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 2-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 2-auxostat, has been used. 15 N tracer studies on thermophilic (53؇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 2 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 2 , 0.6 to 1.4 mmol of N/day (i.e., 20 to 40% of input N) was emitted as N 2. When the pO 2 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 2 > 2% (vol/vol), the free ammonium pool was almost completely oxidized and was converted to nitrite plus nitrate and N 2 gas. Labelling of the auxostat with [ 13 C]carbonate was conducted to reveal whether nitrification was of autotrophic or heterotrophic origin. Incorporation of 13 CO 2 into population-specific cellular compounds was evaluated by profiling the saponifiable phospholipid fatty acids (FAs) by using capillary gas chromatography and subsequently analyzing the 13 C/ 12 C ratios of the individual FAs, after their combustion to CO 2 , 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.

Bacterial populations and processes involved in acetate and propionate consumption in anoxic brackish sediment

“…In addition, the incorporation of 13 C-label into phospholipid-derived fatty acids (PLFA) was determined to study the bacterial populations involved in these processes. Both ourselves and others have recently shown that this approach enables the direct linking of microbial populations to speci¢c processes in environmental samples [1,11,12] and in model communities [2,13]. Our results suggest that propionate was directly taken up from the porewater without the formation of acetate as an intermediate, and that acetate and propionate were predominantly consumed by di¡erent populations or groups of bacteria.…”

“…The limit of detectable increase in N 13 C is about 1^2x, which is mainly the result of natural variation in N 13 C ratios of the unlabelled control incubations and to a lesser extent of analytical methods (0.3x accuracy). Incorporation of label was very speci¢c, as increases in N 13 C ratios (vN 13 C, equivalent to speci¢c incorporation = amount of 13 C/amount of PLFA) were highly variable between bacterial PLFA and showed a range of 10 for 13 C-acetate incorporation and s 100 for 13 C-propionate incorporation (Figs. 3A and 4A).…”

“…Labeling patterns of PLFA were very di¡erent between sediments incubated with 13 C-acetate or 13 C-propionate ( Figs. 3B and 4B).…”

“…Isolates on acetate from anoxic marine sediments mostly belong to the genus Desulfobacter [37^39], which use acetate preferentially or even exclusively [38,39]. However, 13 C-acetate labeling patterns are very di¡erent from the PLFA patterns found in Desulfobacter strains [25,27], showing that this genus was not the dominant acetate-consuming population [1]. Also, 10Me16:0, a suggested speci¢c biomarker PLFA for this genus [25,27], showed very little label incorporation (0.5% of total incorporation, Fig.…”

“…Shown are the average data (with S.D.) for the 13 C-acetate,13 C-propionate and unlabeled control incubations. (IS = internal standard.…”