Denitrification as an adaptive trait in soil and groundwater bacteria

University dissertation from Department of Ecology, Chemical Ecology & Ecotoxicology, Sölvegatan 37, S-223 62 Lund, Sweden. Tel. +46 46-222 93 22, Fax +46 46-222 37 90

Abstract: The focus of the thesis is on selection and adaptation processes in bacteria with emphasis on denitrifying bacteria in groundwater. Other nitrogen transformation processes such as dissimilatory nitrate reduction to ammonium (nitrate ammonification) and nitrification of forest soil bacteria are briefly discussed. Intensive fertilisation of agricultural soils in southern Sweden have been performed during the last 20-30 years and has resulted in accumulation of nitrate in many aquifers. Selection of denitrifiers in environments with high nitrate concentrations may occur since nitrate may act as a selective agent to alter gene frequencies in natural denitrifier populations. Selection may be accompanied by a community shift with a larger part of the community being denitrifiers with enhanced activity. In essence, a denitrifier that can utilise increased nitrate concentrations more efficiently than its neighbours, becomes more competitive and can allocate increasing amounts of energy to growth and reproduction which would result in an increase in fitness. The community shift may be reflected in the isolation frequencies in which about 50% of strains isolated from two nitrate contaminated aquifers with in situ nitrate-nitrogen concentrations of 24.1 and 35.2 mg/l, respectively, were denitrifiers compared to about 20% for a pristine site with an in situ nitrate-nitrogen concentration of 6.3 mg/l. Microcosms with sterile sediment and groundwater were inoculated with single denitrifying strains isolated from the three groundwater aquifers and the denitrifying activity was quantified using gas chromatography. The average denitrification activity for strains from the nitrate contaminated sites were twice as high as the activity of the strains from the pristine site. Denitrification were carbon limited and glucose amendment increased the denitrification activity about a 2-fold for all strains. The strain specific differences in denitrification rates increased to a 2.5-fold after carbon addition indicating that the differences in reduction rates cannot be explained by different carbon utilisation rates but rather reflect innate differences in the reductases of the strains. A preliminary identification of the molecular target for adaptation was performed with artificial electron donors and electron acceptors for all enzymatic steps in the denitrification pathway using a spectrophotometrical method. Nitrous oxide reductase activity was significantly higher in denitrifiers from the nitrate contaminated sites. This suggests that nitrous oxide reductase genes may be the molecular target, possibly by mutation or gene duplication, for adaptation to high nitrate concentrations. Two anaerobic denitrifiers from each of the contaminated sites were capable of aerobic denitrification indicating that high nitrate concentrations may select for strains that denitrifies in the presence of both oxygen and nitrate. Microcosm experiments with fertilised coniferous forest soil were performed to elucidate whether selection of denitrifiers, nitrate ammonifiers and nitrifiers have occurred as a response to nitrogen fertilisation. The activity of the nitrogen transformers were quantified using a gas chromatograph coupled to a mass spectrometer. The dominating fate of added 15NO3- and 15NH4+ was immobilisation in microorganisms or organic matter. The activity of denitrifiers, nitrate ammonifiers and nitrifiers were negligible, suggesting that microorganisms that immobilise or mobilise nitrogen control the major fate of fertiliser nitrogen.

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