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Figure 2 Relative abundance of archaeal OTUs defined using the 16S rRNA gene hyper-variable region V3V4. The bar chart shows the diversity of Archaea at the lowest reliable taxonomic level (where possible the default family is denoted in the key). AD, agricultural ... Abundant non-methanogenic Archaea such as Miscellaneous Crenarchaeotic Group (MCG) (11%) and Halobacteria (7%) represented by Deep Sea Euryarchaeotic Group (DSEG) and Deep Sea Hydrothermal Vent Gp 6 (DHVEG-6) were also detected in the AD sample (Figure  ?(Figure2).2). These groups are phylogenetically diverse and there is a little knowledge of their ecology and metabolism, however it seems that MCG archaeons are able to ferment wide variety of recalcitrant substrates (Kubo et al., 2012) and DSEG are [http://www.selleckchem.com/products/Vorinostat-saha.html Vorinostat] positively correlated with putative ammonia-oxidizing  Thaumarchaeota (Restrepo-Ortiz and Casamayor, 2013). In addition to the 16S rRNA marker, the mcrA gene was used for taxonomic profiling of methanogenic communities in both digesters. The mcrA gene fragments amplified using primers MLf/MLr (Luton et al., 2002) were sequenced and analyzed. More than half of the sequences (57%) amplified from the AD sample were assigned to uncultured Archaea, belonging to the Methanomassiliicoccales (23%), Methanomicrobiales (13%), Methanobacteriales (11%) and Methanosarcinales (10%) orders (Figure ?(Figure3),3), suggesting dominance of hydrogenotrophic methanogens over acetoclastic Archaea. The most abundant genera in AD were Methanobacterium sp. Maddingley MBC34 (11%) followed by Methanosaeta concilli (9%) and Methanoculleus spp. (4%) (Figure ?(Figure3).3). Similarly in WD, the majority of the mcrA amplicons were classified as uncultured Archaea belonging to orders Methanomicrobiales (27%) and Methanomassiliicoccales (7%) (Figure ?(Figure3),3), while at the genus level most of the methanogens were identified as Methanometylovorans hollandica (21%), Methanosaeta concilli (16%), Methonoculleus spp. (12%), or Methanoplanus petrolearius (3%) (Figure ?(Figure33). Figure 3 Phylogenetic placement  of mcrA amplicons from AD (A) and WD (B) samples. The width of the red branches corresponds to the number of unique mcrA amplicon sequence reads in that particular branch (this can be either a leaf or node). The collapse of some ... The results obtained for both marker genes (16S rRNA and mcrA) only partially overlapped, probably due to differences in primer affinities and variation in the gene copy numbers. This observation is in agreement with a previous report showing that these two marker genes generate different taxonomic profiles (Wilkins et al., 2015). Therefore, for a greater insight into the structure of methanogenic communities and to verify the obtained results, novel molecular markers specific for other methanogenesis-linked genes were developed.