Diverse Hydrogen Production and Consumption Pathways Influence
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The ISME Journal (2019) 13:2617–2632 https://doi.org/10.1038/s41396-019-0464-2 ARTICLE Diverse hydrogen production and consumption pathways influence methane production in ruminants 1 2 3 1 3 1 Chris Greening ● Renae Geier ● Cecilia Wang ● Laura C. Woods ● Sergio E. Morales ● Michael J. McDonald ● 3 3 4 5 6 2 Rowena Rushton-Green ● Xochitl C. Morgan ● Satoshi Koike ● Sinead C. Leahy ● William J. Kelly ● Isaac Cann ● 5 3 2 Graeme T. Attwood ● Gregory M. Cook ● Roderick I. Mackie Received: 7 December 2018 / Revised: 3 June 2019 / Accepted: 7 June 2019 / Published online: 26 June 2019 © The Author(s) 2019. This article is published with open access Abstract Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H2 metabolism. Two-thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyse H2 production or fi 1234567890();,: 1234567890();,: consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis con rmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g., Ruminococcus) accounted for half of all hydrogenase transcripts. Various H2 uptake pathways were also expressed, including methanogenesis (Methanobrevibacter), fumarate and nitrite reduction (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis-related transcripts predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic fumarate reducer Wolinella succinogenes in co-culture compared with pure culture. We conclude that H2 metabolism is a more complex and widespread trait among rumen microorganisms than previously recognised. There is evidence that alternative hydrogenotrophs, including acetogenic and respiratory bacteria, can prosper in the rumen and effectively compete with methanogens for H2. These findings may help to inform ongoing strategies to mitigate methane emissions by increasing flux through alternative H2 uptake pathways, including through animal selection, dietary supplementation and methanogenesis inhibitors. Introduction Methane production by livestock accounts for over 5% of These authors contributed equally: Chris Greening and Renae Geier global greenhouse gas emissions annually [1]. These emis- Supplementary information The online version of this article (https:// sions mostly originate from the activity of methanogens doi.org/10.1038/s41396-019-0464-2) contains supplementary within ruminants, which generate methane as an obligate material, which is available to authorized users. * Chris Greening 61801, USA [email protected] 3 Department of Microbiology and Immunology, University of * Roderick I. Mackie Otago, Dunedin 9016, New Zealand [email protected] 4 Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan 1 School of Biological Sciences, Monash University, Clayton, VIC 5 3800, Australia Grasslands Research Centre, AgResearch Ltd., Palmerston North 4410, New Zealand 2 Department of Animal Sciences and Institute for Genomic 6 Biology, University of Illinois at Urbana-Champaign, Urbana, IL Donvis Ltd., Palmerston North 4410, New Zealand 2618 C. Greening et al. end-product of their energy metabolism [2]. Several lineages sufficiently low concentrations for fermentation to remain of methanogenic archaea are core members of the micro- thermodynamically favourable [38]. biome of the ruminant foregut [3–5]. Of these, hydro- Various hydrogenotrophic bacteria are thought to compete genotrophic methanogens are dominant in terms of both with methanogens for the rumen H2 supply. Most attention methane emissions and community composition [6, 7], with has focused on acetogens, which mediate conversion of global surveys indicating that Methanobrevibacter gott- H2/CO2 to acetate using [FeFe]-hydrogenases [39]. Several schalkii and Methanobrevibacter ruminantium comprise genera of acetogens have been isolated from the rumen, 74% of the rumen methanogen community [5]. These including Eubacterium [40], Blautia [41]andAcetitomaculum organisms use molecular hydrogen (H2) to reduce carbon [42]. However, molecular surveys indicate their abundance is dioxide (CO2) to methane through the Wolfe cycle of generally lower than hydrogenotrophic methanogens [43–45]. methanogenesis [8, 9]. Rumen methanogens have also been This is thought to reflect that methanogens outcompete identified that use formate, acetate, methyl compounds and acetogens owing to the higher free energy yield of their ethanol as substrates, but usually do so in conjunction with metabolic processes, as well as their higher affinity for H2. H2 [5, 10–12]. Given their major contribution to greenhouse The dissolved H2 concentration fluctuates in the rumen gas emissions, multiple programs are underway to mitigate depending on diet, time of feeding and rumen turnover rates, ruminant methane production [13, 14]. To date, most stra- but is generally at concentrations between 400 and 3400 nM tegies have focused on direct inhibition of methanogens [46]; these concentrations are typically always above the using chemical compounds or vaccines [15–18]. A promis- threshold concentrations required for methanogens (< 75 nM) ing alternative strategy is to modulate the supply of sub- but often below those of acetogens (< 700 nM)[47]. Despite strates to methanogens, such as H2, for example, through this, it has been proposed that stimulation of acetogens may dietary or probiotic interventions [14, 19, 20]. To achieve be an effective strategy for methane mitigation in this, while maintaining health and productivity of the host methanogen-inhibited scenarios [14, 20, 48, 49]. Various animal, requires an understanding of the processes that microorganisms have also been isolated from cows and sheep mediate substrate supply to methanogens within the rumen. that support anaerobic hydrogenotrophic respiration, includ- H2, the main substrate supporting ruminal methano- ing dissimilatory sulfate reduction (e.g., Desulfovibrio genesis, is primarily produced through fermentation pro- desulfuricans)[50, 51], fumarate and nitrate reduction (e.g., cesses [6]. Various carbohydrate fermentation pathways Selenomonas ruminantium, Wolinella succinogenes)[52– 59] lead to the production of H2 as an end-product, together and trimethylamine N-oxide reduction (e.g., Denitrobacter- with volatile fatty acids and CO2 [21–23]. This process is ium detoxificans)[60]. The first described and most com- supported by hydrogenases, which reoxidise cofactors prehensively studied of these hydrogen oxidisers is reduced during carbohydrate fermentation and dispose of W. succinogenes, which mediates interspecies hydrogen the derived electrons by producing H2. Although it is transfer with R. albus [25]. In all cases, respiratory electron unclear which rumen microorganisms mediate H2 produc- transfer via membrane-bound [NiFe]-hydrogenases and tion in situ, a range of isolates have been shown to produce terminal reductases generates a proton-motive force that H2 in vitro [24–28]. For example, the model rumen bac- supports oxidative phosphorylation [61]. It is generally terium R. albus 7 reoxidises the reduced ferredoxin and assumed that these pathways are minor ones and are limited NADH formed during glucose fermentation by using two by the availability of oxidants. Promisingly, it has been different [FeFe]-hydrogenases depending on environmental observed that dietary supplementation with fumarate, sulfate, conditions [29]. In addition, it is well-established that some or nitrate can significantly reduce methane production rumen fungi and ciliates produce H2 via hydrogenosomes in cattle, likely by stimulating alternative pathways of H2 [30, 31]. A debated source is the nitrogenase reaction, consumption [62, 63]. which produces H2 while fixing N2; whereas numerous We postulate that mitigating methane emissions, while rumen microorganisms encode putative nitrogenases [21], maintaining animal productivity, depends on understanding in situ data indicate that N2 fixation occurs at negligible and controlling H2 utilisation by methanogens. This rates in the rumen [32, 33]. A large proportion of the H2 requires a system-wide perspective of the schemes for produced by hydrogenogenic fermenters is directly production and concomitant utilisation of H2 in the rumen. transferred to hydrogenotrophic methanogens, in an eco- To facilitate this, we determined which organisms and logical process known as interspecies hydrogen transfer enzymes are primarily responsible for H2 production and [25, 34]. Particularly remarkable are the endosymbiotic and consumption in rumen. First, we screened genome, meta- ectosymbiotic associations of methanogens, such as genome and metatranscriptome data sets [21, 64, 65]to M. ruminantium, with rumen ciliates [35–37]. In addition identify microbial genera, metabolic pathways and hydro- to providing a continual substrate supply