Microbial communities in methane dynamics

Methanogenic archaea

Methanogenic archaea are strictly anaerobic producers of CH4 that belong to the phylum Euryarchaeota (Woese et al. 1990). In anaerobic conditions with complex organic compounds, where light and electron acceptors are available in limited amounts, methanogens interact with other chemoheterotrophic bacteria in degrading organic substrates (Garcia 1990). The distribution of methanogens is regulated by the available substrates such as H2, CO2 and acetate (Serrano-Silva et al. 2014). Methanogens can use these substrates directly produced by fermentative bacteria, but they can also use the substrates in syntrophy with hydrogen forming acetogenic bacteria. The latter interaction is referred as interspecies hydrogen transfer (Chaudhary et al. 2013).

Methanogens can be roughly classified by the used substrate (Borrel et al. 2011; Borrel et al. 2013;

Garcia 1990):

1) species using H2/CO2 and formate (e.g. Methanobacterium, Methanobrevibacter, Methanogenium, Methanoregula)

2) species using acetate (Methanosaeta)

3) species using acetate, H2/CO2 and methyl compounds (Methanosarcina) 4) species using methyl compounds (Methanolobus, Methanococcoides)

5) species using H2 and methyl compounds (Methanomassiliicoccus luminyensis, Candidatus, Methanomethylophilus alvus)

The distribution and diversity of methanogens are also affected by the depth, temperature variations, pH and salinity conditions (Chaudhary et al. 2013). Most of the methanogenic species grow within pH range 6.0-8.0 (Serrano-Silva et al. 2014). The optimum temperature for growth of methanogens is between 4-45 C° (Zeikus and Winfrey 1976). Most methanogens are mesophilic (able to function at 20-40 C°), but there are also thermophilic species that require higher temperatures to function (Serrano-Silva et al. 2014). According to Earl et al. (2005), seasonal temperature variation has an influence on the diversity of methanogens, and they have discovered that the diversity of the methanogens in lakes seems to be highest in the autumn.

Studies have shown that Methanomicrobiales is the dominant order of methanogenic organisms and represents about 43 % of the cultured methanogenic population in marine and freshwater sediments.

The second most dominant order is Methanosarcinales, which constitutes about 39 % of the total methanogenic population in sediments (Chaudhary et al. 2013).

Aerobic methanotrophic bacteria

CH4 produced by methanogenesis can be further oxidized by aerobic methane-oxidizing bacteria, methanotrophs. Methanotrophs are a group of aerobic bacteria that can use one-carbon compounds as their source of carbon and energy and assimilate formaldehyde as a major source of cellular carbon (Hanson and Hanson 1996).

Methanotrophs are often grouped as either Type I (ribulose monophosphate, RuMP pathway) or Type II (ribulose-1,5-bisphosphate carboxylase, Serine pathway), based on the pathways for the oxidation of CH4 and assimilation of formaldehyde (Knief 2015). In addition, there are Type X methanotrophs that also use RuMP pathway for formaldehyde assimilation, but can also utilize low levels of enzymes of the Serine pathway (Semrau et al. 2010). Methanotrophs can also be taxonomically grouped into three phylum: Alphaproteobacteria, Gammaproteobacteria and Verrucomicrobia (Borrel et al. 2011;

Table 2). This classification is based on general characteristics such as intracellular membrane structure, pathways for carbon assimilation, phospholipid fatty acid (PLFA) patterns and phylogeny of molecular markers (Semrau et al. 2010).

Table 2. General classification of the known methanotrophic taxa and their metabolic pathways

The first step of methane oxidation is catalysed by methane mono-oxygenase enzymes (MMO). The methanol produced is oxidized to formaldehyde, which is followed by assimilation into cell biomass or further oxidation to CO2 (Theisen et al. 2005). Almost all methanotrophs have the structural genes for the particulate methane mono-oxygenase (pMMO), whereas soluble methane mono-oxygenase (sMMO) has been found only in Methylocella and Methyloferula (Dedysh et al. 2000; Dunfield et al.

2003).

Early studies found that optimal growth conditions for most methanotrophs are at neutral pH and moderate temperature (+25 °C), but also thermotolerant and thermophilic species have been discovered more recently (Semrau et al. 2010). Gammaproteobacteria seems to have a high diversity and good adaptation to variations of temperature, pH, salinity and oxygen, whereas less diverse Alphaproteobacteria show less adaptation to different conditions. Nevertheless, species of the same phylogenetic group can show different adaptation and habitat preferences (Knief 2015).

The availability of nitrogen may have an impact on the methanotrophic community size and composition (Semrau et al. 2010). Type II methanotrophs and Methylococcus species are able to fix

nitrogen (N-fixation pathway), and hence these methanotrophs are dominating in the environments where the concentrations of nitrogen compounds are low (Aronson et al. 2013). Nowadays it is known that some Type I methanotrophs can also fix nitrogen (Semrau et al. 2010).

The aerobic methanotrophic communities have been studied in water columns and sediments of freshwater lakes. According to these studies, the family Methylococcaceae (type I) dominates in boreal lakes (Borrel et al. 2011). For example Taipale et al. (2009) detected both type I and II methanotrophic bacteria in the epilimnion of oxygen-stratified humic Lake Mekkojärvi, but Methylobacter (type I) was the dominant genera in the water column. Low water temperatures seem to favor type I methanotrophs in freshwater lakes, while the growth of type II is limited in these conditions (e.g. Kojima et al. 2009; Tsutsumi et al. 2010).

Anaerobic methane-oxidizing archaea

Anaerobic methane-oxidizing archaea (ANME) perform anaerobic oxidation of CH4 (AOM) through the reversal of the methanogenic pathway (Knittel and Boetius 2009). ANMEs are closely related to methanogenic archaea, and they are able to produce CH4 during net methane oxidation, while methanogens can reverse the methanogenic pathway to CH4 oxidation during net methane production (Timmers et al. 2017).

When AOM is mediated by a consortium of ANME and sulphate-reducing bacteria, there are three distinct methanotrophic clusters responsible for AOM: ANME-1, ANME-2 (subclusters a, b and c) and ANME-3 (Knittel and Boetius 2009). In contrast, ANME-2d can perform nitrate-driven AOM without any bacterial partner transferring electrons directly to a membrane bound NO3- reductase (Timmers et al. 2017). There are also studies showing evidence of AOM coupled to iron and manganese oxide reduction (Sivan et al. 2011), but corresponding organisms have not been identified (Timmers et al. 2017).

Moreover, Ettwig et al. (2010) discovered that AOM can be carried out by Candidatus Methylomirabilis oxyfera (NC10 phylum) in the presence of NO2-. This bacterium can produce oxygen through the reduction of NO2- and use this oxygen to CH4 oxidation. M. oxyfera has been enriched from freshwater sediments (Ettwig et al. 2010).

2.3 STABLE ISOTOPES IN CARBON MEASUREMENTS