Diversity and physiology of syntrophic substrate-oxidizing anaerobes inmethanogenic ecosystems
メタン生成エコシステムにおける嫌気共生細菌の多様性と生態
Ambar SUSILORUKMI1, Hiroyuki IMACHI1, Yuji SEKIGUCHI2, Akiyoshi OHASHI1, Hideki HARADA1
1Nagaoka University of Technology; 2 Institute of Advanced Industrial Science and Technology (AIST)

Introduction
Under methanogenic conditions, degradation of complex organic substances is carried out bycooperation of different trophic groups of anaerobic microorganisms. In this type of microbial ecosystems,
fatty acids and other compounds, such as ethanol and benzoate, are frequently formed as intermediates.
Since such intermediates frequently accumulate in this type of ecosystems, microbes involved in thedegradation of these intermediates are important. The degradation of intermediates is generally carried out by
substrate oxidizing, proton-reducing microbes (syntrophic bacteria), and hydrogenotrophic microbes.
Intermediates are converted by syntrophic bacteria to form acetate, hydrogen and CO2 only when thehydrogen partial pressure is kept extremely low by hydrogenotrophic microbes. The degradation ofintermediates is generally carried out by substrate oxidizing, proton-reducing microbes (syntrophic bacteria),
and hydrogenotrophic microbes. Intermediates are converted by syntrophic bacteria to form acetate,hydrogen and CO2 only when the hydrogen partial pressure is kept extremely low by hydrogenotrophic
microbes [1,2,3,4,5].
Despite their significant metabolic functions in methanogenic ecosystem, information on thephysiological characteristics and phylogenetic diversity of such organisms was limited, especially due to thefastidious characteristics of the syntrophic microbes, such as slow growth, low growth yield and syntrophic
association with hydrogenotrophic microbes. In this study, the diversity and physiology of syntrophic
substrate-oxidizing anaerobes were studied by applying conventional cultivation techniques combined withrRNA-based molecular approaches [6]Material and methods
For cultivation of anaerobic, syntrophic bacteria, two thermophilic (55°C) anaerobic digested
sludges, one mesophilic (35°C), anaerobic digested sludge, a mesophilic, anaerobic granular sludge and rice
paddy soil were used as inoculum for primary enrichments, using ethanol (10 mM), benzoate (5 mM) orpropionate (20 mM) as the sole carbon source. DNA extraction from enrichment cultures was performed bybead-beating method. For 16S rDNA-based clone analysis, a bacterial universal primer set was used.
Fluorescence in situ hybridization (FISH) was carried out according to the method of Amann et al. [7].
Result and discussion
1. Enrichment of anaerobic, syntrophic bacteria
For enrichment of anaerobic, syntrophic bacteria, various types of anaerobic, environmental samples
were used for primary enrichment using ethanol, benzoate or propionate as the sole carbon source. Thegrowth of microbes in these cultures was observed after one to three months of incubation. After 5 times
successive transfers, these cultures produced methane along with substrate depletion. All enrichment
cultures contained F420-autofluorescent microbes and several morphologically distinct microbes. From thisobservation, it was suggested that the substrate-degadation in almost all of the enrichment cultures wascarried out by the syntrophic bacteria and methanogens syntrophically.

2. 16S rRNA-based analysis for syntrophic bacteria in enrichment cultures
To identify bacteria involved in syntrophic substrate-degradation, 16S rDNA-based analysis wasprimary performed for all enrichment cultures. Ten rDNA clones were randomly selected fromeach enrichment culture, and their sequences were determined. Among the predominant clones recoveredfrom each enrichment culture, some clones showed close relation with known bacteria to date as syntroph,such as Desulfovibrio sp. Nonetheless, several clones seemed to indicate novel bacterial lineages that havenever cultivated and isolated so far, such as clones related with the genus Geobacter in two mesophilicethanol enrichments, clones representing a deeply branched lineage of the phylum Firmicutes in athermophilic ethanol enrichment culture, and clones related with the genus Desulfobulbus in a mesophilic
propionate degrading anaerobes.
To determine whether the dominant clones were derived from the dominant microbes in enrichment
cultures, specific DNA probes were designed and applied for the cultures in fluorescence in situ
hybridization analyses. This resulted in the detection of a number of DNA probe-reacted cells in all thecultures, suggesting the probe-positive cells were the dominant microbes in the cultures [8,9]. Table 3 shows
the oligonucleotide probes used in this study, included new probes design of BD202, BD1009 and RPC6.

Both of probe BD were applied to hybridize newly identified bacteria, obtained from benzoate degrading
culture (mesophilic) and ethanol degrading culture (thermophilic). While RPC6 was employed to detect a
new bacteria in mesophilic propionate degrading culture
Table 3. The 16S rRNA-targeted oligonucleotide probes used in this study
Probe nameProbe sequence (5’ to 3’)

Target microbe

ReferenceEUB338
EUB338-II
EUB338-III
D687
Geob420
TDV1011
Ih 820
DEM1164
BD1009BD202
RP452

GCTGCCTCCCGTAGGAGTGCAGCCACCCGTAGGTGT
GCTGCCACCCGTAGGTGTTACGGATTTCACTCCTGACAGAGCTTTACGACCCG
CCCCTAAGGTCGTCCCCCTT
ACCTCCTACACCTAGCACCC
CCTTCCTCCGTTTTGTCAGTCGATTCCGTTTCCGGTCATGACCGCTAAATCGCT
GTCTTTCTCCTTAAGGCC

Bacteria
Bacteria
Bacteria
Desulfovibrio sp
Geobacter sp
Thermodesulfovibrio sp
Pelotomaculum sp
Sporotomaculum sp
Strain BD
Strain JE
Mesophilic
propionate
enrichment culture

Aman et al., 1990
Daims et al., 1999
Daims et al., 1999
Devereux et al., 1992
Small et al., 2001
Sekiguchi et al., 1999
Imachi et al., 2001
Stubner &Meuser, 2001
This study
This study
This study

3. Attempts at isolation of syntrophic substrate-degrading bacteria
Phylogenetic analysis and in situ hybridization experiments identified the dominant, presumably
syntrophic microorganisms in each enrichment culture. To isolate such newly identified bacteria, amolecular-directed isolation strategy was employed; it assumes the physiological properties of novel
identified bacteria based on the information obtained from phylogenetic analysis.
3.1. Uncultured bacteria, phylum Firmicutes

The phylogenetic tree of newly identified bacteria obtained from mesophilic benzoate
enrichment culture (designed as strain BD) and thermophilic ethanol enrichment culture areillustrated in Fig. 3.

These bacteria represent the deeply branched lineage of phylum Firmicutes. Both of these bacteria have been
isolated in pure culture (Fig. 4).

3.2. Thermodesulfovibrio sp-like bacterium
The phylogenetic tree of Thermodesulfovibrio sp-like, isolated from thermophilic ethanol enrichment
culture shown at Fig. 6. Pure culture was obtained at substrate of formate, contained acetate, sulfate andyeast extract (Fig. 5).

3.3. Geobacter sp-like bacteria
Geobacter sp-like were obtained from two mesophilic ethanol enrichment cultures, designed asclone NE1 and FE1. Figure 8 illustrates the phylogenetic of such bacteria. Both of them have beencultivated as well, however, isolation have not been succeed yet.

3.4. Desulfobulbus sp-likeA stable mesophilic propionate degrading culture have been cultivated well after five times
successive transfer. Based on 16S rDNA cone analysis, this bacterium was indicated as a new bacterium,
closely related to Desulfobulbus sp. Phylogenetic tree of this bacterium have been constructed in Fig. 10.

Nevertheless, such bacterium have not been isolated in pure culture yet, although several attempts have beenconducted.

Conclusion
This study strongly suggested that the strategy employing conventional techniques combined with
16S rRNA-based approaches is advantageous to determine the diversity of recalcitrant microbes like
syntrophic microorganisms and to attempt at subsequent isolation of targeted cells. The known syntrophic
microbes obtained from this study were Desulfovibrio sp, Syntrophus sp and Sporotomaculum sp, obtained
from mesophilic cultivation of ethanol and benzoate enrichment cultures, respectively. Instead of those,
several clones represented the bacterial that have never cultivated and isolated so far as syntrophic microbes.
The such newly identidied bacteria obtained from benzoate (mesophilic), propionate (mesophilic) andethanol (mesophilic and thermophilic) enrichment cultures.
To isolate such newly identified bacteria, a molecular-directed isolation strategy was applied.
Through this approach, two novel syntrophic, ethanol-oxidizing bacteria, designed as strain JE and OE; and anovel benzoate-degrading bacterium, designed as strain BD were successfully isolated. Additionally, twobacteria presumably new syntrophic microbes were cultivated as well. They were Geobacter sp-like (frommesophilic ethanol cultures) and Desulfobulbus sp-like (mesophilic propionate culture).

References
1. McInerney, M. J., Bryant, M. P. & Pfennig, N. (1979). Anaerobic bacterium that degrades fatty acids in syntrophic
association with methanogens. Arch Microbiol 122, 129-135.
2. Stams, A. J. M. (1994). Metabolic interaction between anaerobic bacteria in methanogenic environment. Antonie vanLeeuwenhoek 66, 271-294
3. Shinck B. (1992). Syntrophism among prokaryotes. In The Prokaryotes, pp. 272-299. Edited by A. Balows, H. G.
Trupper, M. Dworkin, W. Hardner & K.-H. Schleifer. New York: Springer.
4. Shinck B. (1997). Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev 61,262-280
5. Sekiguchi, Y., Kamagata, Y. & Harada, H. (2001). Recent advances in methane fermentation technology. Current
Opinion in Biotechnology, 12, 277-282
6. Imachi, H., Sekiguchi, Y., Kamagata, Y., Ohashi, A. & Harada, H. (2000). Cultivation and in situ detection of athermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens
in thermophilic methanogenic granular sludge. Appl Environ Microbiol 66, 3608-3615.
7. Amann, R. I. (1995). In situ identification of microorganism by whole cell hybridization with rRNA-targeted nucleic
acid probes, sedction 3.3.6, p3.3.6/1-3.3.6/15. In A. D. L. Akkermans and J.D. van Elsas (ed), Molecular ecology
manual. Kluwer Academic Publishers, London, England.
8. 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
9. Sekiguchi, Y., Kamagata., Nakamura, K., Ohashi, A. & Harada, H. (1999). Fluorescence in situ hybridization
using 16S rRNA-tergeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria inmesophilic and thermophilic sludge granules. Appl Environ Microbiol 65, 1280-1288