Community Structure Analysis of Methanogens Associated with Rumen Protozoa Reveals Bias in Universal Archaeal Primers

Abstract

The diversity of protozoan-associated methanogens in cattle was investigated using five universal archaeal small-subunit (SSU) rRNA gene primer sets. Methanobrevibacter spp. and rumen cluster C (distantly related to Thermoplasma spp.) were predominant. Significant differences in species composition among libraries indicate that some primers used previously to characterize rumen methanogens exhibit biased amplification. Within the rumen, methanogens can exist in a free-living form or in association with protozoa through extracellular attachment (ectosymbionts) or intracellular colonization (endosymbionts) (5). Protozoan-associated methanogens (PAMs) account for up to 37% of methane emitted from ruminants (4), and defaunation generally reduces ruminant methane emissions (7). Despite the fact that a thorough understanding of PAMs is essential for developing strategies to mitigate methane emissions from ruminants, species composition of this community has not been well characterized, particularly in cattle (10). According to a recent review, only four studies have characterized methanogens from isolated protozoa of ruminants, including sheep (three studies), goats (one study), and cattle (one study) (2,9,12,21).Given the inherent difficulty of culturing methanogens, most studies rely on the use of small-subunit (SSU) rRNA gene sequences for elucidating community structures. Numerous universal archaeal primer sets have been used by different groups to characterize ruminant methanogen communities (3,9,16,22,24,25). Sequence libraries created using these primers are subject to bias in which certain taxa are preferentially amplified due to factors such as the number and location of nucleotide mismatches between the primer and the target sequence. Furthermore, many of these primers were derived from outdated sequence databases and are therefore not comprehensive in their coverage. While these potential issues have been acknowledged (1,10,16,17), the impact of primer selection on the diversity of rumen methanogens has yet to be empirically assessed. In this study, we examined the effect of primer bias on the PAM community of cattle by using several primer sets that have been previously used to characterize rumen methanogens. Primers that amplified the same region of the SSU rRNA gene (i.e., V2 to V5) were selected to allow for direct phylogenetic comparison of sequences.Protozoan sampling and DNA extraction. Rumen fluid was obtained from four 10-month-old rumen-cannulated Black Angus heifers that were fed grass hay. Samples (250 ml from each of four sites, including the reticulum, ventral and caudal sacs, and the dorsoventral midline) were pooled and strained twice through two layers of PTEX mesh (355-m pore size; Sefar Inc., Kansas City, MO). To allow for separation, samples were incubated at 39°C for 30 min in a sealed container that was occasionally allowed to vent positive pressure. The top plant debris layer was discarded, and protozoa were separated by filtration through NITEX mesh (11-m pore size; Sefar Inc.). Prot...

Methylotrophic methanogenic Thermoplasmata implicated in reduced methane emissions from bovine rumen

“…The air flow through the chambers was measured with a mass flow meter (Teledyne Hastings Instruments, Hampton, VA, USA), and CO 2 and CH 4 was quantified using infrared sensors (Columbus Instruments, Columbus, OH, USA) exactly as described in Hellwing et al 25 Nucleic acid extraction and double-stranded (ds) cDNA synthesis. Rumen fluid-sampling strategy was developed from previous publications [26][27][28] , taking into account that a significant fraction of the rumen microbiota is adhered to fibers. Hence, representative samples of rumen fluid were collected at the end of each feeding period from the top and bottom part of the frontal, mid and distal section of the rumen (25 ml from each area) through a rumen fistula.…”

“…Primers 958 F and 1100AR were used to quantify 16S rRNA of non-RCC methanogens (Methanobrevibacter, Methanomicrobium, Methanobacterium, Methanomicrococcus and Methanosphera). These primers do not amplify RCC because of 1 and 3 bp mismatches 28 . qPCR was performed using a Mastercycler ep realplex (Eppendorf, Wien, Austria).…”

Few Highly Abundant Operational Taxonomic Units Dominate within Rumen Methanogenic Archaeal Species in New Zealand Sheep and Cattle

“…Whether these different observations are due to differences in community structure or the consequence of technical artifacts remains to be shown, but it is difficult to compare studies that have used different methodologies for data generation. For example, the choice of specific amplification primers may introduce a bias toward some organism groups and may discriminate against others (both by primer sequence and by target region of the 16S rRNA gene for amplification) (34,35). Thus far, studies investigating primer bias have not been performed on rumen methanogen mock communities of known composition.…”

Chemical markers for rumen methanogens and methanogenesis

“…These problems include differences in the rrs gene copy number within a genome (Hook et al, 2009) and differences in genome copy number within a cell (Hildenbrand et al, 2011). There is increasing concern about bias in the PCR reaction (Tymensen and McAllister, 2012), and the small number of sequences related to known methanogens (Firkins and Yu, 2006) may also cause problems. Dehority and Tirabasso (1998) found no relationship between the concentration of celluloytic bacteria and digestion of cellulose in the rumen.…”