The Responses Of Ammonia Oxidizers To Cu And Cd Stress In Fluvo-aquic Soil

Nitrification plays an important role in the natural nitrogen cycle. It is a two-step, with ammonia first oxidized to nitrite by ammonia-oxidizing microorganisms, and then nitrite oxidized to nitrate by nitrite-oxidizing microorganisms. The rate-limiting step is the first step in this process. Ammonia is oxidized to nitrite by ammonia oxidizers (archaea and bacteria) via the intermediate hydroxylamine. In AOB, this process is catalyzed by the enzyme ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). Recent studies have reported that AOA contain amoA, B and C genes for the three subunits of a potential archaeal AMO, but no homologous genes of bacterial HAO, which catalyzes the oxidation of hydroxylamine to nitrite. However, AOA possess numerous Cu-containing proteins, such as multicopper oxidases, small blue copper-containing proteins, which may functionally replace cytochromes. The energy metabolism of AOA possibly relies on Cu rather than on iron-containing electron transfer systems. Therefore, AMO and HAO were key enzyme in the nitrification process. AMO contain amoA, B and C genes and their moleculer weight was27×103(AmoA),38x103(AmoB) and31.4x103(AmoC), respectively. Therefore, it is significant to research the effect of Cu and Cd on ammonia oxidation activity using functional amoA gene.Cu can influx into the ecosystem, particularly into the soil ecosystem, in several ways through pesticides, fertilizers, industry activites, mining and so on. In agricultural soils, Cu has been widely used for many years in bactericides and fungicides, leading to its considerable accumulation. These activities lead to the copper content in the soil was too high, far exceeding soil capacity. The heavy metal contamination have great harm to animals, plants and humans and it can be serious threat to the stability of ecosystem and the health to humans.We selected the fluvo-aquic soil as the tested soil, which is the one of the typical soil in China. The soil is fertile and contains abundant ammonia-oxidizing microorganisms and nitrite-oxidizing microorganisms. We researched the effect of Cu and Cd on ammonia oxidizing communities, in order to determine correlations between heavy metals and ammonia oxidizing communities. Extraction of soil RNA and DNA, fluorescence quantitative PCR were used to quantified the amoA gene copy numbers for AOA and AOB. The sensitivity of ammonia oxidation activity to Cu in soils should be determined and the results of which can be applied as a biological method in environmental monitoring.The main achievements of this paper are as follows:(1) The potential nitrification rate was inhibited quickly and regularly with increasing Cu concentration and reach a significant level. The potential nitrification rate changed with increasing Cd concentration. When the soil samples were incubated for56days, PNR were inhibited significantly (P<0.05).(2) The amoA DNA content did not change significantly after Cu addition for56days and the abundance of archaeal amoA gene was more higher than that of bacterial amoA gene. However, the AOA amoA gene changed significantly after Cd addition for56days in DNA level. Therefore, amoA DNA level cannot ultimately reflect the activity of ammonium oxidizing communities. Isolation of soil RNA and analysis of the abundance and diversity of ammonium oxidizing communities at amoA RNA level are necessary.(3) Cellular mRNA levels were associated with survival and basal metabolism. When the soil samples were incubated after3days at different Cu concentrations, the activity of archaeal amoA gene was inhibited significantly (P<0.05) and decreased evidently at1200mg kg-1Cu concentration. This effect was noticeable at100mg kg-1Cu concentration. It indicated that AOB were more active and sensitive to Cu stress than AOA with respect to ammonium oxidization activity. The archaeal and bacteria amoA gene activity were not inhibited significantly with Cd addtion after incubation for1day and7days in mRNA level. Therefore, the profile of amoA mRNA expression can be used as an indicator of ammonia oxidization activity.(4) The results of qPCR showed that AOA was more abundant than AOB, so AOA play an important role for nitrification in the tested soil. However, the activity of bacterial amoA gene were more active than archaeal amoA gene. AOB were more active in the soil nitrification.