Skip to main content

Back to the Source: Molecular Identification of Methanogenic Archaea as Markers of Colonic Methane Production

Methanogenic archaea (methanogens) are anaerobic microbes that derive their energy from metabolizing simple substrates such as the gaseous H2 and CO2 or the volatile methanol into methane, a unique means of survival absent in bacteria and in eukaryotes [1]. Based on their substrate usage, methanogens are further classified into three primary metabolic groups, hydrogenotrophic using H2 and CO2 or formate, aceticlastic feeding on acetate, and methylotrophic utilizing methanol or other methylated compounds [2]. Regardless of their specific metabolism, all methanogens share the mcrA gene encoding the methyl coenzyme M (CH3–CoM) reductase that converts CH3–CoM to methane. Although methanogens are frequently associated with eukaryotic hosts such as protists, plants, and animals including humans [3], their host-associated biology has scarcely been characterized. Likewise, the impact of methanogens on human health and diseases remains unknown. Nevertheless, growing evidence has suggested that intestinal overgrowth of methanogens is harmful and is associated with several diseases [4]. Prominent among them is GI dysmotility, since luminal methane slows intestinal transit in animal models [5]. Presumably, overgrowth of methanogens overproduces methane, thus contributing to adverse GI-relegated symptoms such as bloating and constipation in humans, an hypothesis supported by the observation that breath methane excretion is elevated in constipation-predominant irritable bowel syndrome (IBS-C) patients, who also have lower postprandial serotonin levels than non-methane excreting IBS patients who excrete elevated H2 [6]. Nonetheless, diagnostic standards for overgrowth of methanogens are ambiguous, as the understanding of this phenomenon is still in its infancy.

Overgrowth of methanogens used to be classified under the umbrella term SIBO (small intestinal bacterial overgrowth). In the latest American College of Gastroenterology (ACG) Guideline for SIBO, it is now defined separately as IMO (intestinal methanogen overgrowth) since methanogens are archaea, not bacteria, and methane (and perhaps methanogens too) exhibits unique clinical relevance [7]. Owing to the relative ease in bacterial cultivation, SIBO can be quantitatively defined as a small intestinal bacterial load ≥ 103 CFUs (colony-forming units) ·mL−1. This CFU diagnostic threshold is highly correlated with an increase of H2 ≥ 20 ppm from baseline within 90 min during a hydrogen breath test, due to rapid post-fasting metabolism of ingested carbohydrates such as glucose or lactulose by the high load of small intestinal bacteria.

Based on these observations, the semi-quantitative hydrogen breath test has been proposed as an alternative to intestinal bacterial culture for SIBO diagnosis. Although its ability to accurately diagnose SIBO in a population with nonspecific GI symptoms has been questioned, the safe and noninvasive breath test is a popular and effective tool in the clinics when used and interpreted appropriately [8]. In contrast, the criteria defining IMO are far from clear. Since methanogens are fastidious, they are not routinely cultivated in clinical laboratories and have no established CFU. Instead, empirical diagnostic recommendations using the methane breath test have been proposed. The ACG recommended the presence of breath methane ≥ 10 ppm at any point during testing after substrate ingestion for a positive IMO diagnosis, although other studies have adopted thresholds between 3 and 10 ppm. Regardless of the threshold, the clinical relevance of a positive methane breath test is not high, since breath methane excretion in health appears highly variable, ranging from below detection (< 2 ppm)–82 ppm [9]. Although the sources of this variation are not well understood, in theory methane emission from any of the ‘hot spots’ including the nasal, oral, and small and large intestinal mucosae, could occur. However, methanogenic activities are not well characterized within these sites (Fig. 1).

Fig. 1

Overview of the hot spots for methanogens and methane production in the human body (upper panel) and the four assays devised by Drs. Teigen, Mathai, and associates for colonic methanogens [10] (bottom panel). Methanogens are often detected in nasal, oral, and in the small and large intestinal sites, all of which in theory contribute to breath methane through the systemic circulation and respiration. Therefore, elevated breath methane could be due to overgrowth of methanogens at one or multiple of the four spots. Currently, quantitative contribution to breath methane from each of the four spots remains elusive. Although elevated breath methane has been linked to several diseases, its quantity varies substantially among healthy adults, compromising the utility of breath methane test for disease diagnosis. Teigen et al. developed molecular assays (16S relative abundance and targeted and untargeted mcrA qPCRs) for methanogens that were sensitive (33% to 100% positive rate) enough to discriminate between positive (30%) and negative methane productions measured by an in vitro fecal methane producing assay

In order to improve the clinical diagnosis of IMO, additional tests will be needed. In this issue of Digestive Diseases and Sciences, Drs. Levi Teigen, Prince Mathai, and associates investigated whether assays of molecular markers for methanogens from stool could serve as surrogates for IMO [10]. They collected stool samples from healthy adults (n = 33) and devised the following four tests in order to infer their methane-producing status (Fig. 1).

  1. 1.

    A 2-h in vitro batch incubation of the samples in gas-tight septum jars aimed at measuring methane production, showing 30% of the samples were methane positive when using the ≥ 10 ppm threshold corrected to per gram of dry stool. The weight correction, not implemented in the breath test, is a practice that should facilitate future test standardization. Yet, this correction renders a direct comparison with breath methane data difficult, since their units are now different (ppm · g−1 dry stool vs. ppm). Nevertheless, the scale of variation (unitless) between the two assays should still be comparable, which is 17-fold (83–1434 ppm · g−1) for the in vitro assay, and 8 to 27-fold (3–82 ppm) depending on the positive cutoff (3–10 ppm) for the breath assay from healthy adults (n = 492) [9]. Therefore, both assays have a comparable but large variation. In order to reliably separate disease samples from healthy ones, this variation must be controlled for in future in vitro assays. Due to the ease of in vitro manipulation, at least two improvements could be reasonably implemented. First, fecal samples may be incubated for extended time and then treated with methanogenic substrates—mimicking a post-fasting measurement of methane production. This would minimize the variation caused by undigested substrates in fecal samples. Second, the incubation may be done with a continuous flow—mimicking either normal or delayed intestinal transit that would better account for the variation in transit that fluctuates considerably for the same healthy individual and is associated with methane production, an advantage over batch incubation.

  2. 2.

    16S rRNA gene amplicon sequencing of the fecal community in order to estimate the relative abundance of methanogens, indicating 66% of the samples were methanogen positive (0.002–9.23% of the total community). The most prevalent and abundant methanogens belong to the hydrogenotrophic Methanobrevibacter, whereas occasional signatures for the methylotrophic Methanosphaera were also significant. At ≥ 0.097%, the relative abundance of 16S rRNA for Methanobrevibacter separated the methane-positive samples from the negative ones with perfect discrimination. Since the β (a measure of variation in microbiome composition) but not the α diversity (a measure of microbiome richness and diversity) was different between the methane-positive and methane-negative samples, a distinct but equally diverse bacterial community was also associated with the methanogens. Indeed, an enrichment of specific bacterial taxa Muribaculaceae, Ruminococcaceae, Christensenellaceae, and Catenibacterium was observed in the methane-positive samples, as opposed to an enrichment of Bacteroides, Blautia, Bacilli, and Lactobacillales in the methane-negative samples. Since the bacteria that co-occur with methanogens may affect gut physiology in their own ways, future studies investigating the effects of these bacteria would increase understanding of the mechanism of methanogen-associated diseases.

  3. 3.

    An untargeted mcrA gene qPCR assay to quantify the absolute abundance of methanogens, delivering an impressive (and surprising) 100% methanogen positive rate (5.3 × 103–7.7 × 107 copies · g−1 wet stool). This confirms mcrA is a superior molecular marker over 16S rRNA for methanogens, which is well documented in environmental studies. Nevertheless, very few studies have used mcrA to survey methanogens in human samples, suggesting an undercount of methanogens in most studies. It is therefore strongly recommended that mcrA assay should be considered for future studies in addition to the standard 16S assay. Sequencing all the mcrA genes is also recommended to exclude any false positives and better stratify the methanogen population. Like the 16S assay, the mcrA assay also effectively separated the methane-positive vs. -negative samples very well. The threshold for positive methane was ≥ 5.2 × 105 copies · g−1 with near perfect discrimination.

  4. 4.

    A targeted mcrA qPCR assay to quantify four known gut methanogen species with five novel primer sets, presenting a 33% methanogen positive rate. The positives only included Methanobrevibacter smithii and Methanosphaera stadtmanae, both already detectable by 16S. This low positive rate suggests most of the diversity revealed by the untargeted mcrA assay was not captured, reinforcing the recommendation to sequence all mcrA genes when using the untargeted assay. The reason for the extra set of primers was that two sets of primers must be developed in order to target all known M. smithii strains. This suggests the current classification of M. smithii may reflect a complex of more than one species, which has not previously been captured by the 16S phylogeny. Indeed, a very recent genome survey proposed a split of M. smithii into two separate species which differed considerably in their mcrA sequences and other genomic features [11]. If the proposal stands, the group closely related to the type strain PS (targeted by the primer set 1A in this study) will continue to represent M. smithii, whereas a second group represented by strain WWM1085 (targeted by this study’s primer set 1B) will have a new candidate species termed Candidatus ‘Methanobrevibacter intestini’. Since data linking M. smithii with diseases are often contradictory according to previous studies, it will be exciting to see if the new classification would solve some of these contradictions which will be facilitated by the targeted qPCR assays developed here.

Collectively, by focusing on the source of methane production—namely the methanogens, this study marks an important methodological advancement toward refining the diagnosis of IMO and its associated diseases. Further advancements in these methods and testing both healthy and diseased samples with a large sample size shall bring promising diagnostic solutions.


  1. 1.

    Lyu Z, Shao N, Akinyemi T, Whitman WB. Methanogenesis. Curr Biol. 2018;28:R727–R732.

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Lyu Z, Whitman WB. Transplanting the pathway engineering toolbox to methanogens. Curr Opin Biotechnol. 2019;59:46–54.

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Borrel G, Brugère J-F, Gribaldo S, Schmitz RA, Moissl-Eichinger C. The host-associated archaeome. Nat Rev Microbiol. 2020.

    Article  PubMed  Google Scholar 

  4. 4.

    Roccarina D, Lauritano EC, Gabrielli M, Franceschi F, Ojetti V, Gasbarrini A. The role of methane in intestinal diseases. Am J Gastroenterol. 2010;105:1250–1256.

    Article  PubMed  Google Scholar 

  5. 5.

    Pimentel M, Lin HC, Enayati P, Van Den Burg B, Lee HR, Chen JH et al. Methane, a gas produced by enteric bacteria, slows intestinal transit and augments small intestinal contractile activity. Am J Physiol Gastrointest Liver Physiol. 2006;290:1089–1095.

    CAS  Article  Google Scholar 

  6. 6.

    Pimentel M, Kong Y, Park S. IBS subjects with methane on lactulose breath test have lower postprandial serotonin levels than subjects with hydrogen. Dig Dis Sci. 2004;49:84–87.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Pimentel M, Saad RJ, Long MD, Rao SSC. ACG clinical guideline: Small intestinal bacterial overgrowth. Am J Gastroenterol. 2020;115:165–178.

    Article  PubMed  Google Scholar 

  8. 8.

    Massey BT, Wald A. Small intestinal bacterial overgrowth syndrome: a guide for the appropriate use of breath testing. Dig Dis Sci. 2020.

    Article  PubMed  Google Scholar 

  9. 9.

    Levitt MD, Furne JK, Kuskowski M, Ruddy J. Stability of human methanogenic flora over 35 years. Clin Gastroenterol Hepatol. 2006;4:123–129

    CAS  Article  Google Scholar 

  10. 10.

    Teigen L, Mathai PP, Matson M, Lopez S, Kozysa D, Kabage AJ, et al. Methanogen abundance thresholds capable of differentiating in vitro methane production in human stool samples. Dig Dis Sci. (Epub ahead of print).

  11. 11.

    Chibani CM, Mahnert A, Borrel G, Almeida A, Gribaldo S, Ruth A. A comprehensive analysis of the global human gut archaeome from a thousand genome catalogue. BioRxiv. 2020:1–53.

Download references


I appreciate the assistance of Robert Britton for his critical comments and suggestions. The image was created with


Zhe Lyu is supported by seed funds provided by the Baylor College of Medicine. Zhe Lyu also received travel support from NSF DEB 1928924 through Brian Hedlund and Co-PIs to participate in the International Conference on Geo-Omics of Archaea and develop research ideas on human-associated archaea.

Author information



Corresponding author

Correspondence to Zhe Lyu.

Ethics declarations

Conflict of interest

The author has no relevant conflicts of interest to disclose.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lyu, Z. Back to the Source: Molecular Identification of Methanogenic Archaea as Markers of Colonic Methane Production. Dig Dis Sci 66, 3661–3664 (2021).

Download citation