Growth of soil ammonia-oxidizing archaea on air-exposed solid surface

Abstract

Soil microorganisms often thrive as microcolonies or biofilms within pores of soil aggregates exposed to the soil atmosphere. However, previous studies on the physiology of soil ammonia-oxidizing microorganisms (AOMs), which play a critical role in the nitrogen cycle, were primarily conducted using freely suspended AOM cells (planktonic cells) in liquid media. In this study, we examined the growth of two representative soil ammonia-oxidizing archaea (AOA), Nitrososphaera viennensis EN76 and "Nitrosotenuis chungbukensis" MY2, and a soil ammonia-oxidizing bacterium, Nitrosomonas europaea ATCC 19718 on polycarbonate membrane filters floated on liquid media to observe their adaptation to air-exposed solid surfaces. Interestingly, ammonia oxidation activities of N. viennensis EN76 and "N. chungbukensis" MY2 were significantly repressed on floating filters compared to the freely suspended cells in liquid media. Conversely, the ammonia oxidation activity of N. europaea ATCC 19718 was comparable on floating filters and liquid media. N. viennensis EN76 and N. europaea ATCC 19718 developed microcolonies on floating filters. Transcriptome analysis of N. viennensis EN76 floating filter-grown cells revealed upregulation of unique sets of genes for cell wall and extracellular polymeric substance biosynthesis, ammonia oxidation (including ammonia monooxygenase subunit C (amoC3) and multicopper oxidases), and defense against H₂O₂-induced oxidative stress. These genes may play a pivotal role in adapting AOA to air-exposed solid surfaces. Furthermore, the floating filter technique resulted in the enrichment of distinct soil AOA communities dominated by the "Ca. Nitrosocosmicus" clade. Overall, this study sheds light on distinct adaptive mechanisms governing AOA growth on air-exposed solid surfaces.

Keywords: H2O2-induced oxidative stress; air-exposed solid surfaces; floating filter cultivation; soil ammonia-oxidizing archaea; soil nitrification; transcriptome.

Figure 1

Comparison of ammonia oxidation activities of AOM grown on floating filters and the control culture in liquid media. The line graphs (A, C, E) and (B, D, F) represent floating filters and liquid cultures, respectively. N. viennensis EN76 (A and B), “N. chungbukensis” MY2 (C and D), and N. europaea ATCC 19718 (E and F) grown on floating filters and liquid media, respectively, with varying inoculum size. The subfigure in the upper right panel shows the specific growth rate (μ_max) with different inoculum sizes. The symbols ^* and × indicate cultures for which growth rates cannot be calculated due to the absence of a log phase and no growth, respectively. All experiments were performed in triplicates. Data are presented as mean ± standard deviation (SD) (n = 3), and the error bars are hidden when they are smaller than the width of the symbols. To avoid overlapping symbols, the value was shifted by −0.03 and 0.02 in (A) for the experiment with 10⁶ cells and 10⁵ cells, respectively, and by −0.09, 0.06, or 0.02 in (C) for the 10⁸ cells, 10⁷ cells, and 10⁶ cells, respectively.

Figure 2

Fluorescent micrographs of N. viennensis EN76 microcolonies grown on floating filters. Micrographs of N. viennensis EN76 with an inoculum size of ~10⁷ cells on polycarbonate filter (A) before and (B) after 1 mM ammonia oxidation (refer to Fig. 1A) and (C) microcolonies of N. viennensis EN76 after oxidation of an additional 1 mM ammonia. (D) Microcolonies of N. viennensis EN76 with an inoculum size of ~10⁶ cells after 20 days of incubation (refer to Fig. 1A). The cells were stained with DAPI for 10 min and dried at 37°C on a glass slide.

Figure 3

Ammonia oxidation activities of N. viennensis EN76 and “N. chungbukensis” MY2 cells under different growth conditions. N. viennensis EN76 (A) and “N. chungbukensis” MY2 (B) with inoculum sizes of ~10⁶ cells and ~ 10⁷ cells, respectively, were used for the experiments. The subfigure in the upper right panel shows the specific growth rate (μ_max) under different culture conditions: 1 = filter without CaCO₃; 2 = filter with CaCO₃; 3 = liquid media without CaCO₃; 4 = inverted filter without CaCO₃. The symbols ^* and × indicate cultures for which growth rates cannot be calculated due to the absence of a log phase and no growth, respectively. All experiments were performed in triplicates. Data are presented as mean ± SD (n = 3), and the error bars are hidden when they are smaller than the width of the symbols. To avoid overlapping symbols, the value was shifted by −0.02 in (A) for the filter without CaCO₃ and by −0.09, 0.05, or 0.01 in (B) for the filter without CaCO₃, filter with CaCO₃, and inverted filter culture, respectively.

Figure 4

Effect of H₂O₂ scavengers on ammonia oxidation activity of N. viennensis EN76 cells grown on floating filters. Growth of N. viennensis EN76 cells with an inoculum size of (A) ~10⁷ cells and (B) ~10⁶ cells on floating filters provided with different concentrations of pyruvate and catalase (10 U ml⁻¹), as compared to the standard pyruvate concentration used in liquid media. The subfigure in the upper right panel shows the specific growth rate (μ_max) under different culture conditions: 1 = 0 mM pyruvate; 2 = 0.1 mM pyruvate; 3 = 1 mM pyruvate; 4 = catalase (10 U ml⁻¹); 5 = 0.1 mM pyruvate (liquid media). The symbols ^* indicate cultures for which growth rates cannot be calculated due to the absence of a log phase. All experiments were performed in triplicates. Data are presented as mean ± SD (n = 3), and the error bars are hidden when they are smaller than the width of the symbols.

Figure 5

Cultivation of AOA from agricultural soil using the floating filter and liquid media cultivation technique. Experiments were conducted with the addition of 50 μM of ATU to inhibit the growth of AOB and comammox. Accumulation of NO₂⁻ + NO₃⁻ indicates ammonia oxidation activity in the cultures. The line graphs (A and B) represent the ammonia oxidation activities of two biological replicates of floating filters and liquid cultures, respectively, during four successive culture transfers. A NMDS analysis of the overall nitrifiers’ ASVs enriched on floating filters and liquid media is shown in (C). The letters and numbers indicate the following: L = liquid culture, F = floating filter culture, a & b = two biological replicates, 1 = first culture, 2 = 2nd culture, 3 = 3rd culture, 4 = 4th culture, and 5 = 5th culture.

Figure 6

A phylogenetic tree of AOA ASVs based on 16S rRNA gene sequences. Representative 16S rRNA sequences of AOA were selected from the NCBI databases. A maximum likelihood tree (A) was inferred with IQ-TREE (IQ-TREE options: -B 1000 -m MFP) using aligned sequences. Bootstrap values ≥70% based on 1000 replications are indicated. The scale bar represents a 0.1 change per nucleotide position. ASVs obtained in this work are indicated. Charts (B, C) and (D, E) represent the composition of nitrifiers of two biological replicates of floating filters and liquid cultures, respectively. The ASV_11 is affiliated with the genus Nitrospira. Details of all nitrifiers’ ASVs taxonomy are provided in Supplementary Table S14.