Microbial aspects of anaerobic methane oxidation with sulfate as electron acceptor

Anaerobic oxidation of methane (AOM) is an important methane sink in the ocean but the microbes responsible for AOM are as yet resilient to cultivation. It was shown that AOM was coupled to sulfate reduction (SR) and this gave rise to current research which aims to develop a biotechnological process in which methane is used an electron donor for SR.
This thesis describes the microbial analysis of an enrichment capable of high rate AOM (286 µmol.gdry weight-1.day-1) coupled to SR using a novel submerged membrane bioreactor system. Initially AOM rates were extremely low (0.004 mmol L-1 d-1), but AOM and SR increased exponential over the course of 884 days to 0.60 mmol L-1 d-1. The responsible organisms doubled every 3.8 months.
By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization (FISH), we showed that the responsible methanotrophs belong to the ANME-2a subgroup of anaerobic methanotrophic archaea, and that sulfate reduction is most likely performed by sulfate reducing bacteria commonly found in association with other ANME related archaea in marine sediments. Another relevant portion of the bacterial sequences can be clustered within the order of Flavobacteriales but their role remains to be elucidated. FISH analyses showed that the ANME-2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with 13C labeled methane showed substantial incorporation of 13C label in the bacterial C16 fatty acids (bacterial; 20, 44 and 49%) and in archaeal lipids, archaeol and hydroxyl-archaeol (21 and 20%, respectively). This confirms that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sediment. To unravel the pathway of this syntrophic conversion, the effect of possible intermediates on AOM and SR was assessed.
To investigate which kind of waste and process streams can be treated by the methanotrophic sulfate-reducing enrichment, the effect of environmental conditions and different substrates was assessed. The optimum pH, salinity and temperature for SR with methane by the enrichment were 7.5, 30‰ and 20°C, respectively. The biomass had a good affinity for sulfate (Km < 1.0 mM), a low affinity for methane (Km > 75 KPa) and AOM was completely inhibited at 2.4 (±0.1) mM sulfide. The enrichment utilized sulfate, thiosulfate, sulfite and elemental sulfur as alternative electron acceptors for methane oxidation and formate, acetate and hydrogen as alternative electron donors for sulfate reduction. As a co-substrate for methane oxidation only methanol stimulated the conversion of 13C labeled CH4 to 13CO2 in batch incubations of Eckernförde bay sediment, other possible co-substrates had a negative effect on the AOM rate.
The research described in this thesis shows the possibility of enriching slow growing methane oxidizing communities but also shows the difficulties in applying this process for a biotechnological purpose because of the extreme slow doubling times and the lack of understanding of the metabolic routes used by these organisms.