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Abstract

Novel high-pressure biotechnical systems that were developed and applied for the study of anaerobic oxidation of methane (AOM) are described. The systems, referred to as high-pressure continuous incubation system (HP-CI system) and high-pressure manifold-incubation system (HP-MI system), allow for batch, fed-batch, and continuous gas-phase free incubation at high concentrations of dissolved methane and were designed to meet specific demands for studying environmental regulation and kinetics as well as for enriching microbial biomass in long-term incubation. Anoxic medium is saturated with methane in the first technical stage, and the saturated medium is supplied for biomass incubation in the second stage. Methane can be provided in continuous operation up to 20 MPa and the incubation systems can be operated during constant supply of gas-enriched medium at a hydrostatic pressure up to 45 MPa. To validate the suitability of the high-pressure systems, we present data from continuous and fed-batch incubation of highly active samples prepared from microbial mats from the Black Sea collected at a water depth of 213 m. In continuous operation in the HP-CI system initial methane-dependent sulfide production was enhanced 10- to 15-fold after increasing the methane partial pressure from near ambient pressure of 0.2 to 10.0 MPa at a hydrostatic pressure of 16.0 MPa in the incubation stage. With a hydraulic retention time of 14 h a stable effluent sulfide concentration was reached within less than 3 days and a continuing increase of the volumetric AOM rate from 1.2 to 1.7 mmol L(-1) day(-1) was observed over 14 days. In fed-batch incubation the AOM rate increased from 1.5 to 2.7 and 3.6 mmol L(-1) day(-1) when the concentration of aqueous methane was stepwise increased from 5 to 15 mmol L(-1) and 45 mmol L(-1). A methane partial pressure of 6 MPa and a hydrostatic pressure of 12 MPa in manifold fed-batch incubation in the HP-MI system yielded a sixfold increase in the volumetric AOM rate. Over subsequent incubation periods AOM rates increased from 0.6 to 1.2 mmol L(-1) day(-1) within 26 days of incubation. No inhibition of biomass activity was observed in all continuous and fed-batch incubation experiments. The organisms were able to tolerate high sulfide concentrations and extended starvation periods.