Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Abstract

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. (13)CO(2)-DNA-stable isotope probing results showed significant assimilation of (13)C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO(2) fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active (13)CO(2)-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.

Figure 1

Changes in nitrate concentrations during incubation of five acidic soils in the presence or absence of DCD. Error bars represent standard errors of triplicate samples.

Figure 2

Changes in abundance of thaumarchaeal amoA genes of five acidic soils in the presence or absence of DCD. Error bars represent standard errors of triplicate samples. Different letters above the bars indicate a significant difference (P<0.05).

Figure 3

Changes in abundance of bacterial amoA genes of five acidic soils in the presence or absence of DCD. Error bars represent standard errors of triplicate samples. Different letters above the bars indicate a significant difference (P<0.05).

Figure 4

Relationship between nitrate concentration and thaumarchaeal (a) or bacterial (b) amoA gene copy numbers in control microcosms. Vertical and horizontal error bars represent standard errors of amoA gene copies and nitrate concentrations from triplicate samples, respectively.

Figure 5

Changes in concentrations of (a) nitrate, (b) ammonium, (c) abundance of thaumarchaeal amoA genes and (d) abundance of bacterial amoA genes during DNA-SIP microcosms of HZ soil for 15 or 30 days. Error bars represent standard errors of triplicate samples. Different letters above the bars indicate a significant difference (P<0.05).

Figure 6

Distribution of the relative abundance of thaumarchaeal and bacterial amoA gene in CsCl gradient for ¹³C-CO₂, ¹²C-CO₂ and ¹³C-CO₂ + DCD treatments during DNA-SIP microcosms of HZ soil. Twenty fractions of genomic DNA extracted from each 4.9-ml centrifuge tube covered a buoyant density from 1.64 to 1.75 g ml⁻¹. The plotted values are the proportion of thaumarchaeal or bacterial amoA gene copy numbers in each fraction to the total abundance across the gradient. Vertical error bars represent standard errors of the relative abundance from triplicate samples. Horizontal error bars represent standard errors of buoyant density of the same order fraction from triplicate samples.

Figure 7

Denaturing gradient gel electrophoresis analysis of thaumarchaeal (a, c) and bacterial (b) amoA genes during DNA-SIP microcosms of HZ soil incubated with ¹³C-CO₂ or ¹²C-CO₂ in the presence or absence of DCD. Thaumrchaeal amoA gene PCR products of twenty fractions of genomic DNA from individual centrifuge tube were subjected to DGGE for changes of different thaumarchaeal communitys for 15 or 30 days (b). Eight arrows marked the bands in heavy fraction around a buoyant density of 1.73 g ml⁻¹ excised for sequencing.

Figure 8

Phylogenetic analysis of archaeal 16S rRNA genes (a) retrieved from ¹³C-CO₂ DNA-SIP heavy fractions (HF) and light fractions (LF) of HZ soil microcosms incubated for 30 days, and thaumarchaeal amoA genes (b) derived from eight DGGE bands in the heavy fractions around a buoyant density of 1.73 g ml⁻¹ from the HZ soil incubated for 30 days. The sequences for the HF and LF were highlighted in red and blue, respectively. HF-Archaea-clone-3-(19) indicates that 19 clones showed above 98% similarities to the clone-3. Bootstrap values (>50%) are indicated at branch points. The scale bar represents 2% and 5% nucleic acid sequence divergence for archaeal 16S rRNA genes and thaumarchael amoA genes, respectively. The letters M and S represent Marine Lineage and Soil and Sediment Lineage, respectively.