For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α-subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers.
Methanogenic archaea (methanogens) are strictly anaerobic microorganisms producing methane as a result of their anaerobic respiration (Schink, 1997; Thauer, 1998). For methanogenesis, they can utilize a limited number of substrates such as carbon dioxide, acetate and methyl-group-containing compounds under anoxic conditions (Liu and Whitman, 2008). Most of the known methanogens are hydrogenotrophs reducing carbon dioxide to form methane; among them, formate is also often utilized as the electron donor instead of hydrogen. Some of the hydrogenotrophic methanogens can also utilize secondary alcohols such as 2-propanol as the electron donor. Acetate is an important intermediate substance in the anaerobic decomposition of organic matter, and is generally exclusively utilized by limited groups of methanogens to form methane under anoxic conditions, where external electron acceptors other than carbon dioxide are unavailable. Methyl-group-containing compounds, such as methanol and methylamines, are also utilized by some methanogens through disproportionation of methyl groups.
To explore the ecological significance of methanogens in these natural and engineered ecosystems, identification and quantification techniques for different methanogen groups are indispensable. For the purpose, analyses of membrane lipid (Weijers et al., 2004; Strapoc et al., 2008), autofluorescence (Neu et al., 2002; Tung et al., 2005; Mochimaru et al., 2007), activity measurement (Lehmann-Richter et al., 1999; Weijers et al., 2004) and immunoenzymatic profiling (Visser et al., 1991; Sorensen and Ahring, 1997) have been used. In addition to these methods, cultivation-independent, nucleic acid-based analysis by using oligonucleotide probe/primers, such as membrane hybridization, fluorescence in situ hybridization (FISH), gene cloning, quantitative polymerase chain reaction (qPCR), and cleavage method with ribonuclease H (RNase H) were most widely and frequently used as means to detect and quantify methanogens more specifically and accurately. In this review, we present a catalogue of previously developed oligonucleotide probes/primers targeting genes of methanogens. Particular emphasis is placed on the probes/primers for 16S rRNA, 16S rRNA gene and the gene for the α-subunit of methyl coenzyme M reductase (mcrA), which are generally used for the taxonomic classification of methanogens (Friedrich, 2005; Liu and Whitman, 2008).
Phylogeny of methanogens
All the methanogens isolated and characterized to date have been classified into the phylum Euryarchaeota of the domain Archaea (Garrity et al., 2007). They are assigned into 33 genera of the classes ‘Methanomicrobia’, Methanobacteria, Methanococci and Methanopyri (Fig. 1, Table 1). The class ‘Methanomicrobia’ is the most phylogenetically and physiologically diverse group of methanogens consisting of three orders (Methanosarcinales, Methanomicrobiales and Methanocellales); 23 genera belonging to seven families (Fig. 1, Table 1). Within the order Methanosarcinales, the genera Methanosarcina and Methanosaeta are known to play a key role in the conversion of acetate into methane in various anaerobic environments, and the rest are known to metabolize relatively broad ranges of substrates, such as hydrogen, methanol and methylamines (Garrity and Holt, 2001). Known members of the order Methanomicrobiales are all hydrogenotrophs, and some of them are often observed in anaerobic environments as important hydrogen scavengers (Liu and Whitman, 2008). Members of the class Methanobacteria, consisting of the families Methanobacteriaceae and Methanothermaceae, are recognized as important hydrogenotrophs that have also been widely found in anaerobic ecosystems (Garrity and Holt, 2001). Methanobacteriaceae comprises four genera, Methanobacterium, Methanosphaera, Methanobrevibacter and Methanothermobacter. The class Methanococci includes the families Methanococcaceae and Methanocaldococcaceae, which are widely distributed in natural ecosystems such as marine sediments and deep sea geothermal sediments (Liu and Whitman, 2008). The class Methanopyri consists of solely the genus Methanopyrus, a hyperthermophilic, hydrogenotrophic methanogen isolated from the deep-sea hydrothermal field (Takai et al., 2008).
Table 1. Oligonucleotide probes and primers targeting the 16S rRNA gene of methanogens.
Despite these efforts in cultivating as yet uncultivable methanogens present in environments, there are still a vast number of uncultivable archaeal taxa that may have similar metabolic functions as those of known methanogens. For example, 16S rRNA gene types assigned into the WSA2 (or ArcI) group were frequently retrieved from methanogenic waste/wastewater treatment systems (Sekiguchi and Kamagata, 2004; Chouari et al., 2005). The WSA2 group is considered to be an archaeal taxon at the class level with no cultured representatives (Hugenholtz, 2002). However, Chouari and colleagues have found that WSA2-related cells can be enriched using formate- or hydrogen-containing culture media, suggesting that they harbour methanogenic activity (Chouari et al., 2005). Another example similar to the Rice Cluster I group is Rice Cluster II (RC-II). Members of the RC-II group were also considered to be methanogens, because the 16S rRNA gene clones affiliated with this group were frequently observed in methanogenic enrichment cultures containing ethanol as an electron donor, and because the RC-II group is a lineage within the phylogenetic radiation of the orders Methanosarcinales and Methanomicrobiales (Lehmann-Richter et al., 1999). As can be noted from these examples, there is no doubt that the actual biodiversity of methanogens will be much expanded in the future as the number of isolated and described methanogens continues to increase. However, in this review, we mainly focus on the quantitative monitoring tools for previously cultured methanogens.
Oligonucleotide probes/primers for 16S rRNA and its gene
The 16S rRNA gene has been best used for the identification of methanogens in environments. However, because archaeal 16S rRNA genes other than those of methanogens can also often be detected using PCR primer sets for a wide range of methanogen taxa, it has limitation in exclusively describing the population structure of methanogens. Therefore, there is a need to detect methanogens on the basis of functional genes that are found to be unique in methanogenesis. Such a functional gene frequently used is mcrA. Methyl coenzyme M reductase (mcr) is the terminal enzyme involved in methanogenesis, which reduces the methyl group bond of methyl coenzyme M with the release of methane (Friedrich, 2005). Because the α-subunit of mcr (mcrA) and its isoenzyme gene (mrtA) are highly conserved among methanogens, and that these genes are almost exclusively found in methanogens, mcrA/mrtA-based detection of methanogens has been used. The phylogeny of methanogens determined using mcrA/mrtA (or translated amino acid) sequences is in good accordance with those determined using 16S rRNA gene sequences (Friedrich, 2005). Previously reported, frequently used probes/primers for mcrA/mrtA are categorized into three primer sets, namely, MCR (Springer et al., 1995), ME (Hales et al., 1996) and ML (Luton et al., 2002) (Table 2). The targeted regions of the forward primers of these sets are considerably different, whereas those of the reverse primers are almost the same. The MCR primer set was originally designed to determine the phylogeny of the family Methanosarcinaceae (Springer et al., 1995). The ME primer set was designed to describe methanogenic populations in wetlands (Hales et al., 1996), for which the difficulty in amplifying mcrA/mrtA relevant to Methanosarcinaceae and Methanobacteriaceae was pointed out later (Lueders et al., 2001; Juottonen et al., 2006). The ML primer set was developed on the basis of the mcrA sequences obtained from five orders, comprising Methanosarcinales, Methanomicrobiales, Methanobacteriales, Methanococcales and Methanopyrales (Luton et al., 2002). Four other primer sets and probes for specific taxonomic groups have also been developed recently (Table 2).
Table 2. Oligonucleotide PCR primers and probes targeting the mcrA gene.
Assessing the biodiversity of methanogens in complex communities by PCR detection and cloning of methanogen genes
Some of the noted primers for 16S rRNA and methyl coenzyme M reductase genes have often been used for the detection and identification by PCR to explore the diversity of methanogens in environmental samples (Table 3). For example, the 146f/1324r primer set for most of all the known methanogens was designed for the 16S rRNA gene clone analysis of deep sediment gas hydrate deposit, and the results showed that gene clones (phylotypes) affiliated with Methanosarcina and Methanobrevibacter predominated in the sediments (Marchesi et al., 2001). Similarly, some of these primers shown in Table 1 have been used for PCR to profile methanogen populations by denaturing gradient gel electrophoresis (DGGE) (e.g. (Casamayor et al., 2001; 2002; Yu et al., 2005; 2006; 2008). As examples, Wright and Pimm (2003) developed PCR and sequencing primers for the 16S rRNA gene of methanogens, and used them for the ribotyping of members of the classes ‘Methanomicrobia’ and Methanobacteria. The detection of methanogens by PCR in lamb rumen samples was performed using methanogen-specific primers targeting different taxonomic levels (Skillman et al., 2004). Banning and colleagues (2005) designed novel reverse primers to provide specific amplification of the 16S rRNA genes of ‘Methanomicrobia’ (Methanomicrobiales and Methanosarcinales), Methanobacteriales and Methanococcales, and successfully used them for the identification of methanogenic population structures in lake sediments.
Table 3. Examples of oligonucleotide primer sets for PCR-based analyses for methanogens.
Massive parallel sequencing of PCR-amplified 16S rRNA genes using next generation sequencers (such as the FLX pyrosequencers) allows us to obtain a huge number of community sequence tags (for example c. 10 000–100 000 16S pyrotags for each sample), which is more than any Sanger-based cloning study to date, and have been used for characterizing archaeal populations (including methanogens) in hydrothermal chimneys (Brazelton et al., 2010a,b). The methodological advancements of 16S rRNA gene pyrosequencing include higher resolution (more sequences) for gene-based community structure analysis, analysis of multiple related samples and use of metadata (Tringe and Hugenholtz, 2008). Because of these advancements, as well as recent development of analytical tools for massive sequence data such as QIIME (Caporaso et al., 2010), the method may be further used for characterizing diversity of methanogens in ecosystems.
Polymerase chain reaction-based molecular techniques, such as PCR-cloning, pyrosequencing, DGGE and T-RFLP are adequate to gain entire community composition and diversity of methanogens in ecosystems. Based on the frequency of retrieval of phylotypes in gene library (or relative intensity of DGGE or T-RF bands in electropherogram), relative abundance of phylotypes of interest can be inferred. However, it should be noted that entire microbial community structure analysis based on bulk cell lysis, DNA extraction, PCR and cloning are often suspect because of several biases involved in each of the steps (Dahllof, 2002). Therefore, one should be careful to discuss on the abundance of phylotypes in samples based solely on the data obtained by these methods. More reliable methods to carry out quantitative detection of different groups of methanogens in samples would be to use the following quantitative molecular techniques.
Identification and quantification of methanogens in complex communities by membrane hybridization method
Quantitative membrane hybridization of labelled DNA probes to community rRNAs has been applied to various environmental rRNAs for the quantitative detection of specific groups of microbes present in complex communities (Stahl et al., 1988; Raskin et al., 1994a). RNA-dependent community analysis is known to indicate the in situ activity of individual members in ecosystems, because of the reasons that RNA synthesis is known to reflect the in situ growth rates of organisms (Poulsen et al., 1993; Amann et al., 1995), and that the turnover of RNA is thought to be much higher than that of DNA. Therefore, rRNA-dependent molecular techniques like the present one provide precise information about the dynamic nature of individual microbes in systems. In 1994, Raskin and colleagues carried out the first leading studies on the development of eight oligonucleotide probes for the quantitative detection of methanogens in anaerobic wastewater treatment sludges (Stahl and Amann, 1991; Raskin et al., 1994a,b). In these studies, they established the group-specific oligonucleotide probes targeting Methanomicrobiales (probes MG1200 and MSMX860), Methanobacteriaceae (probes MB310 and MB1174) and Methanococcales (probe MC1109). Because of the importance of methane production from acetate in anaerobic bioreactors, specific probes for aceticlastic methanogens, such as the members of Methanosarcinaceae (probes MS1414 and MS821) and Methanosaeta (probe MX825), were also developed.
These probes have been successfully applied to the quantification of methanogens in laboratory- and full-scale anaerobic bioreactors based on rRNA (Raskin et al., 1995; Griffin et al., 1998; Liu et al., 2002; McMahon et al., 2004; Zheng et al., 2006). Although membrane hybridization enables the sensitive quantification of individual species of rRNA molecules, this method requires several laborious experimental steps, often radioactively labelled DNA probes, and reference rRNA samples as external standards for each experiment. Thus, the method itself may be replaced by similar but much rapid and simpler methods, such as real-time RT-PCR and RNase H methods. However, the probes used for membrane hybridization experiments may be also used as probes/primers in other experiments shown below.
FISH for methanogens
Whole-cell FISH based on 16S rRNA is now commonly used to detect specific groups of microbes and to quantify populations of interest in environments by direct counting under a microscope (Amann et al., 1995). In addition, FISH is used for visualizing the spatial distribution of the population of interest in biofilms, such as those of methanogens in sludge granules in methanogenic wastewater treatment systems (Sekiguchi et al., 1999). Basically, the probes developed for membrane hybridization of methanogen 16S rRNAs or reverse primers for PCR amplification of methanogen 16S rRNA genes can directly be used as oligonucleotide probes for in situ hybridization studies, the probes previously designed by Raskin and colleagues (Raskin, et al., 1994b) have frequently be used for the purpose of FISH studies as well. These probes have been used for the quantitative detection of methanogens using the FISH technique in various anaerobic ecosystems, such as peat bog (e.g. Horn et al., 2003), aquifer (e.g. Kleikemper et al., 2005), landfills (e.g. Laloui-Carpentier et al., 2006) and anaerobic wastewater treatment processes (e.g. Sekiguchi et al., 1999; Plumb et al., 2001; Boonapatcharoen et al., 2007; Chen et al., 2009). Recently, the improvement of the specificity and sensitivity of the probes designed by Raskin and colleagues (1994b) has been reported. Crocetti and colleagues (2006) refined the experimental conditions of such probes for FISH analysis to accurately and sensitively detect methanogens.
Although FISH is a powerful method for visualizing the cells of interest, there are some drawbacks in detecting cells; one of such problems is concerned with the penetration of oligonucleotide probes into the cells (Amann et al., 1995). For methanogens, FISH staining is often difficult for some Methanobacterium and Methanobrevibacter cells, for which oligonucleotide probes do not penetrate into their cells (Sekiguchi et al., 1999; Yanagita et al., 2000; Nakamura et al., 2006). To solve this problem, fixed cells were subjected to freeze-thaw cycles before hybridization, resulting in the improvement of probe penetration (Sekiguchi et al., 1999). Another way to solve this problem is the use of recombinant pseudomurein endoisopeptidase, which increases the permeability of oligonucleotide probes into cells, and allows a better visualization of methanogens in anaerobic granular sludge and the endosymbiotic methanogens in the anaerobic ciliate Trimyema compressum (Nakamura et al., 2006). An improved protocol of catalysed reporter deposition-FISH for methanogens with recombinant pseudomurein endoisopeptidase has also been reported, which can increase fluorescence signal intensity in FISH for detecting cells with a low rRNA content (Kubota et al., 2008).
Recently, mcrA-based in situ detection of methanogens has been performed using the two-pass tyramide signal amplification-FISH approach combined with locked nucleic acids (Kubota et al., 2006; Kawakami et al., 2010). These attempts were, at this point, only partially successful in detecting methanogen cells, because mcrA is generally present as a single copy gene on their chromosome, which results in a low sensitivity of detection.
For the qPCR detection of mcrA, the ME primer set was used for the quantification of methanogenic and methanotrophic populations in methane seep sediments (Inagaki et al., 2004; Nunoura et al., 2006). Afterwards, Nunoura and colleagues (2008) slightly modified the ME primer series, and showed that the mixture of the ME3MF and ME3MF-e forward primers and the ME2' reverse primer is most suitable for the qPCR detection of the methanogens and ANMEs in the environments. The results showed that a significant amount of methanogens and ANMEs was found in anaerobically digested sludge and methane seep sediments. The ML primer set was also used for the quantitation of methanogenic archaeal populations in the rumen (Denman et al., 2007) and human subgingival plaque (Vianna et al., 2008). Moreover, Steinberg and Regan (2008; 2009) developed the mlas/mcrA-rev primer set, which is a derivative of the ML primer set, for the clone library construction and qPCR analyses of methanogens in oligotrophic fen and anaerobic digester sludge. In addition, the genus-specific TaqMan probes for the mcrA-based quantitative detection of the Methanosaeta, Methanosarcina and Methanoculleus resident in acetate-fed chemostats, and the results showed that dilution rate is a key factor in the acetate bioconversion pathway (Shigematsu et al., 2004).
Quantitative PCR method provides sensitive, quantitative data of gene of interest with a sufficiently high dynamic range of quantification (Zhang and Fang, 2006). Therefore, in addition to the use of digital PCR (Ottesen et al., 2006), qPCR may be further used for quantitative monitoring of methanogen taxa of interests in complex microbial communities. However, it should be noted that the method is PCR-based and hence their data can be suspect because of biases involved in DNA extraction and primer/probe mismatches.
Assessing methanogen population by RNase H method
Although the above-mentioned quantitative methods such as membrane hybridization and qPCR are becoming general means to determine the abundance of the population of interest in a complex microbial community, there is a need to develop more simple and rapid techniques that meet the needs for real-time monitoring of the population of interest in a complex community. Recently, a simple and rapid quantification method, namely, the RNase H method, has been developed (Uyeno et al., 2004). This method is based on the sequence-specific cleavage of 16S rRNA with ribonuclease H (RNase H) and oligonucleotide (scissor) probes. RNAs from a complex community were first mixed with an oligonucleotide and subsequently digested with RNase H. Because RNase H specifically degrades the RNA strand of RNA : DNA hybrid heteroduplexes, the targeted rRNAs are cleaved at the hybridization site in a sequence-dependent manner and are consequently cut into two fragments. In contrast, non-targeted rRNAs remain intact under the same conditions. For the detection of cleaved rRNAs, the resulting RNA fragment patterns can be resolved by gel electrophoresis using RNA-staining dyes. The relative abundance of the targeted species of 16S rRNA fragments in total 16S rRNA can also be quantified by determining the signal intensity of individual 16S rRNA bands in an electropherogram (without the use of external standards). Because this method does not require an external RNA standard for each experiment, as is required in membrane hybridization, and because the present method is relatively easy to perform within a short time (i.e. within 2–3 h), this technique may provide direct, rapid and easy means of the quantitative detection of particular groups of anaerobes based on their rRNA, such as those of methanogens as well.
This method has been successfully applied to the quantification of active methanogens in anaerobic biological treatment processes (Uyeno et al., 2004; Sekiguchi et al., 2005; Narihiro et al., 2009b). In general, oligonucleotide probes used in FISH and membrane hybridization methods can directly be used as scissor probes in the RNase H method. Recently, a total of 40 probes, including newly designed and previously reported probes listed in Table 1, have been optimized for the specific quantification of methanogens at different taxonomic levels for use in the RNase H method and have been applied to quantitative and comprehensive detection of methanogens in various types of anaerobic biosystems (Narihiro et al., 2009b). As a result, methanogen populations were identified at different taxonomic levels and were influenced by the process temperature and wastewater compositions. Because of the reasons that this method is based on rRNA and that the RNA (rRNA) level is often dependent on the in situ activity of individual cells as described above, this method may be used for real-time monitoring of active methanogens and other important bacteria in engineered ecosystems such as waste/wastewater treatment systems to better control such bioreactors.
Stable isotope probing (SIP)-based detection of active methanogen populations in environments
To identify metabolically active populations in environments, SIP of DNA (Radajewski et al., 2000) and RNA (Manefield et al., 2002) has been used in recent years. In principle, SIP technology is based on the incorporation of 13C-labelled substrates into the nucleic acids. The separation of isotopically labelled (active) fractions from unlabeled (inactive) fractions is generally performed with density gradient centrifugation. The substrate-assimilated microorganisms in the labelled fractions are identified by a set of PCR-based molecular techniques such as gene cloning, DGGE and other methods. Therefore, for the purpose of identifying active methanogens that are responsible for particular metabolisms in environments, the probes/primers listed in Tables 1 and 2 can be used.
As examples, active methanogen populations involved in the syntrophic propionate oxidation in anoxic soil were analysed on the basis of rRNA-SIP, and it was found that the members of the genera Methanobacterium, Methanosarcina and Methanocella play a key role in scavenging hydrogen/formate/acetate in syntrophic association with propionate-oxidizing bacteria (Lueders et al., 2004). Conrad and coworkers have studied the detection of active methanogen populations using DNA-SIP combined with 13C-labelled CO2, and the results of T-RFLP profiling and phylogenetic analysis for clonal 16S rRNA gene fragments suggest that members of the RC-I group (Methanocellales) serve as important methanogens in rice paddy fields (Lu and Conrad, 2005; Lu et al., 2005). The active methanogenic populations in enrichment culture of municipal solid waste digester residues spiked with 13C-labelled substrates (such as cellulose, glucose and sodium acetate) were determined by DNA-SIP followed by cloning analysis (Li et al., 2009).
Other methods and future perspectives
DNA microarray platform, like PhyloChip, is becoming an important tool for parallel detection of different community members of microbes in ecosystems. For high throughput and comprehensive detection of methanogens in parallel, ANAEROCHIP (Franke-Whittle et al., 2009b) and GeoChip (Wang et al., 2009) have been developed recently. The primers/probes summarized in this review may be integrated into such a platform for parallel and hierarchical detection of methanogens. These primer/probes for methanogens can also be used in novel PCR-based techniques, such as the hierarchical oligonucleotide primer extension method (Wu and Liu, 2007), which has recently been developed for quantitative, multiplex detection of targeted microbial genes among PCR-amplified genes. SIP technology has been noted as an important pretreatment step for functional microbial community analyses, such as Raman microscopy-FISH (Huang et al., 2007; 2009) and metagenomic approaches (Kalyuzhnaya et al., 2008; Sul et al., 2009). Moreover, recent advances in analytical chemistry, such as isotope ratio mass spectrometry (Penning et al., 2006; Vavilin et al., 2008) and secondary ion mass spectrometry (Orphan et al., 2001), hold great promise for the highly sensitive determination of targeted microbes. Thus, in addition to describing the diversity of methanogens in particular environments of interest on the basis of DNA and RNA, such function-related analyses of methanogens may become important in the fields of environmental, determinative and applied microbiology.
As described in this minireview, a vast number of probe/primers have been developed for describing and quantifying methanogen populations, covering most parts of the known culturable methanogens described so far. A variety of molecular methods have also been developed that are used in combination with the probe/primers. Because these molecular methods have their own advancements and drawbacks, researchers need to select appropriate combinations of methods and probe/primers depending on what the researchers need to know. For details, recent reviews may be helpful for the selection of molecular techniques to be used (Talbot et al., 2008; Tabatabaei et al., 2010). In molecular ecology, multiple approaches are best to gain a complete picture of methanogen populations in environments. Therefore, the use of appropriate (multiple) molecular techniques in combinations with other non-molecular based methods like membrane lipid, autofluorescence, activity measurement and immunoenzymatic profiling should be considered. It should also be noted that there are still a number of uncultivated methanogens in various environments, and that they should be further isolated and characterized in detail. Monitoring tools for such uncultured methanogens remain to be developed to further increase in the coverage of methanogens present in environments.
This work was supported by the Environment Research and Technology Development Fund (S2-03) and the Global Environment Research Fund (RF-076) of the Ministry of the Environment, Japan.