. In addition, fluorescent in situ hybridization (FISH) with oligonucleotide probes targeting the 16S rRNA of micro-organisms can be used to evaluate the abundance and distribution of specific phylogenetic groups in their natural microhabitats (Amann et al., 1992
). These molecular techniques have been successfully applied to identify syntrophic propionate-oxidizing bacteria (Harmsen et al., 1996
), and to localize methanogens in anaerobic granular sludge systems (Rocheleau et al., 1999
; Sekiguchi et al., 1999
). In this study, a molecular approach was used in combination with electron microscopy to characterize the microbial consortia in a laboratory-scale terephthalate-degrading UASB reactor.
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METHODS |
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Anaerobic batch experiment.
A 305 ml serum bottle was charged with 250 ml disrupted granular sludge (final volatile suspended solids, 300 mg l-1), oxygen-free medium and 3 mM terephthalate. The oxygen-free medium (pH 7·2) consisted of vitamins, trace metals and 50 mM carbonate buffer (Owen et al., 1979
). During the anaerobic experiment at 37 °C for 20 d, 1 ml liquid sample was withdrawn every 14 d from the serum bottle with a syringe, and filtered through a 0·45 µm disposable filter. Volatile fatty acid concentrations were analysed using a gas chromatograph equipped with a flame-ionization detector (Shimaza 8A) and a Thermon-3000 (Shincarbon 60/80 mesh)-packed glass column. The temperatures at injection, oven and detector ports were controlled at 180, 150 and 180 °C, respectively. Aromatic compounds (terephthalate and benzoate) were determined by HPLC using a Hitachi L-3000 instrument equipped with a photodiode array detector and a separation column (LiChroCART 250-4 RP-18e, Merck). Mixed solvents, which were used as the mobile solutions in HPLC, were acetonitrile and water containing 1% (v/v) acetic acid. Final gaseous products (methane and hydrogen) were analysed by GC with a thermal conductivity detector (China GC 8900). The 2 m stainless column was packed with Hayesep Q (60/80 mesh) and installed inside a 60 °C oven. Pure N2 was used as the carrier gas at a constant flow rate of 10 ml min-1.
Transmission electron microscopy (TEM).
Sludge granules were first fixed in 0·1 M phosphate buffer solution (PBS) containing 2·5% (v/v) glutaraldehyde and 4% (w/v) paraformaldehyde for at least 12 h at 4 °C and washed three times with 0·1 M PBS. The granules were subsequently fixed in 0·1 M PBS containing 1% (w/v) osmium tetroxide for 2 h at 4 °C and washed three times with 0·1 M PBS. The samples were dehydrated with a series of ethanol washes (50, 75, 85, 95 and 100%), and exposed to acetone for 15 min twice. The dehydrated sample was gradually washed in a series of acetone solutions containing 50% (v/v) and then 75% (v/v) Spurrs resin at room temperature with gentle shaking (12 h each wash). The mixture was decanted and replaced with 100% Spurrs resin, followed by incubation for 24 h at room temperature. The specimens were embedded in fresh resin and cured at 70 °C for 14 h. A diamond knife was used to produce 6070 nm ultra-thin sections. The granule section was retrieved onto an uncoated 300-mesh copper grid (Ted Pella). The grid was stained in 50% ethanol solution containing 2% (w/v) uranyl acetate for 20 min, and then in 0·4% (w/v) lead citrate solution for 5 min and air-dried. The sectioned samples were analysed with a transmission electron microscope (JEOL Jem 1200EX).
DNA extraction and microbial community analysis.
Granular sludge samples were homogenized with a tissue grinder and suspended in 750 µl lysis buffer (100 mM Tris/HCl, 100 mM EDTA and 0·75 M sucrose, pH 8·0). Following cell lysis, phenol/chloroform extraction and ethanol precipitation procedures (Liu et al., 1997 ), total DNA from the sludge sample was obtained.
16S rDNA clone libraries for the domains Bacteria and Archaea were constructed to reveal the microbial community structure in the granular sludge. Initially, the 16S rDNAs from members of the domains Bacteria and Archaea were PCR-amplified with two frequently used primer sets, eu11F/eu1512R (Bond et al., 1995 ) and A23F/A1392R (Barns et al., 1994
), respectively. The PCR reaction was performed in 1x PCR buffer (Gibco-BRL) containing 200 µM (each) of deoxynucleoside triphosphates, 1·5 mM MgCl2, 0·1 µM (each) of primers and 2·5 U Taq polymerase (Gibco-BRL) in a final volume of 100 µl, using a PCR Express programmable thermal cycler (Hybaid). The thermal cycling programs used were described previously (Barns et al., 1994
; Bond et al., 1995
). After PCR amplification, PCR products were cloned into the pPCR-Script AmpSK(+) vector using a commercial cloning kit (Stratagene) according to the manufacturers instructions.
For rapid clone screening, white colonies containing 16S rDNA inserts of the correct size (1·5 kb) in vectors were identified using PCR amplification with vector-specific primers (M13F and M13R). Products of the correct length (
1·6 kb) were diluted and used as the DNA template in a subsequent DGGE-PCR reaction. The DGGE primers used for the amplification of 16S rDNA from members of the domain Bacteria were U968F-gc (Heuer et al., 1997
) and U1392R (Ferris et al., 1996
). The primers for the amplification of 16S rDNA from members of the domain Archaea were A934F (5'-AGGAATTGGCGGGGGAGCA-3') and 1390R-gc (5'-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGCGGTGTGTGCAA-3'), which were designed in this study (underlined sequences are the gc-clamp region). The forward primer perfectly matched at least 91·2% of the archaeal 16S rRNA gene sequences in the Ribosomal Database Project (RDP) database (Maidak et al., 1997
). The reverse primer matched more than 85% of all sequences in the current RDP database at one-mismatch specificity. The thermal program used for amplification of bacterial 16S rRNA genes was described previously (Nielsen et al., 1999
). For members of the domain Archaea, a touch-down thermal program was used. An initial denaturation step (95 °C, 3 min), followed by five cycles of touch-down amplification [denaturation (95 °C, 45 s), annealing (68 °C, 45 s with 1 °C decrease each cycle to 63 °C) and extension (72 °C, 2 min)], 23 cycles of amplification [denaturation (95 °C, 45 s), annealing (63 °C, 45 s) and extension (72 °C, 2 min)], and a final extension (72 °C, 3 min) was used. Nested DGGE-PCR products were verified by electrophoresis in a 0·8% agarose gel, and were screened by DGGE using a gel with a denaturant gradient from 40% to 60%.
DGGE was performed using the DCode system (Bio-Rad) at 200 V, 60 °C for 3·5 h, as previously described (Nielsen et al., 1999 ). The separated DNA fragments were visualized by silver staining (Riesner et al., 1989
) and photographed using an image-capture system equipped with a Kodak DC-120 digital camera. Clones that produced PCR products which migrated to different positions in the DGGE gel were identified and sequenced with a Taq Dye-Deoxy terminator cycle sequencing kit (Applied Biosystems). Sequencing was performed with an ABI DNA Sequencer model 377 (Applied Biosystems) at Mission Biotech (Taipei, Taiwan). In addition to the amplification primers, bacterial primer 926R (Amann et al., 1992
) and archaeal primer 934R (Amann et al., 1995
) were used as primers in the sequencing reaction.
Phylogenic analysis and probe design.
Approximate phylogenetic affiliations of the inferred 16S rRNA gene sequences obtained were initially compared to the GenBank database using the NCBI BLAST program. Sequences closely related (sequence identity >90%) to the clone sequences were retrieved and imported into the sequence alignment program of ARB (provided by Oliver Strunk & Wolfgang Ludwig, Technical University of Munich, Munich, Germany) for further phylogenetic analysis. The 16S rRNA gene sequences obtained in this study and the most closely related sequences from the GenBank database were aligned using the sequence alignment editor in ARB and then manually corrected. Phylogenetic trees were built from these aligned sequences using the neighbour-joining method provided in the ARB program. For distance correction, the algorithm of Jukes & Cantor (1969) was used. Alignment positions at which less than 50% of the sequences in the entire data set shared the same residues were excluded from the calculations. 16S rRNA-targeted oligonucleotide probes were designed from the cloned sequences using the probe design function in ARB. Probe specificity was checked using the Check Probe analysis in the RDP (Maidak et al., 1997
). Fluorescently labelled probes were synthesized by Operon Technologies (Alameda, CA).
Fixation, sectioning and FISH of sludge granules.
Sludge granules were gently washed three times with 0·1 M PBS, fixed in 0·1 M PBS containing 4% (w/v) paraformaldehyde at 4 °C overnight, and washed three times with 0·1 M PBS. To improve the penetration efficiency of oligonucleotide probes, the granule was subjected to at least five cycles of freeze-and-thaw (-80 °C to +60 °C) after fixation (Sekiguchi et al., 1999 ). The fixed sample was then dehydrated in a 50% ethanol solution at 4 °C overnight, and washed with a series of ethanol/water solutions (50, 80 and 96%, 3 min each), then with an ethanol/xylene mixture (50:50, v/v), and finally with 100% xylene. The xylene solution was gradually replaced with an equal volume of paraffin/xylene mixture with the paraffin content varying from 25%, 50%, 75% to 100% at 62 °C (12 h for each replacement). The granule-embedded paraffin block was cooled and sectioned into 23 µm slices with a rotary microtome (Microm type HM310). The centre sections of sludge granules were collected and floated on 0·8% (v/v) formaldehyde solution on the surface of a poly-L-lysine-coated microscope slide at 40 °C, and air-dried. The paraffin in the granule sections was removed by washing twice with 100% xylene for 40 min and then the sections were washed twice more with 100% ethanol for 40 min. The slides with granule sections were air-dried at room temperature.
In situ hybridization was conducted according to protocols previously described (Nielsen et al., 1999 ; Sekiguchi et al., 1999
). Initially, 10 µl hybridization buffer (0·9 M NaCl, 1% SDS, 100 mM Tris/HCl, pH 7·2) containing 50 ng of each labelled probe (see Table 1
) was added to each well on the slide and hybridized at 45 °C for at least 12 h. The hybridization stringency was controlled by adding different amounts of formamide to the hybridization buffer (10% for MG1200; 15% for EUB338; 20% for MX825 and delta-TA1; 30% for delta402; and 35% for ARC915, MB1174 and delta-TA2). For washing, the slide was briefly rinsed with double-distilled water and incubated in the same hybridization solution without the addition of probes for 30 min at 48 °C. The slide was briefly rinsed with milliQ water, and air-dried prior to examination using an epifluorescence microscope equipped with a cooled CCD camera (Quntax; Photometrics). When multiple probes were used, the probes with higher-stringency hybridization conditions were applied first, followed by probes with lower-stringency hybridization conditions.
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RESULTS AND DISCUSSION |
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The phylogenetic analysis indicated that archaeal OTUs found in the syntrophic granule were all close relatives of methanogens in the Euryarchaeota (Fig. 3a). Three OTUs (TA1, TA4 and TA5), which accounted for 81·7% of the total clones, were closely affiliated with Methanosaeta concilii (formerly known as Methanothrix soehngenii) (99% sequence identity). The other two OTUs (TA2 and TA3) were closely related to the genera Methanospirillum and Methanogenium, in the family Methanospirillaceae and the order Methanomicrobiales, respectively. Both acetoclastic Methanosaeta spp. and hydrogenotrophic methanogens (Methanospirillum, Methanobacterium and Methanobrevibacter) are frequently found in anaerobic sludge granules (Sekiguchi et al., 1998
; Grotenhuis et al., 1991
), and the former was suggested to be important for sludge granulation (Grotenhuis et al., 1992
). The TEM results also support the clone library finding that Methanosaeta-like organisms were the most predominant archaea. In contrast, methanogens putatively from the family Methanobacteriaceae were detected by TEM, but no archaeal clone related to this family was found.
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In the GNS division, OTU TA17 (7·5% of the total clones of Bacteria) is closely associated with subdivision I (Hugenholtz et al., 1998 ). Since this subdivision is currently composed of only environmental 16S rDNA clones obtained from various ecosystems, members of this subdivision were suggested to play an important role in the environment (Hugenholtz et al., 1998
). Sekiguchi et al. (1998
) retrieved several 16S rRNA gene sequences within this subdivision from mesophilic and thermophilic UASB systems treating sucrose, acetate and propionate. Their results based on in situ hybridization with a specific probe targeting this subdivision further revealed that members of this bacterial group are filamentous, and presumably were responsible for degradation of carbohydrates (Sekiguchi et al., 1999
). Taking this together with the observation of the GNS-I 16S rRNA gene sequences in the terephthalate-degrading UASB system, it may be suggested that members of this group are important in the degradation of substrate under methanogenic conditions. The exact metabolic traits of the GNS-I in UASB systems warrant further studies.
In the Synergistes division, one OTU (TA19) was found, but it only accounted for 0·9% of the total bacterial clones. Clone TA19 was closely related to an environmental clone, vandinHA73, obtained from an anaerobic digester (Godon et al., 1997 ), and formed a cluster that was deeply branched and phylogenetically distant from the Synergistes genus. Furthermore, three OTUs (TA6, TA8 and TA18), accounting for 13·1% of the total bacterial clones, were found to have a low degree of similarity to any known 16S rRNA sequences in the database. Very few environmental 16S rDNA clones were found to affiliate with OTUs TA19, TA6, TA8 and TA18. As a result, no conclusions could be drawn on the metabolic and physiological traits of microbial populations represented by these OTUs.
Microbial topography as revealed by FISH
The distribution of archaeal and bacterial populations in the granular consortium was determined using in situ hybridization with Archaea (ARC915)- and Bacteria (EUB338)-specific probes, respectively. As with the TEM observations (Fig. 2), most of the archaeal (green) and bacterial (red) cells observed were randomly distributed and of equal abundance (bacterial cells=52·9±5·4%) in the sludge granule (Fig. 4a
). In some areas where bacterial cells and archaeal cells were closely associated, yellow signals were observed. This is due to the inability of epifluorescence microscopy to differentiate signals from close but different focal planes in a granular thin section thicker than 12 µm. Microcolonies of archaeal and bacterial cells were observed to be denser at the outer region than at the inner region of the granule. A fraction of the granule at the inner region was composed of void areas or micro-channels, which have been suggested to facilitate diffusive transport of substrates, nutrients and by-products within the granule (Wu et al., 1991
). This so-called nonlayered architecture observed in the terephthalate-degrading granule was similar to that observed in granular sludges that degraded propionate (Grotenhuis et al., 1991
) or glutamate (Fang et al., 1994a
) and hydrolysed proteins (Fang et al., 1994b
). The formation of a nonlayered granular structure was suggested to be highly dependent on the degradation mechanism, which usually includes an acetogenesis step followed by a methanogenesis step (Fang et al., 1995
). Because the acetogenesis step is often reported as being rate-limiting (Fang et al., 1995
; Rocheleau et al., 1999
), a slow but steady flux of intermediates (i.e. acetate and hydrogen) into the granule can be provided. A terephthalate-degrading granule with a relatively small size (
0·6 mm) could easily provide constant flux of terephthalate, degradation intermediates, nutrients and final gaseous products into and out of the granule, leading to the formation of a nonlayered structure.
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The cloning results did not reveal any sequences from the family Methanobacteriaceae, but cells morphologically resembling Methanobacteriaceae were observed using TEM. This discrepancy was addressed by using in situ hybridization with probes targeting the Methanobacteriaceae (probe MB1174) and Methanosaeta (probe MX825). Fig. 4(c) shows that a significant fraction of cells hybridized with the MB1174 probe (red), and the cells were closely clustered with cells that hybridized with the Methanosaeta-specific probe (green). The inability to retrieve 16S rRNA gene sequences from Methanobacteriaceae was likely due to the bias associated with total community DNA extraction (Sekiguchi et al., 1998
), PCR amplification (Suzuki & Giovannoni, 1996
) and 16S rDNA cloning (Amann et al., 1995
).
Syntrophic bacterial populations
The 16S rDNA cloning analysis revealed that the novel group found in the -Proteobacteria was the predominant group (Fig. 3b
). Thus, the abundance and localization of this novel bacterial group in the granular consortium were further determined by FISH. Two 16S rRNA-targeted oligonucleotide probes (Table 1
) were designed to encompass this novel bacterial group at different levels of specificity as shown in Fig. 3(b)
. Probe delta-TA1, a group-specific probe, targeted the novel
-proteobacterial group TA. It had at least three mismatches with the 16S rRNA gene sequence of any known bacterium (e.g. S. erythraea) outside the novel group, and more than three mismatches with other bacterial sequences in the
-Proteobacteria. Probe delta-TA2 was a subgroup-specific probe that targeted the most abundant OTUs, TA11/TA12 (27·3% of the total bacterial clones), within the novel bacterial group. This probe had one mismatch with TA11, three mismatches with TA7, TA10, TA14 and TA16 within the novel group, and more than four mismatches with any known bacterial species from the
-Proteobacteria. The specificity of these two probes was tested under different hybridization conditions, and the optimal hybridization condition showed no reaction to the non-target organisms, S. erythraea and Desulfovibrio sp.
Fig. 4(d) shows the combined FISH of the granular consortium with a bacterial-domain probe (EUB338) and a group-specific probe (delta-TA1). At least 87±7·6% of the clustered bacterial cells hybridized with the probe delta-TA1 and showed as yellow coloured as a result of overlapping red (EUB338) and green (delta-TA1) probes. The red cells represent bacterial populations outside the novel bacterial group. Unlike cells belonging to the novel bacterial group, these red cells did not form dense microcolonies, but were randomly scattered throughout the sludge granule. The predominance of the novel group TA was further confirmed by the combined use of a probe (delta402) specific for the
-Proteobacteria and probe delta-TA1 (data not shown). This finding closely agreed with the 16S rDNA cloning analysis result.
The population diversity within the novel -proteobacterial group TA was further determined by the combined use of probes delta-TA1 and delta-TA2. At least two different populations with similar rod-shaped morphology were observed (Fig. 4e
). Based on the hybridization signal, the microbial population targeted by probe TA2 made up a significant proportion (72±9·6%) of the novel bacterial group. This result also verified that the optimal hybridization specificity was obtained for probes delta-TA1 and delta-TA2. Thus, the novel
-proteobacterial group should include at least two or more than two members, considering that at least 10 different 16S rRNA gene sequences have been found within this novel group from various environments (Dojka et al., 1998
; Wintzingerode et al., 1999
).
Conclusions
In summary, a not-yet-cultured, rod-shaped novel group in the -Proteobacteria was presumed to be the primary population responsible for degrading terephthalate to acetate and hydrogen; it formed a close spatial association with different methanogenic populations (i.e. Methanosaeta, Methanospirillum and Methanobacteriaceae) that convert acetate, hydrogen and carbon dioxide into methane. Enrichment culture and characterization of the terephthalate-degrading consortium are being undertaken to further confirm the role of the novel
-proteobacterial group in terephthalate degradation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Amann, R. I., Stromley, J., Devereux, R., Key, R. & Stahl, D. A.(1992). Molecular and microscopic identification of sulfate-reducing bacteria in multispecies biofilms. Appl Environ Microbiol 58, 614-623.[Abstract]
Amann, R. I., Ludwig, W. & Schleifer, K. H.(1995). Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59, 143-169.[Abstract]
Barns, S. M., Fundyga, R. E., Jeffries, M. W. & Pace, N. R.(1994). Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc Natl Acad Sci USA 91, 1609-1613.[Abstract]
Bond, P. L., Hugenholtz, P., Keller, J. & Blackall, L. L.(1995). Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Appl Environ Microbiol 61, 1910-1916.[Abstract]
Cheng, S. S., Ho, C. T. & Wu, J. H.(1997). Pilot study of UASB process treating PTA manufacturing wastewater. Water Sci Technol 36, 73-82.
Dojka, M. A., Hugenholtz, P., Haack, S. & Pace, N. R.(1998). Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation. Appl Environ Microbiol 64, 3869-3877.
Fang, H. H. P., Chui, H. K. & Li, Y. Y.(1994a). Microbial structure and activity of UASB granules treating different wastewaters. Water Sci Technol 30, 87-96.
Fang, H. H. P., Chui, H. K., Li, Y. Y. & Chen, T.(1994b). Performance and granule characteristics of UASB process treating wastewater with hydrolyzed proteins. Water Sci Technol 30, 55-63.
Fang, H. H. P., Chui, H. K. & Li, Y. Y.(1995). Effect of degradation kinetics on the microstructure of anaerobic biogranules. Water Sci Technol 32, 165-172.
Ferris, M. J., Muyzer, G. & Ward, D. M.(1996). Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62, 340-346.[Abstract]
Franck, H. G. & Stadelhofer, L. W.(1988). p-Xylene and its derivatives: terephthalic acid. In Industrial Aromatic Chemistry , pp. 283-290. Edited by H. G. Franck. New York:Springer.
Godon, J., Zumstein, E., Dabert, P., Habouzit, F. & Moletla, R.(1997). Molecular microbial diversity of an anaerobic digester as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol 63, 2802-2813.[Abstract]
Grotenhuis, J. T. C., Smit, M., Plugge, C. M., Yuansheng, X. U., van Lammeren, A. A. M., Stams, A. J. M. & Zehnder, A. J. B.(1991). Bacteriological composition and structure of granular sludge adapted to different substrates. Appl Environ Microbiol 57, 1942-1949.[Medline]
Grotenhuis, J. T. C., Plugge, C. M., Stams, A. J. M. & Zehnder, A. J. B.(1992). Hydrophobicities and electrophoretic mobilities of anaerobic bacterial isolates from methanogenic granular sludge. Appl Environ Microbiol 58, 1054-1056.[Abstract]
Harmsen, H. M., Kengen, H. M. P., Akkermans, A. D. L., Stams, A. J. M. & de Vos, W. M.(1996). Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes. Appl Environ Microbiol 62, 1656-1663.[Abstract]
Heuer, H., Krsek, M., Baker, P., Smalla, K. & Wellington, E. M. H.(1997). Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl Environ Microbiol 63, 3233-3241.[Abstract]
Hugenholtz, P., Goeber, B. M. & Pace, N. R.(1998). Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180, 4765-4774.
Jukes, T. H. & Cantor, C. R.(1969). Evolution of protein molecules. In Mammalian Protein Metabolism , pp. 21-132. Edited by H. M. Munro. New York:Academic Press.
Kleerebezem, R., Mortier, J., Hulshoff Pol, L. W. & Lettinga, G.(1997). Anaerobic pretreatment of petrochemical effluents: terephthalic acid wastewater. Water Sci Technol 36, 237-248.
Kleerebezem, R., Hulshoff Pol, L. W. & Lettinga, G.(1999a). Anaerobic degradation of phthalate isomers by methanogenic consortia. Appl Environ Microbiol 65, 1152-1160.
Kleerebezem, R., Hulshoff Pol, L. W. & Lettinga, G.(1999b). The role of benzoate in anaerobic degradation of terephthalate. Appl Environ Microbiol 65, 1161-1167.
Lau, C. M.(1977). Staging aeration for high-efficiency treatment of aromatics plant wastewater. Proc Ind Waste Conf Purdue Univ 32, 63-74.
Lettinga, G.(1995). Anaerobic digestion and wastewater treatment systems. Antonie Leeuwenhoek 67, 3-28.[Medline]
Liu, W. T., Marsh, T. L., Cheng, H. & Forney, L. J.(1997). Characterization of microbial diversity by determining terminal restriction fragment length polymorphism of 16S ribosomal DNA. Appl Environ Microbiol 63, 4516-4522.[Abstract]
Maidak, B. L., Olsen, G. L., Larsen, N., Overbeek, R., McCaughey, M. J. & Woese, C. R.(1997). The RDP (Ribosomal Database Project). Nucleic Acids Res 25, 109-110.
Mountfort, D. O., Brulla, W. J., Krumholz, L. R. & Bryant, M. P.(1984). Syntrophus buswellii gen. nov., sp. nov.: benzoate catabolizer from methanogenic ecosystems. Int J Syst Bacteriol 34, 216-217.
Muyzer, G. & Smalla, K.(1998). Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Leeuwenhoek 73, 127-141.
Nielsen, A. T., Liu, W. T., Filipe, C., Grady, L., Molin, J. R. S. & Stahl, D. A.(1999). Identification of a novel group of bacteria in sludge from a deteriorated biological phosphorus removal reactor. Appl Environ Microbiol 65, 1251-1258.
Owen, W. F., Stuckey, D. C., Herly, J. B.Jr, Young, L. Y. & McCarty, P. L.(1979). Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Water Res 13, 485-492.
Pereboom, J. H. F., DeMan, G. & Su, Y. T. (1994). Start-up of full scale UASB reactor for the treatment of terephthalic acid wastewater. In 7th Int Symp on Anaerobic Digestion, vol. 3, pp. 307312. Cape Town.
Raskin, L., Stomley, J. M., Rittmann, B. E. & Stahl, D. A.(1994). Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 60, 1232-1240.[Abstract]
Riesner, D., Steger, G., Zimmat, R., Owens, R. A., Wagenhofer, M., Hillen, W., Vollbach, S. & Henco, K.(1989). Temperature-gradient gel electrophoresis of nucleic acids: analysis of conformational transitions, sequence variations, and proteinnucleic acid interactions. Electrophoresis 10, 377-389.[Medline]
Rocheleau, S., Greer, C. W., Lawrence, J. R., Cantin, C., Larsme, L. & Guiot, S. R.(1999). Differentiation of Methanosaeta concilii and Methanosarcina barkeri in anaerobic mesophilic granular sludge by fluorescence in situ hybridization and confocal scanning laser microscopy. Appl Environ Microbiol 65, 2222-2229.
Schink, B.(1997). Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev 61, 262-280.[Abstract]
Sekiguchi, Y., Kamagata, Y., Syutsubo, K., Ohashi, A., Harada, H. & Nakamura, K.(1998). Phylogenetic diversity of mesophilic and thermophilic granular sludges determined by 16S rRNA gene analysis. Microbiology 144, 2655-2665.[Abstract]
Sekiguchi, Y., Kamagata, Y., Nakamura, K., Ohashi, A. & Harada, H.(1999). Fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules. Appl Environ Microbiol 65, 1280-1288.
Stahl, D. A. & Amann, R.(1991). Development and application of nucleic acid probes. In Nucleic Acid Techniques in Bacterial Systematics , pp. 205-248. Edited by E. Stackebrandt & M. Goodfellow. New York:Wiley.
Suzuki, M. T. & Giovannoni, S. J.(1996). Bias caused by template annealing in the amplification of mixture of 16S rRNA genes by PCR. Appl Environ Microbiol 62, 625-630.[Abstract]
Theile, J. H. & Zeikus, J. G.(1988). Interactions between hydrogen-and formate-producing bacteria and methanogens during anaerobic digestion. In Handbook on Anaerobic Fermentations , pp. 537-595. Edited by L. E. Erickson & D. Y. C. Fung. New York:Marcel Dekker.
Whitman, W. B., Bowen, T. L. & Boone, D. R.(1992). The methanogenic bacteria. In The Prokaryotes , pp. 719-767. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York:Springer.
Wintzingerode, V. F., Selent, B., Hegemann, W. & Göbel, U. B. (1999). Phylogenetic analysis of an anaerobic, trichlorobenzene-transforming microbial consortium. Appl Environ Microbiol 65, 283-286.
Wu, W. M., Hickey, R. F. & Zeikus, J. G.(1991). Characterization of metabolic performance of methanogenic granules treating brewery wastewater: role of sulfate-reducing bacteria. Appl Environ Microbiol 57, 3438-3449.[Medline]
Wu, W. M., Jain, M. K. & Zeikus, J. G.(1996). Formation of fatty acid-degrading, anaerobic granules by defined species. Appl Environ Microbiol 62, 2037-2044.[Abstract]
Received 12 July 2000;
revised 22 September 2000;
accepted 20 October 2000.