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 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. <br/

Enrichment of ANME-1 from Eckernförde Bay sediment on thiosulfate, methane and short-chain fatty acids

The microorganisms involved in sulfate-dependent anaerobic oxidation of methane (AOM) have not yet been isolated. In an attempt to stimulate the growth of anaerobic methanotrophs and associated sulfate reducing bacteria (SRB), Eckernförde Bay sediment was incubated with different combinations of electron donors and acceptors. The organisms involved in AOM coupled to sulfate reduction (ANME-1, ANME-2, and Desulfosarcina/Desulfococcus) were monitored using specific primers and probes. With thiosulfate as sole electron acceptor and acetate, pyruvate or butyrate as the sole electron donor, ANME-1 became the dominant archaeal species. This finding suggests that ANME-1 archaea are not obligate methanotrophs and that ANME-1 can grow on acetate, pyruvate or butyrat

Enrichment of anaerobic methanotrophs in sulfate-reducing membrane bioreactors

Anaerobic oxidation of methane (AOM) in marine sediments is coupled to sulfate reduction (SR). AOM is mediated by distinct groups of archaea, called anaerobic methanotrophs (ANME). ANME co-exist with sulfate-reducing bacteria, which are also involved in AOM coupled SR. The microorganisms involved in AOM coupled to SR are extremely difficult to grow in vitro. Here, a novel well-mixed submerged-membrane bioreactor system is used to grow and enrich the microorganisms mediating AOM coupled to SR. Four reactors were inoculated with sediment sampled in the Eckernförde Bay (Baltic Sea) and operated at a methane and sulfate loading rate of 4.8 L L(-1) day(-1) (196 mmol L(-1) day(-1)) and 3.0 mmol L(-1) day(-1). Two bioreactors were controlled at 15 degrees C and two at 30 degrees C, one reactor at 30 degrees C contained also anaerobic granular sludge. At 15 degrees C, the volumetric AOM and SR rates doubled approximately every 3.8 months. After 884 days, an enrichment culture was obtained with an AOM and SR rate of 1.0 mmol g(volatile suspended solids) (-1) day(-1) (286 micromol g(dry weight) (-1) day(-1)). No increase in AOM and SR was observed in the two bioreactors operated at 30 degrees C. The microbial community of one of the 15 degrees C reactors was analyzed. ANME-2a became the dominant archaea. This study showed that sulfate reduction with methane as electron donor is possible in well-mixed bioreactors and that the submerged-membrane bioreactor system is an excellent system to enrich slow-growing microorganisms, like methanotrophic archae

Trace methane oxidation and the methane dependency of sulfate reduction in anaerobic granular sludge

This study investigates the oxidation of labeled methane (CH(4)) and the CH(4) dependence of sulfate reduction in three types of anaerobic granular sludge. In all samples, (13)C-labeled CH(4) was anaerobically oxidized to (13)C-labeled CO(2), while net endogenous CH(4) production was observed. Labeled-CH(4) oxidation rates followed CH(4) production rates, and the presence of sulfate hampered both labeled-CH(4) oxidation and methanogenesis. Labeled-CH(4) oxidation was therefore linked to methanogenesis. This process is referred to as trace CH(4) oxidation and has been demonstrated in methanogenic pure cultures. This study shows that the ratio between labeled-CH(4) oxidation and methanogenesis is positively affected by the CH(4) partial pressure and that this ratio is in methanogenic granular sludge more than 40 times higher than that in pure cultures of methanogens. The CH(4) partial pressure also positively affected sulfate reduction and negatively affected methanogenesis: a repression of methanogenesis at elevated CH(4) partial pressures confers an advantage to sulfate reducers that compete with methanogens for common substrates, formed from endogenous material. The oxidation of labeled CH(4) and the CH(4) dependence of sulfate reduction are thus not necessarily evidence of anaerobic oxidation of CH(4) coupled to sulfate reductio

Microbial diversity and community structure of a highly active anaerobic methane-oxidizing sulfate-reducing enrichment

Summary 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. Here we describe the microbial analysis of an enrichment obtained in a novel submerged-membrane bioreactor system and capable of high-rate AOM (286 mumol g(dry weight) (-1) day(-1)) coupled to sulfate reduction. By constructing a clone library with subsequent sequencing and fluorescent in situ hybridization, 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. Fluorescent in situ hybridization analyses showed that the ANME-2a cells occur as single cells without close contact to the bacterial syntrophic partner. Incubation with (13)C-labelled methane showed substantial incorporation of (13)C label in the bacterial C(16) fatty acids (bacterial; 20%, 44% and 49%) and in archaeal lipids, archaeol and hydroxyl-archaeol (21% and 20% respectively). The obtained data confirm that both archaea and bacteria are responsible for the anaerobic methane oxidation in a bioreactor enrichment inoculated with Eckernförde bay sedimen