Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

Abstract

In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products.

Comparative analysis of deep sequenced methanogenic communities: identification of microorganisms responsible for methane production

“…S3H). This is in agreement with other studies [50, 51] and suggests the importance of acetoclastic and methylotrophic methanogenesis in industrial wastewater plants.…”

Microbiota of anaerobic digesters in a full-scale wastewater treatment plant

“…Methanoculleus, Methanobrevibacter and Methanobacterium were the predominant microorganisms in anaerobic digesters operated at high loadings and under thermophilic conditions, during co-digestion of manure and waste whey at an organic loading rate of 60.4 g COD/(m 3 ·day) Methanoculleus abundance reached over 40% , Lv et al 2013). Methanosarcina sp., which are very versatile microorganisms that often occur in anaerobic digesters (Wu et al 2013, Cydzik-Kwiatkowska et al 2012, were not identifi ed in the investigated samples. Methanosarcina sp.…”

Community and Proteomic Analysis of Anaerobic Consortia Converting Tetramethylammonium to Methane

“…The Methanomethylovorans populations are methylotrophic and frequently thrive in terrestrial (freshwater) ecosystems over a broad temperature range [ 30 – 35 ]. The proteomics data of this study suggested the methylotrophic activities of M. hollandica associated with QMA degradation ( Figure 5 ).…”

“…However, the concentration effects cannot account for the constantly low abundance of Methanomethylovorans and Methanolobus in the UASB reactor or the dominance of Methanosarcina in the zone near the outlet (height, 400 cm). In a previous study, quantitative analysis of samples taken from a UASB reactor on different dates yielded a similar distribution of methylotrophic methanogens but with higher abundance of Methanomethylovorans than Methanosarcina [ 35 ]. This suggested dynamic competition between Methanomethylovorans and Methanosarcina .…”

“…also dominated the UASB reactor, particularly in the center space (100–200 cm) (approximately 76.5% of total archaeal 16S rRNA sequence), suggesting that in addition to methylotrophic methanogenesis by Methanosarcina , a major fraction of methane in that zone could be formed through a hydrogenotrophic methanogenesis pathway. Interestingly, the percentage was extraordinarily higher than that of the hydrogenotrophic methanogens detected in the CMSS reactor (2.0%) and in the bottom and top of UASB reactor, as well as other anaerobic reactors degrading industrial wastewaters and sludge biomass (<45.6%) [ 35 , 44 ]. This unusual was also related to the contrast of archaeal to bacterial cell ratios analyzed by using Q-PCR.…”