Culturing Methanogenic Archaea from Tropical Peatlands: Identifying Alternative Approaches to Retrieve Broader Isolate Diversity

Methanogens anaerobically metabolize simple carbon compounds coupled with an electron donor and produce methane in a process known as methanogenesis. While their importance in anoxic ecosystems and their greenhouse gas emissions are known, less is known about their diverse members. This is in part due to limited culture-dependent studies as a consequence of the difficulty to culture and isolate them under laboratory conditions. Current methods in methanogen isolation require lengthy protocols, expensive equipment, can be easily contaminated, and even if a successful isolation is completed, traditional methods are biased towards only a few species of methanogens- leaving much of this community unsampled and thus unrepresented. New approaches in the isolation of methanogens need to be investigated in order to circumvent these obstacles. Here, I evaluated the effects of different strategies and alternative methods with the goal of increasing the diversity of recovered methanogens from Amazon peatlands as a study case. The results show that: a) through the use of different antibiotics the bacterial community makeup can be altered and lead to different methanogenic enrichments, some antibiotics reliably increase methanogenesis in all study sites, others only enhance it in some sites, while some have a low rate of methanogenesis enriching novel slow growers, b) the use of different substrates has less of an effect on methane production rates, however the complex substrate butyrate leads to consistent late stimulation, c) altering media components (reducing agent and overall geochemical background) for Amazon conditions would lead to a shorter time to isolation, d) and multiple methanogenic enrichments were achieved building on variable conditions and can lead to novel Amazon lineages. Molecular data is offering a more detailed view of bacteria and methanogens increasing or decreasing in response to treatments. Overall, it is shown that combining alternative approaches that manipulate interactions, metabolic substrate availability and culturing conditions could lead to more diverse isolation outputs from methanogenic cultures.