Detection of Active Butyrate-Degrading Microorganisms in Methanogenic Sludges by RNA-Based Stable Isotope Probing

Hatamoto, Masashi; Imachi, Hiroyuki; Yashiro, Yuto; Ohashi, Akiyoshi; Harada, Hideki

doi:10.1128/aem.00045-08

Cited by 47 publications

(38 citation statements)

References 29 publications

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“…Moreover, PCR performed with the universal bacterial primer pairs 9f/1490R and EUB338F/1490R (Hatamoto et al, 2007) failed to detect 16S rRNA genes. We also generated an archaeal 16S rRNA gene clone library, by using the universal archaeal primer pair Ar109f/1490R (Großkopf et al, 1998) for PCR amplification.…”

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Methanocella arvoryzae sp. nov., a hydrogenotrophic methanogen isolated from rice field soil

Sakai

Conrad

Liesack

et al. 2010

International Journal of Systematic and Evolutionary Microbiology

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A novel hydrogenotrophic methanogen, designated strain MRE50 T , was isolated from a methanogenic consortium, which was originally established from an Italian rice field soil. Cells were non-motile rods, 1.3-2.8 mm long and 0.4-0.7 mm wide. Coccoid cells were also observed in cultures at the late-exponential phase of growth. Strain MRE50 T grew at 37-55 6C (optimally at 45 6C), at pH 6-7.8 (optimally at pH 7.0) and in the presence of 0-20 g NaCl l "1 . The isolate utilized H 2 /CO 2 and formate for growth and methane production. Phylogenetic analyses of the 16S rRNA gene and the methanogen-specific marker gene mcrA showed that strain MRE50 T is affiliated with the order Methanocellales, previously known as uncultured archaeal group Rice Cluster I. Based on both 16S rRNA gene and mcrA gene sequences, strain MRE50 T was related most closely to Methanocella paludicola SANAE T . Levels of sequence similarity were 92.5 and 86.1 %, respectively, indicating that strains MRE50 T and Methanocella paludicola SANAE T represent different species within the genus Methanocella. In addition, although these strains shared phenotypic properties including cell morphology and substrate utilization, they differed with respect to susceptibility to antibiotics, and temperature and NaCl ranges for growth. Given the phenotypic differences and the distinct phylogenetic placement of the new isolate relative to the type species of the genus Methanocella, strain MRE50 T is considered to represent a novel species of the genus Methanocella, for which the name Methanocella arvoryzae sp. nov. is proposed. The type strain is MRE50 T (5NBRC 105507 T 5DSM 22066 T ).The order Methanocellales is a recently proposed euryarchaeal order within the class 'Methanomicrobia' (Sakai et al., 2008). The order comprises just a single isolate, Methanocella paludicola SANAE T , a mesophilic hydrogenotrophic methanogen isolated from a rice field soil in Japan (Sakai et al., 2007). Before the taxonomic description of Methanocella paludicola and proposal of the order Methanocellales, this euryarchaeal lineage had long been recognized as uncultured archaeal group 'rice cluster I' (RC-I). Members of RC-I are known to play a key role in methane production in rice paddy fields (Conrad et al., 2006;Lu & Conrad, 2005). However, despite their ubiquity and abundance in rice paddy field environments, only one isolate, representing Methanocella paludicola, has been described so far. Here we report the isolation of a second member of the order Methanocellales, designated strain MRE50 T . We describe the physiology and phylogeny of this methanogen and propose that it represents a novel species of the genus Methanocella.Basal medium was prepared as described previously with the slight modification that both 1.5 mM Na 2 S . 9H 2 O and 1.5 mM cysteine hydrochloride were added as reducing agents (Chin et al., 1998;Lueders et al., 2001). The standard medium normally contains both 1 mM acetate and 0.01 % (w/v) yeast extract (Difco). However, we isolated strain MRE50 T and performe...

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mentioning

confidence: 99%

Methanocella arvoryzae sp. nov., a hydrogenotrophic methanogen isolated from rice field soil

Sakai

Conrad

Liesack

et al. 2010

International Journal of Systematic and Evolutionary Microbiology

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“…Butyrate is one of the important intermediates in the degradation of organic matter in anoxic environments (3)(4)(5)22). The degradation of butyrate to H 2 , formate, and acetate is endergonic under standard conditions.…”

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“…Studies on natural environments, however, are very scarce (1,5). Acetate, propionate, and butyrate are the most important intermediates during the degradation of organic residues in paddy field soils (4,22).…”

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Syntrophomonadaceae-Affiliated Species as Active Butyrate-Utilizing Syntrophs in Paddy Field Soil

Liu

Qiu

2011

Appl Environ Microbiol

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DNA-based stable-isotope probing was applied to identify the active microorganisms involved in syntrophic butyrate oxidation in paddy field soil. After 14 and 21 days of incubation with [U-13 C]butyrate, the bacterial Syntrophomonadaceae and the archaeal Methanosarcinaceae and Methanocellales incorporated substantial amounts of 13 C label into their nucleic acids. Unexpectedly, members of the Planctomycetes and Chloroflexi were also labeled with 13 C by yet-unclear mechanisms.Butyrate is one of the important intermediates in the degradation of organic matter in anoxic environments (3)(4)(5)22). The degradation of butyrate to H 2 , formate, and acetate is endergonic under standard conditions. This thermodynamic barrier, however, can be overcome by the syntrophic interaction between butyrate-oxidizing bacteria and methanogenic archaea, which keep the products H 2 , formate, and acetate at low concentrations (23). A few strains involved in syntrophic butyrate oxidation have been isolated into pure cultures (e.g., see references 15, 16, and 30). These organisms represent a thermodynamically extreme lifestyle, since even in the optimum syntrophic association with methanogens, the Gibbs free energy available for syntrophic butyrate oxidizers is still close to the thermodynamic limit (⌬G 0Ј ϭ Ϫ20 kJ per reaction). Recently, genome sequences of two syntrophic butyrate oxidizers (Syntrophomonas wolfei and Syntrophus aciditrophicus) (17, 24) revealed that they have limited fermentation and respiration mechanisms but possess multiple copies of ␤-oxidation genes and sets of reverse electron transfer machineries which are essential for the syntrophic oxidation of butyrate.Studies on natural environments, however, are very scarce (1, 5). Acetate, propionate, and butyrate are the most important intermediates during the degradation of organic residues in paddy field soils (4, 22). Only two studies known to us so far, however, have been conducted to determine the syntrophic degradation of propionate (12) and acetate (7), and none have been done on syntrophic butyrate oxidation in paddy field soil. Therefore, our objectives were to investigate syntrophic butyrate oxidation and identify the active organisms responsible for this process in a Chinese paddy field soil using nucleic acid-based stable-isotope probing (SIP), which has been proven to be powerful in linking the identities of microorganisms in environments with their specific functions (2).Soil sample and anoxic incubation. Paddy field soil was collected from an experimental station of the China National Rice Research Institute in Hangzhou, China (30 o 04Ј37ЉN, 119 o 54Ј37ЉE). The soil was a clay loam and had the following properties as measured by standard methods (18): pH 6.7, a cation exchange capacity of 14.4 cmol kg Ϫ1 , an organic C content of 24.2 g kg Ϫ1 , and a total N content of 2.3 g kg Ϫ1 . Soil was air dried and passed through 2-mm sieves. Three-gram soil samples were weighed in 15-ml serum bottles and mixed with 4.5 ml distilled anoxic water. The vials were c...

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“…In the PCR amplification, we used the primer pair Ar109f (Großkopf et al, 1998) and 1490R (Weisburg et al, 1991) or EUB338* (Amann et al, 1990;Daims et al, 1999;Hatamoto et al, 2007) and 1490R for the construction of 16S rRNA gene-based archaeal and bacterial clone libraries, respectively. We also used primers Arch21F (DeLong, 1992) and 1490R to obtain the nearly full-length 16S rRNA gene of the isolate.…”

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Methanolinea tarda gen. nov., sp. nov., a methane-producing archaeon isolated from a methanogenic digester sludge

Imachi

Sakai

Sekiguchi

et al. 2008

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLO

182

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Methanolinea tarda gen. nov., sp. nov., a methaneproducing archaeon isolated from a methanogenic digester sludge A novel methane-producing archaeon, strain NOBI-1 T was isolated from an anaerobic, propionate-degradation enrichment culture, which was originally obtained from a mesophilic methanogenic sludge digesting municipal sewage sludge. Cells were non-motile, rod-shaped, 0.7-1.0 mm by 2.0 mm, and formed multicellular filaments longer than 8 mm. Growth was observed between 35 and 55 6C (optimum 50 6C) and pH 6.7 and 8.0 (optimum pH 7.0). The G+C content of the genomic DNA was 56.3 mol%. The strain utilized H 2 and formate for growth and methane production. Based on comparative sequence analyses of the 16S rRNA gene and mcrA gene (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane-production pathway), strain NOBI-1 T was affiliated with the order Methanomicrobiales, but it was significantly distant from any other known species within the order. The most closely related species based on 16S rRNA and mcrA gene sequence similarity were respectively 'Candidatus Methanoregula boonei' (93.7 % 16S rRNA gene sequence similarity) and Methanocorpusculum parvum (74.2 % deduced McrA amino acid sequence similarity to the type strain). These phenotypic and genetic properties justified the creation of a novel species of a new genus for the strain, for which we propose the name Methanolinea tarda gen. nov., sp. nov. The type strain of Methanolinea tarda is strain NOBI-1 T (5DSM 16494 T 5JCM 12467 T 5NBRC 102358 T ).Methane-producing archaea (methanogens) are one of the key populations in methanogenic waste and wastewater treatment processes because they are responsible for the final step of the degradation of organic substances (Anderson et al., 2003;Garcia et al., 2000). So far, a number of methanogens, placed in a wide range of taxonomic groups within the phylum Euryarchaeota, have been isolated from various methanogenic treatment processes and characterized as novel species belonging to genera such as Methanobacterium, Methanoculleus and Methanosaeta (Garcia et al., 2000;Garrity & Holt, 2001). In addition, recent molecular surveys targeting the 16S rRNA gene and mcrA gene, encoding the alpha subunit of methyl coenzyme M reductase, have revealed that numerous unidentified methanogens may also exist in such ecosystems (Chouari et al., 2005;Sekiguchi & Kamagata, 2004;Sekiguchi, 2006; Shigematsu et al., 2004). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain NOBI-1 T is AB162774 (other 16S rRNA gene clonal sequences have been deposited as accession numbers AB233309, AB233010 and AB233299-AB233302). The GenBank/EMBL/DDBJ accession numbers for the mcrA gene sequences of strains NOBI-1 T and SANAE are respectively AB300466 and AB300467.Details of 16S rRNA gene phylotypes obtained from the sludge, information on relative abundance of various groups of organisms and a graph of methane production by strain NOBI-1 T are available as supplementary material with the onl...

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Detection of Active Butyrate-Degrading Microorganisms in Methanogenic Sludges by RNA-Based Stable Isotope Probing

Cited by 47 publications

References 29 publications

Methanocella arvoryzae sp. nov., a hydrogenotrophic methanogen isolated from rice field soil

Methanocella arvoryzae sp. nov., a hydrogenotrophic methanogen isolated from rice field soil

Syntrophomonadaceae-Affiliated Species as Active Butyrate-Utilizing Syntrophs in Paddy Field Soil

Methanolinea tarda gen. nov., sp. nov., a methane-producing archaeon isolated from a methanogenic digester sludge

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