Exploring the ecophysiology of anaerobic communities of methanotrophic archaea and sulfate-reducing bacteria

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is a widespread occurring process in anoxic marine sediments. The process is performed by ANaerobic MEthane oxidizing archaea (ANME) and associated sulfate reducing bacteria (SRB). The ANME presumably oxidize methane through reverse methanogenesis. The associated SRB were thought to reduce sulfate using an interspecies electron carrier (IEC) derived from AOM. The product of methane oxidation that is transferred to the SRB is either a less reduced compound that acts as IEC or electrons are transferred directly (through nanowires or pili) or indirectly (through extracellular quinones). However, recent evidence emerged that ANME could perform both methane oxidation and sulfate reduction to produce sulfur, where the SRB disproportionate the produced sulfur. Little is known on the physiology and ecology of these ANME and associated SRB. The main reasons for this are the difficulties in lab cultivation and to perform <em>in situ </em>studies. Anaerobic methane oxidation is a process that is at the border of what is energetically possible for sustaining life, which makes it hard to cultivate the responsible organisms. Estimates of the Gibbs free energy yields are between -18 and -35 kJ mol-1 and growth rates between 1.1 and 7.5 months, depending on the environment. AOM therefore operates close to thermodynamic equilibrium and is highly dependent on substrate and product concentrations. In <strong>chapter 2</strong>, we obtained faster growth rates at elevated methane partial pressure as compared to ambient pressure. The increase in partial pressure increased the solubility of methane and thus the energy yield for the organisms. In <strong>chapter 6</strong>, we showed higher AOM activity and growth of ANME under thermodynamically favorable sulfate and sulfide concentrations. The problems in studying the process <em>in situ </em>in complex environments comes from difficulties in differentiation of reversible processes. In most studies, methane oxidation is monitored by labelled CO2 formation from labelled methane. Methanogens can perform trace methane oxidation (TMO)during net methanogenesis, which also results in the production of labelled CO2 from labelled methane. When AOM becomes less favorable, the anaerobic back flux of AOM becomes significant, leading to the production of measurable amounts of methane. In <strong>chapter 2 </strong>and <strong>chapter 3</strong>, we were able to differentiate between AOM and TMO in long-term incubations. Another challenge is related to the detection of ANME in complex environments. The phylogenetic distance between and within ANME clades is large. In <strong>chapter 5</strong>, we discussed the difficulties in primer and probe design for selective detection of ANME without targeting closely related methanogens. Furthermore, it is not known if even more ANME species and clades have yet to be discovered that are not detected with the primers and probes used thus far. In <strong>chapter 3</strong>, we found indications that besides ANME-2a/b, ANME-2d archaea were also able to perform AOM coupled to sulfate reduction in freshwater conditions. The finding of ANME-2a/b in freshwater shows that ANME archaea are ubiquitously distributed and not only occur in marine sediments. In <strong>chapter 6</strong>, we confirmed that different ANME clades show niche separation based on the presence of methane and different sulfate and sulfide concentrations. In <strong>chapter 2</strong>, we obtained indications that ANME-2c grows at high methane partial pressure. More research on the ecophysiology could help in understanding occurrence and activity of ANME in different environments. Many different SRB have been found so far to form close associations with ANME. Most fall within the <em>Desulfosarcina/Desulfococcus </em>(DSS) clade and only for two enrichment cultures the dominant partner of ANME-2a/b was determined to belong to a specific group with the DSS named SEEP-SRB1. In <strong>chapter 2</strong>, we found more evidence that a group outside the DSS clade, SEEP-SRB2, could also associate with ANME-2a/b and that Eel-1 members are not directly involved in AOM. In <strong>chapter 4</strong>, we enriched for SRB within the DSS clade on alternative substrates besides methane, but we were unable to show that these are involved in AOM. Therefore, more research on the sulfate-reducing partner is needed to understand the metabolic interactions between ANME and SRB.