Anaerobic methane oxidation in fresh water lake sediments
Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (AOM-SR) is a biological process mediated by anaerobic methanotrophs (ANME) and sulfate reducing bacteria. Due to its relevance in regulating the global carbon cycle and potential biotechnological application for treating sulfate-rich wastewater, AOM-SR has drawn attention from the scientific community. However, the detailed knowledge on ANME community, its physiology and metabolic pathway are scarcely available, presumably due to the lack of either pure cultures or the difficulty to enrich the biomass. To enhance the recent knowledge on ANME distribution and enrichment conditions, this research investigated AOM-SR with the following objectives: (i) characterize the microbial communities responsible for AOM in marine sediment, (ii) enrich ANME in different bioreactor configurations, i.e. membrane bioreactor (MBR), biotrickling filter (BTF) and high pressure bioreactor (HPB), and (iii) assess the AOM-SR activity under different pressure and temperature conditions.
The microbes inhabiting coastal sediments from Marine Lake Grevelingen (the Netherlands) was characterized and the ability of the microorganisms to carry out AOM-SR was assessed. By performing batch activity tests for over 250 days, AOM-SR was evidenced by sulfide production and the concomitant consumption of sulfate and methane at approximately equimolar ratios and a sulfate reduction rate of 5 µmol gdw-1 d-1 was attained. Sequencing of 16S rRNA genes showed the presence of ANME-3 in the Marine Lake Grevelingen sediment.
Two bioreactor configurations, i.e. MBR and BTF were operated under ambient conditions for 726 days and 380 days, respectively, to enrich the microorganisms from Ginsburg mud volcano performing AOM. The reactors were operated in fed-batch mode for the liquid phase with a continuous supply of gaseous methane. In the MBR, an external ultra-filtration membrane was used to retain the biomass, whereas, in the BTF, biomass retention was achieved via biomass attachment to the packing material. AOM-SR was recorded only after ~ 200 days in both bioreactor configurations. The BTF operation showed the enrichment of ANME in the biofilm by Illumina Miseq method, especially ANME-1 (40%) and ANME-2 (10%). Interestingly, in the MBR, aggregates of ANME-2 and Desulfosarcina were visualized by catalyzed reported deposition–fluorescence in situ hybridization (CARD-FISH). Acetate production was observed in the MBR, indicating that acetate was a possible intermediate of AOM. Although both bioreactor configurations showed good performance and resilience capacities for AOM enrichment, the sulfate reduction rate was slightly higher and faster in the BTF (1.3 mM d-1 on day 280) than the MBR (0.5 mM d-1 on day 380).
In order to simulate cold seep conditions and differentiate the impact of environmental conditions on AOM activities, sediment highly enriched with the ANME-2a clade was incubated in HPB at different temperature (4, 15 and 25oC at 10 MPa) and pressure (2, 10, 20 and 30 MPa at 15oC) conditions. The incubation at 10 MPa pressure and 15oC was observed to be the most suitable condition for the ANME-2a phylotype, which is similar to in situ conditions where the biomass was sampled, i.e. Captain Aryutinov mud volcano, Gulf of Cadiz. The incubations at 20 MPa and 30 MPa pressures showed the depletion in activities after 30 days of incubation. Incubation of AOM hosting sediment at in situ condition could be a preferred option for achieving high AOM activities and sulfate reduction rates.
In this thesis, it has been experimentally demonstrated that biomass retention and the continuous supply of methane can favor the growth of the slow growing anaerobic methane oxidizing community in bioreactors even under ambient conditions. Therefore, locating ANME habitats in shallow environments and enriching them at ambient conditions can be advantageous for future environmental biotechnology applications.