Biotechnological aspects of anaerobic oxidation of methane coupled to sulfate reduction

R.J.W. Meulepas

Research output: Thesisinternal PhD, WU


Sulfate reduction (SR) can be used for the removal and recovery of metals and oxidized sulfur compounds from waste streams. Sulfate-reducing bacteria reduce oxidized sulfur compounds to sulfide. Subsequently, sulfide can precipitate dissolved metals or can be oxidized to elemental sulfur. Both metal sulfides and elemental sulfur can be reused in various applications. SR with hydrogen or ethanol as electron donor is an established biotechnological process. However, the costs of these electron donors limit the application possibilities. Methane would be a cheaper and more attractive electron donor. SR coupled to the anaerobic oxidation of methane (AOM) occurs in marine sediments. Uncultured archaea, distantly related to methanogens, and bacteria are involved in this process. The in vitro demonstration of SR coupled to AOM gave rise to this research, which aims to develop a biotechnological process in which methane is used as electron donor for SR.
Three types of anaerobic granular sludge were screened for the ability to reduce sulfate with methane as electron donor. To do so, incubations were done with 13C-labeled methane. All three sludge types anaerobically oxidized 13C-labeled methane to 13C-labeled carbon dioxide. Moreover, the presence of methane enhanced the SR rate. However, AOM by sludge was not coupled to SR, but coincides with net methanogenesis. The methane-dependent SR was caused by the inhibitory effect of methane on methanogens competing (possibly in syntrophic consortia with acetogenic bacteria) with sulfate reducers for the same endogenous substrate. Therefore, anaerobic granular sludge does not form a suitable inoculum for sulfate-reducing bioreactors fed with methane.
Well-mixed ambient-pressure submersed-membrane bioreactors, fed with sulfate and methane, were inoculated with sediment from Eckernförde Bay (Baltic Sea). Initially AOM rates were extremely low (0.004 mmol L-1 day-1), but at 15ºC AOM and SR rates increased over the course of 884 days to 0.60 mmol L-1 day-1 or 1.0 mmol gVSS-1 day-1. The AOM rate doubled approximately every 3.8 months. Molecular analyses revealed that the archaea in the obtained enrichment belonged predominately to the anaerobic methanotroph ANME-2a. Both bacteria and archaea incorporated carbon derived from 13C-labeled methane into their lipids, indicating that both were involved in AOM coupled to SR. To investigate which kind of waste streams can be treated by the methane-oxidizing sulfate-reducing enrichment, the effect of environmental conditions and alternative substrates on AOM and SR 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 by 2.4 (±0.1) mM sulfide. The enrichment utilized sulfate, thiosulfate and sulfite as electron acceptors for methane oxidation, and methane, formate, acetate and hydrogen as electron donors for SR.
This study shows that methane can be used as electron donor for sulfate reduction in bioreactors. However, the low growth rate of the responsible microorganisms still forms a major bottleneck for biotechnological applications.

Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
  • Buisman, Cees, Promotor
  • Lens, Piet, Co-promotor
  • Stams, Fons, Co-promotor
Award date19 Jun 2009
Place of Publication[S.l.
Print ISBNs9789085853978
Publication statusPublished - 2009


  • anaerobic conditions
  • oxidation
  • methane
  • microbiology
  • biotechnology
  • anaerobic microbiology
  • sulfate reduction


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