Stable-Isotope Analysis of a Combined Nitrification-Denitrification Sustained by Thermophilic Methanotrophs under Low-Oxygen Conditions

Abstract

To simulate growth conditions experienced by microbiota at O(inf2)-limited interfaces of organic matter in compost, an experimental system capable of maintaining dual limitations of oxygen and carbon for extended periods, i.e., a pO(inf2)-auxostat, has been used. (sup15)N tracer studies on thermophilic (53(deg)C) decomposition processes occurring in manure-straw aggregates showed the emission of dinitrogen gas from the reactor as a result of simultaneous nitrification and denitrification at low pO(inf2) values (0.1 to 2.0%, vol/vol). The N loss was confirmed by nitrogen budget studies of the system. Depending on the imposed pO(inf2), 0.6 to 1.4 mmol of N/day (i.e., 20 to 40% of input N) was emitted as N(inf2). When the pO(inf2) was raised, the rates of both nitrification and denitrification increased instantaneously, indicating that ammonia oxidation was limited by oxygen. In auxostats permanently running at pO(inf2) >= 2% (vol/vol), the free ammonium pool was almost completely oxidized and was converted to nitrite plus nitrate and N(inf2) gas. Labelling of the auxostat with [(sup13)C]carbonate was conducted to reveal whether nitrification was of autotrophic or heterotrophic origin. Incorporation of (sup13)CO(inf2) into population-specific cellular compounds was evaluated by profiling the saponifiable phospholipid fatty acids (FAs) by using capillary gas chromatography and subsequently analyzing the (sup13)C/(sup12)C ratios of the individual FAs, after their combustion to CO(inf2), by isotope ratio mass spectrometry. Apart from the observed label incorporation into FAs originating from a microflora belonging to the genus Methylococcus (type X group), supporting nitrification of a methylotrophic nature, this analysis also corroborated the absence of truly autotrophic nitrifying populations. Nevertheless, the extent to which ammonia oxidation continued to exist in this thermophilic community suggested that a major energy gain could be associated with it.