Grazing weakens competitive interactions between active methanotrophs and nitrifiers modulating greenhouse-gas emissions in grassland soils

Abstract

Grassland soils serve as a biological sink and source of the potent greenhouse gases (GHG) methane (CH₄) and nitrous oxide (N₂O). The underlying mechanisms responsible for those GHG emissions, specifically, the relationships between methane- and ammonia-oxidizing microorganisms in grazed grassland soils are still poorly understood. Here, we characterized the effects of grazing on in situ GHG emissions and elucidated the putative relations between the active microbes involving in methane oxidation and nitrification activity in grassland soils. Grazing significantly decreases CH₄ uptake while it increases N₂O emissions basing on 14-month in situ measurement. DNA-based stable isotope probing (SIP) incubation experiment shows that grazing decreases both methane oxidation and nitrification processes and decreases the diversity of active methanotrophs and nitrifiers, and subsequently weakens the putative competition between active methanotrophs and nitrifiers in grassland soils. These results constitute a major advance in our understanding of putative relationships between methane- and ammonia-oxidizing microorganisms and subsequent effects on nitrification and methane oxidation, which contribute to a better prediction and modeling of future balance of GHG emissions and active microbial communities in grazed grassland ecosystems.

Fig. 1. Total N₂O and CH₄ emissions from August 2014 to October 2015 in ungrazed and grazed soils.

The vertical bars indicate the standard error of the mean (S.E.M.).

Fig. 2. Interactions between methane and ammonia oxidation in grassland soils.

Methane oxidation (a) and nitrification (b) after incubation for 21 days in the ungrazed and grazed soils at weekly urea addition of 0 (U0), 20 (U20), and 100 μg N g^-1 (U100) in presence or absence of methane addition (1% CH₄). Methane oxidation was calculated by methane consumption in the microcosms in presence or absence of urea addition, while nitrification was assessed by nitrate production. The error bars represent standard errors of three replicates for the U0 + CH₄ treatments, and six replicates for the U20 and U100 treatments (¹²C + ¹³C-labeled treatments). Different lower-case letters indicate significant differences among different treatments. Asterisks indicate significant difference between ungrazing and grazing under the same treatment. d.w.s. refers to dry weight of soil.

Fig. 3. Changes of abundance of methylotrophs and AOB amoA genes in soil microcosms over a 21-days incubation in ungrazed and grazed soils following weekly urea addition of 0 (U0), 20 (U20), and 100 μg N g⁻¹ (U100) in presence or absence of methane addition (1% CH₄).

The pmoA gene abundance (a), the relative abundance of targeted 16S rRNA genes affiliating with methane-oxidizing bacteria (b), the amoA gene copy numbers in total DNA (c), relative frequencies of targeted 16S rRNA genes affiliating with ammonia-oxidizing bacteria in total reads (%) (d) in the ungrazed and grazed soils after 21-day incubation were represented. The error bars represent standard errors of three replicates for the U0 + CH₄ treatments, and 6 replicates for the U20 and U100 treatments (¹²C + ¹³C-labeled treatments) (a–c). The error bars represent standard errors of six replicates for the U20 and U100 treatments (¹²C + ¹³C-labeled treatments) (d, e). Different lower-case letters indicate significant differences among different treatments. Asterisks indicate significant difference between ungrazing and grazing under the same urea treatment. d.w.s. refers to dry weight of soil.

Fig. 4. Distribution of pmoA and amoA gene copy numbers in fractionated DNA across the buoyant density.

Quantitative distribution of pmoA genes (a-c, f-h), and bacterial amoA genes (d, e, i, j) across the entire buoyant density gradient of the fractionated DNA from the ungrazed (a-e) and grazed (f–j) soils at weekly urea addition of 0 (U0), 20 (U20), and 100 μg N g^-1 (U100), in presence or absence of methane addition (1% CH₄) after incubation for 21 days. The normalized data are shown as the ratio of the gene copy number in each fraction to the maximum quantities in each treatment. The dotted and plain lines represent samples in ungrazed and grazed soils, respectively.

Fig. 5. Alpha-diversity indices of active methanotrophs and nitrifiers in grassland soils.

Alpha-diversity measurements of Shannon index (a, c, e) and Simpson index (b, d, f) of active MOB (a, b), AOB (c, d) and NOB (e, f) in ungrazed and grazed soils. Different letters indicate significant differences (P < 0.05) based on the analysis of variance.

Fig. 6. Phylogenetic tree of the ¹³C-labeled 16S rRNA genes affiliated with MOB and AOB from the labeled microcosms after incubation for 21 days.

Phylogenetic analysis of the 16S rRNA genes affiliated with MOB (a, b) and AOB (c, d) in ¹³C-labeled DNA. The designation “HF” indicates the ¹³C-DNA in the active fraction after the ultracentrifugation of the total DNA extract from the labeled microcosms. The designation “U0 + CH₄-HF-OTU-1-13,312-31.8%” indicates that OTU-1 contains 13,312 reads with > 97% sequence similarity, accounting for 31.8% of the total MOB 16S rRNA gene reads in the ¹³C-DNA from the CH₄-treated soil microcosms. The scale bars represent 1% nucleic acid sequences divergence for the 16SrRNA genes in ungrazed (a) and grazed (b) soils, respectively. The designation “HF” indicates the ¹³C-DNA in the active fraction after the ultracentrifugation of the total DNA extract from the labeled microcosms. The designation “U20 + CO₂-HF-OTU-1-1,087-67.0%” indicates that OTU-1 contains 1087 reads with > 97% sequence similarity, accounting for 67.0% of the total bacterial AOB 16S rRNA gene reads in the ¹³C-DNA from the U20-treated soil microcosms. The designation “U20 + CO₂ + CH₄-HF-OTU-1-1,096-65.5%” indicates that OTU-1 contains 1096 reads with > 97% sequence similarity, accounting for 65.5% of the total bacterial AOB 16S rRNA gene reads in the ¹³C-DNA from the U20 + CO₂ + CH₄-treated soil microcosms. The scale bars represent 2% nucleic acid sequences divergence for the 16SrRNA genes in ungrazed (c) and grazed (d) soils, respectively.

Fig. 7. The estimate absolute abundance (EEA) of active MOB and AOB in grassland soils.

Comparison of the EAA of the major active genera affiliated with MOB (a, b) and AOB (c, d) in the ungrazed (a, c) and grazed (b, d) soils after 21-day incubation with weekly urea addition of 0 (U0), 20 (U20), and 100 μg N g⁻¹ (U100), and 1% methane (CH₄).

Fig. 8. Network analysis of co-occurring active phylotypes of methanotrophs and nitrifiers in grassland soils.

The co-occurring network of active MOB, AOB and NOB after 21-day incubation in the ungrazed (a) and grazed (b) soils at weekly urea addition of 20 (low) and 100 μg N g⁻¹ (high) with and without 1% methane addition. Nodes represent the labeled OTUs of each of the three functional guilds, which were actively labeled with ¹³C. Isolated nodes were removed. Connecting lines (edges) correspond to significantly (P < 0.05) positive (red) or negative (green) correlations between nodes, where thicker lines represent stronger correlations. Labeled MOB: OTU 2 (Methylobacter); Labeled AOB: OTU 9 (Nitrosospira), OTU 466 (Nitrosococcus), OTU 703 (Nitrosococcus); Labeled NOB: OUT 5, OUT 12, OTU 55, OTU 75, OTU 126, OTU 133, OTU 191, OTU 622, OTU 816, OTU 919, OTU 2489, OTU 7401 (Nitrospira). The size of each node is proportional to the number of connections (i.e., degree), and the thickness of each connection between two nodes (i.e., edge) is proportional to the value of correlation coefficients. Green edges indicate positive relationships between two individual nodes, while red edges indicate negative relationships.