Growth of anaerobic methane-oxidizing archaea and sulfate reducing bacteria in a high pressure membrane-capsule bioreactor

Communities of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB) grow slowly, which limits the ability to perform physiological studies. High methane partial pressure was previously successfully applied to stimulate growth, but it is not clear how different ANME subtypes and associated SRB are affected by it. Here, we report on the growth of ANME-SRB in a membrane capsule bioreactor inoculated with Eckernförde Bay sediment that combines high-pressure incubation (10.1 MPa methane) and thorough mixing (100 rpm) with complete cell retention by a 0.2-m-pore-size membrane. The results were compared to previously obtained data from an ambient-pressure (0.101 MPa methane) bioreactor inoculated with the same sediment. The rates of oxidation of labeled methane were not higher at 10.1 MPa, likely because measurements were done at ambient pressure. The subtype ANME-2a/b was abundant in both reactors, but subtype ANME-2c was enriched only at 10.1 MPa. SRB at 10.1 MPa mainly belonged to the SEEP-SRB2 and Eel-1 groups and the Desulfuromonadales and not to the typically found SEEP-SRB1 group. The increase of ANME-2a/b occurred in parallel with the increase of SEEP-SRB2, which was previously found to be associated only with ANME-2c. Our results imply that the syntrophic association is flexible and that methane pressure and sulfide concentration influence the growth of different ANME-SRB consortia. We also studied the effect of elevated methane pressure on methane production and oxidation by a mixture of methanogenic and sulfate-reducing sludge. Here, methane oxidation rates decreased and were not coupled to sulfide production, indicating trace methane oxidation during net methanogenesis and not anaerobic methane oxidation, even at a high methane partial pressure.This work was supported in part by the EET program of the Dutch Ministries of Economic Affairs; Education, Culture and Science; and Environment and special planning through the Anaerobic Methane Oxidation for Sulfate Reduction project. This research was also supported by the Dutch Technology Foundation STW, which is part of the Netherlands Organization for Scientific Research (NWO) and which is partly funded by the Ministry of Economic Affairs. The research of A.J.M.S. is supported by an ERC grant (project 323009) and a Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

Evaluation and optimization of PCR primers for selective and quantitative detection of marine ANME subclusters involved in sulfate-dependent anaerobic methane oxidation

Since the discovery that anaerobic methanotrophic archaea (ANME) are involved in the anaerobic oxidation of methane coupled to sulfate reduction in marine sediments, different primers and probes specifically targeting the 16S rRNA gene of these archaea have been developed. Microbial investigation of the different ANME subtypes (ANME-1; ANME-2a, b, and c; and ANME-3) was mainly done in sediments where specific subtypes of ANME were highly enriched and methanogenic cell numbers were low. In different sediments with higher archaeal diversity and abundance, it is important that primers and probes targeting different ANME subtypes are very specific and do not detect other ANME subtypes or methanogens that are also present. In this study, primers and probes that were regularly used in AOM studies were tested in silico on coverage and specificity. Most of the previously developed primers and probes were not specific for the ANME subtypes, thereby not reflecting the actual ANME population in complex samples. Selected primers that showed good coverage and high specificity for the subclades ANME-1, ANME-2a/b, and ANME-2c were thoroughly validated using quantitative polymerase chain reaction (qPCR). From these qPCR tests, only certain combinations seemed suitable for selective amplification. After optimization of these primer sets, we obtained valid primer combinations for the selective detection and quantification of ANME-1, ANME-2a/b, and ANME-2c in samples where different ANME subtypes and possibly methanogens could be present. As a result of this work, we propose a standard workflow to facilitate selection of suitable primers for qPCR experiments on novel environmental samples.This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009). Research of PHATand AJMS is supported by the SIAM Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO).info:eu-repo/semantics/publishedVersio

Relating MEC population dynamics to anode performance from DGGE and electrical data

<p>The microbial electrolysis cell (MEC) is a promising system for H-2 production, but little is known about the active microbial population in MEC systems. Therefore, the microbial community of five different MEC graphite felt anodes was analyzed using denaturing gradient gel electrophoresis (DGGE) profiling. The results showed that the bacterial population was very diverse and there were substantial differences between microorganisms in anolyte and anode samples. The archaeal population in the anolyte and at the anodes, and between the different MEC anodes, was very similar. SEM and FISH imaging showed that Archaea were mainly present in the spaces between the electrode fibers and Bacteria were present at the fiber surface, which suggested that Bacteria were the main microorganisms involved in MEC electrochemical activity. Redundancy analysis (RDA) and QR factorization-based estimation (QRE) were used to link the composition of the bacterial community to electrochemical performance of the MEC. The operational mode of the MECs and their consequent effects on current density and anode resistance on the populations were significant. The results showed that the community composition was most strongly correlated with current density. The DGGE band mostly correlated with current represented a Clostridium sticklandii strain, suggesting that this species had a major role in current from acetate generation at the MEC anodes. The combination of RDA and QRE seemed especially promising for obtaining an insight into the part of the microbial population actively involved in electrode interaction in the MEC. (C) 2013 Elsevier GmbH. All rights reserved.</p>