BACKGROUND
methanogen diversity has been widely investigated across a range of ruminants by using clone library sequence approaches and many unknown methanogen 16s rrna sequences have been uncovered.
tajima et al.
<cit>  investigated the diversity of bovine rumen fluid using two different archaea-specific primer sets, and for the first time reported the existence of a novel cluster of uncultured archaeal sequences which were distantly associated with thermoplasma.
however, the authors concluded that these novel sequences were likely from transient microbiota contaminating the animal feed, probably scavenging in an ecological niche in the rumen.
wright et al.
<cit>  was the first to verify that these novel thermoplasma-affiliated sequences were derived from the rumen when they investigated the diversity of rumen methanogens from sheep.
the authors suggested a new order of methanogens for these novel sequences in the new cluster.
the same authors  <cit>  further found that over 80% of the total methanogen clones  from the rumen of merino sheep in australia were 72–75% similar to thermoplasmaacidophilum and thermoplasmavolcanium.
they  <cit>  also found that about 50% of the total clones from methanogen 16s rrna gene library of potato-fed feedlot cattle were present in the new cluster, and 38% for corn-fed feedlot cattle.
huang et al.
<cit>  found that thermoplasmatales-affiliated sequences dominated in the yak and cattle methanogen clone libraries, accounting for  <dig> % and  <dig> % of the sequences in the two libraries, respectively.
our previous study  <cit>  on the diversity of methanogens in the rumen of jinnan cattle showed that thermoplasmatales-affiliated sequences were widely distributed in the rumen epithelium, rumen solid and fluid fractions.
in addition, ruminant-derived sequences in this new cluster were also found in other studies  <cit> .
based on the analysis of the global data set, janssenand kirs  <cit>  placed the majority  of rumen archaea detected in total rumen contents into three genus-level groups: methanobrevibacter , methanomicrobium, and a large group of uncultured rumen archaea affiliated with thermoplasmatales , and named the uncultured archaea group in the rumen, for the first time, as rumen cluster c .
using rcc specific dgge, clone library analysis and quantitative real-time pcr, jeyanathan et al.
<cit>  investigated the composition of archaeal communities in the rumens of farmed sheep, cattle and red deer, and found that the 16s rrna gene sequences of rcc were highly diverse and made up an average of  <dig> % of the total archaea.
to date, the rcc has been found in many ruminants, including cattle  <cit> , sheep  <cit> , goats  <cit> , water buffalo  <cit> , and red deer  <cit> .
further the proportion of rcc within the total methanogen populations is high   <cit> .
however, most of these studies have been conducted using sequencing-based culture-independent molecular methods.
the role of rcc in the rumen remains unclear in the absence of cultivated isolates.
further, although rcc has been labeled as a group of methanogens, there is little evidence to support that the rcc is methanogen  <cit> .
recently, poulsen et al.
<cit>  investigated the impact of rapeseed oil on the abundance of rumen microorganisms and their gene expression by metatranscriptomics, and found that methylamines might be the substrates for rcc.
they further verified this by in vitro experiment which was composed of adding trimethylamine  to bovine rumen fluids and incubating for 24 hours.
the results showed that methane production increased 22%, accompanied by a three fold increase for the abundance of rcc.
moreover, the recently reported methanomassiliicoccus luminyensis from human feces, which was clustered within rcc clade in our present study, could use hydrogen to reduce methanol to methane  <cit> .
borrel et al.
<cit>  published the genome sequence of another rcc related isolate  from human gut and reported this isolate contains genes needed for methylotrophic methanogenesis from methanol and methylamines.
padmanabha et al.
<cit>  reported that a chicken gut isolate  belonging to rcc clade could strictly use hydrogen to reduce both methylamines and methanol to methane.
in agreement with wright et al.
<cit>  suggesting a new order, paul et al.
<cit>  strongly proposed that these unclassified thermoplasmatales sequences  represents the seventh order of methanogenic archaea, based on the comparative phylogenetic analysis of the 16s rrna genes and mcra gene sequences, together with the enriched cultures from the higher termites and millipedes and the recently reported isolate m. luminyensis.
thus, the methanogenic archaeon in this order are widely distributed in marine habitat, soil, and in the intestinal tracts of termites and mammals.
although the exact contribution of rcc to rumen methane production still remains unclear, they possibly play an important role in the methanogenesis, due to their high percentage in the rumen methanogen population  <cit> .
therefore, the cultivation and isolation of these unique rccs from rumen has become increasingly important for understanding the role of rcc in the rumen.
however, many attempts have been made, but the isolation of anoxic pure rcc from the rumen still remains unsuccessful.
our previous studies showed that some rumen methanogens were indigenously associated with anaerobic fungi, which could result in a strong degradation of lignocellulosic materials  <cit> .
through an approach using a co-culture derived from a mixed-culture, our study further found that a novel species belonging to rcc grew in the anaerobic fungal subcultures.
therefore, the present study aimed to identify this novel species and investigate its features in the anaerobic fungal cultures.
pcr specific primers were designed to monitor the novel rcc species growing in the fungal cultures and its distribution in the rumen.
to better understand the novel rcc species, purification was also conducted.
RESULTS
presence of methanogens in the anaerobic fungal subcultures
the methanogen diversity in the fungal cultures during transfers was shown in dgge in figure  <dig>  as the consecutive transfer proceeded there was a reduction in the diversity of methanogens, resulting in only two strong bands on the gel of the 62nd subcultures.
in order to understand the composition of the methanogens in the enriched mixed cultures, a clone library targeting the 16s rrna gene was constructed for the methanogens in the 25th subcultures.
a total of  <dig> clones were examined by riboprint analysis, and  <dig> phylotypes were revealed .
two of these  <dig> phylotypes, represented by two clones, were  <dig> %,  <dig> % similar to methanobrevibacter sp.
30y, respectively.
ten phylotypes, represented by  <dig> clones, were  <dig> % to  <dig> % similar to methanobrevibacter sp.
z <dig>  one phylotype , represented by two clones, was  <dig> % similar to ca.
m. alvus m ×  <dig>  as shown in figure  <dig>   <dig> of the  <dig> phylotypes were clustered into the “ro” cluster of the genus methanobrevibacter.
the phylotype lgm-af <dig> was clustered with sequences representing rcc.
a total of  <dig> clones were examined.
further, in order to understand the methanogens which survived in the long-term transferred fungal subcultures, the two strong bands from the 62nd subcultures were excised from the dgge gel for further cloning.
five clones generated from each band were sequenced and showed to be identical.
one band had its sequence  99% similar to lgm-af <dig>  and the other had its sequence  98% similar to methanobrevibacter sp.
z <dig>
transfer frequency affects the abundance of the novel rcc species in the fungal subcultures
to monitor the abundance of the novel rcc species, pcr specific primers  to this novel rcc were designed.
blast searches of the primer sequences showed homology to sequences within the novel rcc species only.
their specificity was further confirmed by running pcr, and results showed that the primers only targeted the novel rcc species, and did not target other methanogen isolates or clones, or bacteria species tested in this study .
the effects of the transfer frequency on the abundance of the novel rcc species in the anaerobic fungal subculture were investigated using the specific primers.
the results showed that, as the transfer proceeded, the16s rrna gene copy numbers of the novel rcc species significantly increased in the mixed cultures with the five-day transfer frequency and the seven-day transfer frequency , while it decreased in the three-day subcultures .
this finding suggested that low transfer frequency might benefit the enrichment of the novel rcc species in the mixed cultures.
distribution of the novel rcc species in the rumen
the distribution of the novel rcc species in the rumen epithelium, in the liquid and solid fractions of goats fed with diets of different concentrate levels is shown in table  <dig>  the16s rrna gene copy numbers of the novel rcc species in the rumen epithelium, the liquid and solid fraction ranged from  <dig>  to  <dig> ,  <dig>  to  <dig>  and  <dig>  to <dig>  , respectively.
the total archaea ranged from <dig>  to  <dig> ,  <dig>  to  <dig>  and  <dig>  to  <dig>  , respectively.
the abundance of the novel rcc species in the rumen of goats fed low concentrate diet was numerically higher than that of goats fed high concentrate diet.
but, the abundance of the total archaea was not affected by the high concentrate feeding.
the relative abundance of the novel rcc species within total archaea  in the liquid fraction was numerically higher than in the other two fractions  in each diet group.
a, b, c, means with different letters in the same column are different p <  <dig> ; n =  <dig>
*, the ph value of rumen content,  <dig>  ±  <dig>  ;  <dig>  ±  <dig>  ;  <dig>  ±  <dig>  .
purification of the novel rcc species with anaerobic fungus
one fungal culture containing the novel rcc species was obtained after purification with trimethylamine to support the novel rcc and with lumazine to inhibit the growth of methanobrevibacter sp.
the anaerobic fungus was identified as belonging to piromyces sp.
as revealed by morphological examination .
the sequencing results showed only one 16s rrna gene sequence from the total dna extracted from the supernatant of the fungal culture, and this sequence was 100% identical to lgm-af <dig>  and 99% to the clone from jinnan cattle rumen .
further confirmation was also performed by sequencing the mcra gene coding the alpha subunit of the methyl-coenzyme m reductase that plays a crucial role in the methanogenesis, and the results showed that only one mcra gene sequence  was present.
these findings strongly supported the hypothesis that the novel rcc species in the co-culture with rumen anaerobic fungi is a methanogen.
discussion
in this study, a novel rcc species was found growing in the anaerobic fungal subcultures.
many studies have shown that a large group of rcc inhabited the rumen of a variety of ruminant species on various diets  <cit> .
thus, the rcc species grown in the anaerobic fungal cultures in the present study just represented a small group of the total rcc.
it has been proposed that the rcc in the rumen and its relatives in other environments could constitute the seventh order of the methanogens   <cit> .
methanogens within this new methanogenic order distantly related to the thermoplasmatales, have been shown to be present in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals, suggesting their ubiquitous in various environments.
the whole order was proposed to form three big clusters, ca.
m. alvus cluster, m. luminyensis cluster and lake pavin cluster  <cit> .
the novel rcc species in the present study was grouped in the ca.
m. alvus cluster.
the present study reported the first account for rcc species grown in the fungal cultures from the goat rumen.
nevertheless this single species may not represent the whole rcc community in the rumen.
therefore, further research is needed to uncover this community and its features in the rumen.
interestingly, this novel species could survive in the long-term transferred fungal subcultures .
thus, there must be an underlying mechanism supporting the growth of this novel rcc species in the fungal subcultures.
a similar phenomenon for protozoa was reported by irbis and ushida  <cit> .
when testing a single protozoa cell for the 16s rrna gene sequences of archaea, they found that the cultured rumen protozoa isotricha intestinalis and ophryoscolex purkynjei from goats carried thermoplasma sp.
related sequences .
recent studies showed that methanogens belonging to this group  <cit>  could strictly use hydrogen to reduce methanol and methylamines to methane.
it is well known that both anaerobic fungi and protozoa could produce hydrogen, which is one of the substrates for methanogens  <cit> .
this may make it possible for anaerobic fungi to provide rcc species with hydrogen.
methanol and methylamines could be derived from the microbial degradation of pectin, betaine, and choline from diets in the rumen  <cit> .
ametaj et al.
<cit>  demonstrated that there were methanol and methylamines in the rumen fluid of lactating dairy cows fed graded amounts of barley grain.
in this study, the medium for the co-culture of anaerobic fungi and methanogens contained rice straw and clarified rumen fluid.
anaerobic fungi could degrade the pectin of rice straw by pectinolytic enzymes  <cit> , accompanying the release of methanol.
thus, it is possible that the novel rcc species obtained a certain amount of tma and methanol from the rumen fluid and the degradation of rice straw.
when methanol was used to enrich rcc in the fungal cultures, methanosphaera sp.
was obtained instead of rcc species , which implied that methanosphaera sp.
may compete for the same substrate  with rcc.
in addition to the competition for the available substrates, there might be other underlying mechanisms enabling the novel rcc species to survive in the in vitro and in vivo niches.
apparently, further research is necessary to reveal the underlying mechanisms.
the novel rcc exhibited apparent enrichment with less frequent transfer, with relatively higher proportion in 7 day transfer culture than in  <dig> d or 5d transfer cultures .
in our previous study, cheng et al.
<cit>  investigated the effects of transfer frequencies on the diversity of anaerobic fungi and methanogens in the enriched mixed cultures.
they found that anaerobic fungal diversity was related to transfer frequencies and appeared to be simplified as transfer proceeded.
in contrast, the methanogen population generally remained diverse, regardless of the transfer frequencies.
thus, the survival and the shift of the abundance of the novel rcc species in fungal cultures might be related to the changes of the composition of the anaerobic fungal community.
on the other hand, it seems that the rcc grew slowly in the in vitro culture, while the methanobrevibacter tended to grow more rapidly.
thus longer incubation interval between transfers would allow the rcc populations to increase while the methanobrevibacter populations were declining.
therefore, the approach using long incubation intervals would allow the enrichment of the novel rcc.
however, how much the transfer frequency effect may be due to the specific co-culture with an anaerobic fungus remains an open question.
the present study quantified the abundance of the novel rcc species and the total archaea in the rumen.
it seems that the abundance of the novel rcc species was also affected by the diet composition, with the value in the rumen of goats fed low concentrate diet numerically higher than that of goats fed high concentrate diet .
but the abundance of the total archaea seems not affected by the levels of concentrate in the diets .
similarly, hook et al.
<cit>  reported that high-concentrate feeding did not affect the density of the total rumen methanogens, but they found that high-concentrate feeding mitigated the methane production and altered the methanogen diversity and community structure.
they also suggested that ph sensitive methanogens might be lost when the rumen ph decreased.
it was possible that the novel rcc species was sensitive to low ph caused by high-concentrate feeding.
it is also possible that some unaffected methanogens occupied the vacated niche of the novel rcc species in the rumen of goats fed with high-concentrate diet.
nevertheless, it is still unclear whether all the rcc populations are sensitive to the high-concentrate feeding and/or low ph.
the present study provided the first estimation of this rcc species distribution in the rumen.
the abundance of the novel rcc species was different in the rumen epithelium, rumen liquid and solid fractions .
the relative abundance of the novel rcc species as indicated by its proportion within total archaea populations in their respective fraction was higher in liquid fraction as compared to epithelium and solid fraction.
previous study suggested that it was difficult to detach all of the microbes associated with the solid fraction  <cit> , thus the abundance of rcc and archaea in this fraction may be grossly underrepresented.
our previous study  <cit>  showed that the composition of the methanogens were different in the rumen epithelium, solid and liquid fractions of jinnan cattle, especially for the unidentified archaea.
we compared these unidentified archaeal sequences with rcc sequences  in this study and found that  <dig> % of the total clones in the liquid fraction was clustered within rcc clade, and  <dig> % in the solid,  <dig> % in the epithelium.
the clones  that were most similar to the novel rcc species were from the rumen epithelium fraction.
moreover, gu et al.
<cit>  reported that  <dig> % of the clones in the goat rumen fluid library belonged to the thermoplasmatales family , and  <dig> % in the rumen solid library; however, no clones were > 95% similar to the novel rcc species.
in this study, the relative density of the novel rcc species was numerically higher in the rumen liquid fraction  than in the other two fractions , which might be due to the specific characteristics of the novel rcc species.
in the rumen, liquid, solid and epithelium fractions have different turnover rates.
janssen and kirs  <cit>  proposed that the methanogens associated with different rumen fractions could be expected to have different growth rates since they would be removed from the rumen at different rates.
thus, the novel rcc species might have a relatively higher growth rate than other rccs in the rumen liquid fraction.
in the present study, the novel rcc species was co-isolated with anaerobic fungus.
most recently, a tri-culture with a rcc member, a clostridium sp.
and a bacteroides sp.
was enriched from bovine rumen .
further attempts to obtain pure rcc species were made but unsuccessful.
it seems that there is a close relationship between the novel rcc species and anaerobic fungus.
two isolates  related to rcc had been obtained from human feces.
most recently, another rcc related isolate m. gallocaecorum strain dok- <dig>  <cit>  from chicken gut was reported.
the three isolates exhibited similar nutritional requirements for methyl-group chemicals.
in the current study, the phylogenetic analysis showed that the novel rcc species were clustered into the same clade with ca.
m. alvus mx <dig> .
however, the 16s rrna gene sequence of the novel rcc species showed 93% similarity to ca.
m.alvusmx <dig> , and 87% to m. luminyensis .
the mcra gene sequences of the novel rcc species  showed 84% similarity to ca.
m. alvus mx <dig> , and 78% to m. luminyensis .
thereby, though clustered into the rcc clade, the novel rcc species in this study were phylogenetically distant with the two human isolates, the recently reported rcc isolates, suggesting that the new order for rcc and its relatives may be highly diverse.
CONCLUSIONS
a novel rcc species was found surviving in the long-term transferred anaerobic fungal subcultures and closely associated with anaerobic fungi.
the results verified that the quantification of the novel rcc species in vivo and in vitro is possible by real-time pcr using its specific primers.
the relative abundance of the novel rcc species in the anaerobic fungal subcultures was affected by the transfer frequencies, with the seven day transfer frequency suitable for its enrichment.
the high concentrate feeding did not affect the abundance of the total archaea population, but numerically reduced the abundance of the novel rcc species in the goat rumen.
the relative abundance of the novel rcc species was numerically higher in the rumen liquid fraction than in the epithelium and solid fractions.
a novel rcc species was co-isolated with an anaerobic fungus, and was identified as being a methanogen.
the finding in the present study may help to culture and investigate the unknown methanogens in the rumen.

 methanogen diversity has been widely investigated across a range of ruminants by using clone library sequence approaches and many unknown methanogen 16s rrna sequences have been uncovered. tajima et al. investigated the diversity of bovine rumen fluid using two different archaea-specific primer sets, and for the first time reported the existence of a novel cluster of uncultured archaeal sequences which were distantly associated with thermoplasma. however, the authors concluded that these novel sequences were likely from transient microbiota contaminating the animal feed, probably scavenging in an ecological niche in the rumen. wright et al. was the first to verify that these novel thermoplasma-affiliated sequences were derived from the rumen when they investigated the diversity of rumen methanogens from sheep. the authors suggested a new order of methanogens for these novel sequences in the new cluster. the same authors further found that over 80% of the total methanogen clones from the rumen of merino sheep in australia were 72–75% similar to thermoplasmaacidophilum and thermoplasmavolcanium. they also found that about 50% of the total clones from methanogen 16s rrna gene library of potato-fed feedlot cattle were present in the new cluster, and 38% for corn-fed feedlot cattle. huang et al. found that thermoplasmatales-affiliated sequences dominated in the yak and cattle methanogen clone libraries, accounting for % and % of the sequences in the two libraries, respectively. our previous study on the diversity of methanogens in the rumen of jinnan cattle showed that thermoplasmatales-affiliated sequences were widely distributed in the rumen epithelium, rumen solid and fluid fractions. in addition, ruminant-derived sequences in this new cluster were also found in other studies. based on the analysis of the global data set, janssenand kirs placed the majority of rumen archaea detected in total rumen contents into three genus-level groups: methanobrevibacter, methanomicrobium, and a large group of uncultured rumen archaea affiliated with thermoplasmatales, and named the uncultured archaea group in the rumen, for the first time, as rumen cluster c. using rcc specific dgge, clone library analysis and quantitative real-time pcr, jeyanathan et al. investigated the composition of archaeal communities in the rumens of farmed sheep, cattle and red deer, and found that the 16s rrna gene sequences of rcc were highly diverse and made up an average of % of the total archaea. to date, the rcc has been found in many ruminants, including cattle, sheep, goats, water buffalo, and red deer. further the proportion of rcc within the total methanogen populations is high. however, most of these studies have been conducted using sequencing-based culture-independent molecular methods. the role of rcc in the rumen remains unclear in the absence of cultivated isolates. further, although rcc has been labeled as a group of methanogens, there is little evidence to support that the rcc is methanogen. recently, poulsen et al. investigated the impact of rapeseed oil on the abundance of rumen microorganisms and their gene expression by metatranscriptomics, and found that methylamines might be the substrates for rcc. they further verified this by in vitro experiment which was composed of adding trimethylamine to bovine rumen fluids and incubating for 24 hours. the results showed that methane production increased 22%, accompanied by a three fold increase for the abundance of rcc. moreover, the recently reported methanomassiliicoccus luminyensis from human feces, which was clustered within rcc clade in our present study, could use hydrogen to reduce methanol to methane. borrel et al. published the genome sequence of another rcc related isolate from human gut and reported this isolate contains genes needed for methylotrophic methanogenesis from methanol and methylamines. padmanabha et al. reported that a chicken gut isolate belonging to rcc clade could strictly use hydrogen to reduce both methylamines and methanol to methane. in agreement with wright et al. suggesting a new order, paul et al. strongly proposed that these unclassified thermoplasmatales sequences represents the seventh order of methanogenic archaea, based on the comparative phylogenetic analysis of the 16s rrna genes and mcra gene sequences, together with the enriched cultures from the higher termites and millipedes and the recently reported isolate m. luminyensis. thus, the methanogenic archaeon in this order are widely distributed in marine habitat, soil, and in the intestinal tracts of termites and mammals. although the exact contribution of rcc to rumen methane production still remains unclear, they possibly play an important role in the methanogenesis, due to their high percentage in the rumen methanogen population. therefore, the cultivation and isolation of these unique rccs from rumen has become increasingly important for understanding the role of rcc in the rumen. however, many attempts have been made, but the isolation of anoxic pure rcc from the rumen still remains unsuccessful. our previous studies showed that some rumen methanogens were indigenously associated with anaerobic fungi, which could result in a strong degradation of lignocellulosic materials. through an approach using a co-culture derived from a mixed-culture, our study further found that a novel species belonging to rcc grew in the anaerobic fungal subcultures. therefore, the present study aimed to identify this novel species and investigate its features in the anaerobic fungal cultures. pcr specific primers were designed to monitor the novel rcc species growing in the fungal cultures and its distribution in the rumen. to better understand the novel rcc species, purification was also conducted.  presence of methanogens in the anaerobic fungal subcultures the methanogen diversity in the fungal cultures during transfers was shown in dgge in figure  as the consecutive transfer proceeded there was a reduction in the diversity of methanogens, resulting in only two strong bands on the gel of the 62nd subcultures. in order to understand the composition of the methanogens in the enriched mixed cultures, a clone library targeting the 16s rrna gene was constructed for the methanogens in the 25th subcultures. a total of clones were examined by riboprint analysis, and phylotypes were revealed. two of these phylotypes, represented by two clones, were %, % similar to methanobrevibacter sp. 30y, respectively. ten phylotypes, represented by clones, were % to % similar to methanobrevibacter sp. z one phylotype, represented by two clones, was % similar to ca. m. alvus m × as shown in figure  of the phylotypes were clustered into the “ro” cluster of the genus methanobrevibacter. the phylotype lgm-af was clustered with sequences representing rcc. a total of clones were examined. further, in order to understand the methanogens which survived in the long-term transferred fungal subcultures, the two strong bands from the 62nd subcultures were excised from the dgge gel for further cloning. five clones generated from each band were sequenced and showed to be identical. one band had its sequence 99% similar to lgm-af and the other had its sequence 98% similar to methanobrevibacter sp. z transfer frequency affects the abundance of the novel rcc species in the fungal subcultures to monitor the abundance of the novel rcc species, pcr specific primers to this novel rcc were designed. blast searches of the primer sequences showed homology to sequences within the novel rcc species only. their specificity was further confirmed by running pcr, and results showed that the primers only targeted the novel rcc species, and did not target other methanogen isolates or clones, or bacteria species tested in this study. the effects of the transfer frequency on the abundance of the novel rcc species in the anaerobic fungal subculture were investigated using the specific primers. the results showed that, as the transfer proceeded, the16s rrna gene copy numbers of the novel rcc species significantly increased in the mixed cultures with the five-day transfer frequency and the seven-day transfer frequency, while it decreased in the three-day subcultures. this finding suggested that low transfer frequency might benefit the enrichment of the novel rcc species in the mixed cultures. distribution of the novel rcc species in the rumen the distribution of the novel rcc species in the rumen epithelium, in the liquid and solid fractions of goats fed with diets of different concentrate levels is shown in table  the16s rrna gene copy numbers of the novel rcc species in the rumen epithelium, the liquid and solid fraction ranged from to, to and to, respectively. the total archaea ranged from to, to and to, respectively. the abundance of the novel rcc species in the rumen of goats fed low concentrate diet was numerically higher than that of goats fed high concentrate diet. but, the abundance of the total archaea was not affected by the high concentrate feeding. the relative abundance of the novel rcc species within total archaea in the liquid fraction was numerically higher than in the other two fractions in each diet group. a, b, c, means with different letters in the same column are different p < ; n =  *, the ph value of rumen content,  ± ;  ± ;  ± . purification of the novel rcc species with anaerobic fungus one fungal culture containing the novel rcc species was obtained after purification with trimethylamine to support the novel rcc and with lumazine to inhibit the growth of methanobrevibacter sp. the anaerobic fungus was identified as belonging to piromyces sp. as revealed by morphological examination. the sequencing results showed only one 16s rrna gene sequence from the total dna extracted from the supernatant of the fungal culture, and this sequence was 100% identical to lgm-af and 99% to the clone from jinnan cattle rumen. further confirmation was also performed by sequencing the mcra gene coding the alpha subunit of the methyl-coenzyme m reductase that plays a crucial role in the methanogenesis, and the results showed that only one mcra gene sequence was present. these findings strongly supported the hypothesis that the novel rcc species in the co-culture with rumen anaerobic fungi is a methanogen. discussion in this study, a novel rcc species was found growing in the anaerobic fungal subcultures. many studies have shown that a large group of rcc inhabited the rumen of a variety of ruminant species on various diets. thus, the rcc species grown in the anaerobic fungal cultures in the present study just represented a small group of the total rcc. it has been proposed that the rcc in the rumen and its relatives in other environments could constitute the seventh order of the methanogens. methanogens within this new methanogenic order distantly related to the thermoplasmatales, have been shown to be present in various environments, including marine habitats, soil, and also the intestinal tracts of termites and mammals, suggesting their ubiquitous in various environments. the whole order was proposed to form three big clusters, ca. m. alvus cluster, m. luminyensis cluster and lake pavin cluster. the novel rcc species in the present study was grouped in the ca. m. alvus cluster. the present study reported the first account for rcc species grown in the fungal cultures from the goat rumen. nevertheless this single species may not represent the whole rcc community in the rumen. therefore, further research is needed to uncover this community and its features in the rumen. interestingly, this novel species could survive in the long-term transferred fungal subcultures. thus, there must be an underlying mechanism supporting the growth of this novel rcc species in the fungal subcultures. a similar phenomenon for protozoa was reported by irbis and ushida. when testing a single protozoa cell for the 16s rrna gene sequences of archaea, they found that the cultured rumen protozoa isotricha intestinalis and ophryoscolex purkynjei from goats carried thermoplasma sp. related sequences. recent studies showed that methanogens belonging to this group could strictly use hydrogen to reduce methanol and methylamines to methane. it is well known that both anaerobic fungi and protozoa could produce hydrogen, which is one of the substrates for methanogens. this may make it possible for anaerobic fungi to provide rcc species with hydrogen. methanol and methylamines could be derived from the microbial degradation of pectin, betaine, and choline from diets in the rumen. ametaj et al. demonstrated that there were methanol and methylamines in the rumen fluid of lactating dairy cows fed graded amounts of barley grain. in this study, the medium for the co-culture of anaerobic fungi and methanogens contained rice straw and clarified rumen fluid. anaerobic fungi could degrade the pectin of rice straw by pectinolytic enzymes, accompanying the release of methanol. thus, it is possible that the novel rcc species obtained a certain amount of tma and methanol from the rumen fluid and the degradation of rice straw. when methanol was used to enrich rcc in the fungal cultures, methanosphaera sp. was obtained instead of rcc species, which implied that methanosphaera sp. may compete for the same substrate with rcc. in addition to the competition for the available substrates, there might be other underlying mechanisms enabling the novel rcc species to survive in the in vitro and in vivo niches. apparently, further research is necessary to reveal the underlying mechanisms. the novel rcc exhibited apparent enrichment with less frequent transfer, with relatively higher proportion in 7 day transfer culture than in d or 5d transfer cultures. in our previous study, cheng et al. investigated the effects of transfer frequencies on the diversity of anaerobic fungi and methanogens in the enriched mixed cultures. they found that anaerobic fungal diversity was related to transfer frequencies and appeared to be simplified as transfer proceeded. in contrast, the methanogen population generally remained diverse, regardless of the transfer frequencies. thus, the survival and the shift of the abundance of the novel rcc species in fungal cultures might be related to the changes of the composition of the anaerobic fungal community. on the other hand, it seems that the rcc grew slowly in the in vitro culture, while the methanobrevibacter tended to grow more rapidly. thus longer incubation interval between transfers would allow the rcc populations to increase while the methanobrevibacter populations were declining. therefore, the approach using long incubation intervals would allow the enrichment of the novel rcc. however, how much the transfer frequency effect may be due to the specific co-culture with an anaerobic fungus remains an open question. the present study quantified the abundance of the novel rcc species and the total archaea in the rumen. it seems that the abundance of the novel rcc species was also affected by the diet composition, with the value in the rumen of goats fed low concentrate diet numerically higher than that of goats fed high concentrate diet. but the abundance of the total archaea seems not affected by the levels of concentrate in the diets. similarly, hook et al. reported that high-concentrate feeding did not affect the density of the total rumen methanogens, but they found that high-concentrate feeding mitigated the methane production and altered the methanogen diversity and community structure. they also suggested that ph sensitive methanogens might be lost when the rumen ph decreased. it was possible that the novel rcc species was sensitive to low ph caused by high-concentrate feeding. it is also possible that some unaffected methanogens occupied the vacated niche of the novel rcc species in the rumen of goats fed with high-concentrate diet. nevertheless, it is still unclear whether all the rcc populations are sensitive to the high-concentrate feeding and/or low ph. the present study provided the first estimation of this rcc species distribution in the rumen. the abundance of the novel rcc species was different in the rumen epithelium, rumen liquid and solid fractions. the relative abundance of the novel rcc species as indicated by its proportion within total archaea populations in their respective fraction was higher in liquid fraction as compared to epithelium and solid fraction. previous study suggested that it was difficult to detach all of the microbes associated with the solid fraction, thus the abundance of rcc and archaea in this fraction may be grossly underrepresented. our previous study showed that the composition of the methanogens were different in the rumen epithelium, solid and liquid fractions of jinnan cattle, especially for the unidentified archaea. we compared these unidentified archaeal sequences with rcc sequences in this study and found that % of the total clones in the liquid fraction was clustered within rcc clade, and % in the solid, % in the epithelium. the clones that were most similar to the novel rcc species were from the rumen epithelium fraction. moreover, gu et al. reported that % of the clones in the goat rumen fluid library belonged to the thermoplasmatales family, and % in the rumen solid library; however, no clones were > 95% similar to the novel rcc species. in this study, the relative density of the novel rcc species was numerically higher in the rumen liquid fraction than in the other two fractions, which might be due to the specific characteristics of the novel rcc species. in the rumen, liquid, solid and epithelium fractions have different turnover rates. janssen and kirs proposed that the methanogens associated with different rumen fractions could be expected to have different growth rates since they would be removed from the rumen at different rates. thus, the novel rcc species might have a relatively higher growth rate than other rccs in the rumen liquid fraction. in the present study, the novel rcc species was co-isolated with anaerobic fungus. most recently, a tri-culture with a rcc member, a clostridium sp. and a bacteroides sp. was enriched from bovine rumen. further attempts to obtain pure rcc species were made but unsuccessful. it seems that there is a close relationship between the novel rcc species and anaerobic fungus. two isolates related to rcc had been obtained from human feces. most recently, another rcc related isolate m. gallocaecorum strain dok- from chicken gut was reported. the three isolates exhibited similar nutritional requirements for methyl-group chemicals. in the current study, the phylogenetic analysis showed that the novel rcc species were clustered into the same clade with ca. m. alvus mx. however, the 16s rrna gene sequence of the novel rcc species showed 93% similarity to ca. m.alvusmx, and 87% to m. luminyensis. the mcra gene sequences of the novel rcc species showed 84% similarity to ca. m. alvus mx, and 78% to m. luminyensis. thereby, though clustered into the rcc clade, the novel rcc species in this study were phylogenetically distant with the two human isolates, the recently reported rcc isolates, suggesting that the new order for rcc and its relatives may be highly diverse.  a novel rcc species was found surviving in the long-term transferred anaerobic fungal subcultures and closely associated with anaerobic fungi. the results verified that the quantification of the novel rcc species in vivo and in vitro is possible by real-time pcr using its specific primers. the relative abundance of the novel rcc species in the anaerobic fungal subcultures was affected by the transfer frequencies, with the seven day transfer frequency suitable for its enrichment. the high concentrate feeding did not affect the abundance of the total archaea population, but numerically reduced the abundance of the novel rcc species in the goat rumen. the relative abundance of the novel rcc species was numerically higher in the rumen liquid fraction than in the epithelium and solid fractions. a novel rcc species was co-isolated with an anaerobic fungus, and was identified as being a methanogen. the finding in the present study may help to culture and investigate the unknown methanogens in the rumen. 
the novel archaea belonging to rumen cluster c, which may play an important role in methane production in the rumen have received increased attention. in addition, the relative abundance of the rcc species was numerically higher in the rumen liquid fraction than in the rumen epithelium and solid fractions. a novel rcc species existing in the fungal subcultures was identified and demonstrated by the 16s rrna gene clone library. interestingly, the novel rcc species survived in the fungal cultures over all the subculture transferring, even in the 62nd subculture, in contrast to the other methanogens, which disappeared during subcultures. further work showed that subculture transfer frequency significantly affected the relative abundance of the novel rcc species in the fungal subcultures. in addition, quantitative real-time pcr revealed that high concentrate diets did not affect the abundance of archaea, but numerically reduced the abundance of the novel rcc species in the rumen. pcr and sequencing analysis showed that the novel rcc species contained a mcra gene, which is known to play a crucial role in methanogenesis, and thus could be identified as a methanogen. in this study, a novel rcc species was identified as a methanogen and closely associated with anaerobic fungi. in the present study, rcc grown in anaerobic fungal subcultures was identified by the molecular and culture methods. the five-day and seven-day transfer frequencies increased the relative abundance of the rcc species. finally, a purified fungal culture containing the rcc species was successfully obtained. however, the present information on rcc in the rumen is limited by the unsuccessful isolation of axenic pure rcc from the rumen. this novel approach by using co-culture with anaerobic fungi may provide a feasible way to culture and investigate not yet identified methanogens.
 the novel archaea belonging to rumen cluster c, which may play an important role in methane production in the rumen have received increased attention. however, the present information on rcc in the rumen is limited by the unsuccessful isolation of axenic pure rcc from the rumen. in the present study, rcc grown in anaerobic fungal subcultures was identified by the molecular and culture methods.  a novel rcc species existing in the fungal subcultures was identified and demonstrated by the 16s rrna gene clone library. interestingly, the novel rcc species survived in the fungal cultures over all the subculture transferring, even in the 62nd subculture, in contrast to the other methanogens, which disappeared during subcultures. further work showed that subculture transfer frequency significantly affected the relative abundance of the novel rcc species in the fungal subcultures. the five-day and seven-day transfer frequencies increased the relative abundance of the rcc species. in addition, quantitative real-time pcr revealed that high concentrate diets did not affect the abundance of archaea, but numerically reduced the abundance of the novel rcc species in the rumen. in addition, the relative abundance of the rcc species was numerically higher in the rumen liquid fraction than in the rumen epithelium and solid fractions. finally, a purified fungal culture containing the rcc species was successfully obtained. pcr and sequencing analysis showed that the novel rcc species contained a mcra gene, which is known to play a crucial role in methanogenesis, and thus could be identified as a methanogen.  in this study, a novel rcc species was identified as a methanogen and closely associated with anaerobic fungi. this novel approach by using co-culture with anaerobic fungi may provide a feasible way to culture and investigate not yet identified methanogens. 