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 butyrate.

Effect of methanogenic substrates on anaerobic oxidation of methane and sulfate reduction by an anaerobic methanotrophic enrichment.

Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (SR) is assumed to be a syntrophic process, in which methanotrophic archaea produce an interspecies electron carrier (IEC), which is subsequently utilized by sulfate-reducing bacteria. In this paper, six methanogenic substrates are tested as candidate-IECs by assessing their effect on AOM and SR by an anaerobic methanotrophic enrichment. The presence of acetate, formate or hydrogen enhanced SR, but did not inhibit AOM, nor did these substrates trigger methanogenesis. Carbon monoxide also enhanced SR but slightly inhibited AOM. Methanol did not enhance SR nor did it inhibit AOM, and methanethiol inhibited both SR and AOM completely. Subsequently, it was calculated at which candidate-IEC concentrations no more Gibbs free energy can be conserved from their production from methane at the applied conditions. These concentrations were at least 1,000 times lower can the final candidate-IEC concentration in the bulk liquid. Therefore, the tested candidate-IECs could not have been produced from methane during the incubations. Hence, acetate, formate, methanol, carbon monoxide, and hydrogen can be excluded as sole IEC in AOM coupled to SR. Methanethiol did inhibit AOM and can therefore not be excluded as IEC by this study.

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 reduction.

Effect of environmental conditions on sulfate reduction with methane as electron donor by an Eckemförde Bay enrichment.

Sulfate reduction (SR) coupled to anaerobic oxidation of methane (AOM) is meditated by marine microorganisms and forms an important process in the global sulfur and carbon cycle. In this research, the possibility to use this process for the removal and recovery of sulfur and metal compounds from waste streams was investigated. A membrane bioreactor was used to enrich for a community of methane-oxidizing sulfate-reducing microorganisms from Eckernförde Bay sediment The AOM and SR rate of the obtained enrichment were 1.0 mmol gvss(-1) d(-1). The operational window and optimal environmental conditions for SR with methane as electron donor were assessed. The optimum pH, salinity, and temperature were 7.5, 30% per hundred and 20 degrees C, respectively. The enrichment had a good affinity for sulfate (Km < 0.5 mM) and a low affinity for methane (Kn > 0.075 MPa). A0M coupled to SR was completely inhibited at 2.4 (L0.1) mM sulfide. AOM occurred with sulfate, thiosulfate, and sulfite as electron accepters. Sulfate reduction with methane as electron donor can be applied for the removal of sulfate or for the production of sulfide,for metal precipitation. However, the low optimal temperature and the high salt requirement limit the operational window of the process.

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 archaea.

Solid phase extraction and liquid chromatography-tandem mass spectrometry for the evaluation of 4-hydroxy-2-nonenal in pork products.

This research was aimed at setting up an analytical method for the determination in pork products of 4-hydroxy-2-nonenal (4-HNE), an aldehyde produced from the oxidation of omega-6-polyunsaturated fatty acids. Such a compound mediates various biological effects, but it is considered to be very toxic to mammalian cells at levels higher than physiological ones. The methods used for the determination of 4-HNE in biological fluids, such as blood, were found to be unsuitable for meat samples because both the repeatability and the recovery in spiked samples were unsatisfactory. A new method, based on solid phase extraction and HPLC-MS/MS, was therefore developed and validated. The limit of detection of 4-HNE in spiked samples was 0.043 mg/kg, and the recovery was approximately 60% depending on the concentration. Good linearity was observed in the range of 0.1-10 mg/kg, and repeatability and interday and intraday precision expressed as relative standard deviation were <10%. The method has been successfully applied to the determination of the aldehyde in samples of various pork products. 4-HNE was present in some products, especially the smoked and/or cooked ones, at levels that might not be a real risk for human health.