Changes in denitrifier abundance, denitrification gene mRNA levels, nitrous oxide emissions, and denitrification in anoxic soil microcosms amended with glucose and plant residues

Abstract

In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification. The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene mRNA levels, nitrous oxide (N(2)O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast, the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirS(P)) increased only in glucose-amended soil: the nirS(P) guild abundance increased 5-fold over the 72-h incubation period (P < 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P < 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen (C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 x 10(7) gene copies and 6.9 x 10(7) transcripts g(-1) dry soil. Cumulative N(2)O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirS(P) and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic C and different C:N ratios.

FIG. 1.

Soil concentrations of NO₃⁻ (A), NO₂⁻ (B), NH₄⁺ (C), and EOC (D). Treatments consisted of 0 mg C kg⁻¹ dry soil (C0), 1,000 mg glucose-C kg⁻¹ dry soil (G1000), 1,000 mg red clover-C kg⁻¹dry soil (RC1000), 1,000 mg soybean-C kg⁻¹ dry soil (S1000), and 1,000 mg barley straw-C kg⁻¹ dry soil (B1000). Values are means (n = 6). Error bars are ±1 standard error. Significant differences among mean values are represented in two ways based on Tukey's test (P < 0.05): (i) differences among treatment means for individual time points are represented by letters adjacent to the time points but only for sampling dates in which significant differences among treatments means exist, and (ii) differences among treatment means averaged across time points are shown by letters in the figure legend.

FIG. 2.

Total denitrification (N₂O plus N₂) (A), cumulative N₂O emissions (B), and respiration (C). Treatment mixtures were incubated with C₂H₂ to measure cumulative denitrification or without C₂H₂ to measure cumulative N₂O emissions. Respiration was calculated from the average between soils incubated with and those incubated without C₂H₂. Treatments consisted of 0 mg C kg⁻¹ dry soil, 1,000 mg glucose-C kg⁻¹ dry soil, 1,000 mg red clover-C kg⁻¹ dry soil, 1,000 mg soybean-C kg⁻¹ dry soil, and 1,000 mg barley-C kg⁻¹ dry soil. Values are means (n = 6). Error bars are ±1 standard error. Significant differences among mean values are represented in two ways based on Tukey's test (P < 0.05): (i) differences among treatment means for individual time points are represented by letters adjacent to the time points but only for sampling dates in which significant differences among treatments means exist, and (ii) differences among treatment means averaged across time points are shown by letters in the figure legend.

FIG. 3.

Quantification of nosZ gene numbers (A) and nosZ transcripts (B) using qPCR and RT-qPCR, respectively. Treatments consisted of 0 mg C kg⁻¹ dry soil (C0), 1,000 mg glucose-C kg⁻¹ dry soil (G1000), 1,000 mg red clover-C kg⁻¹ dry soil (RC1000), 1,000 mg soybean-C kg⁻¹ dry soil (S1000), and 1,000 mg barley straw-C kg⁻¹ dry soil (B1000). Values are means (n = 6). Error bars are ±1 standard error. Standard curve descriptors and detection levels are as follows: for nosZ gene numbers, y = −3.33x + 39.14, R² = 0.999, E = 99.9%, and NTC = undetected; for nosZ transcripts, y = −3.27x + 38.41, R² = 0.999, E = 102%, and NTC = undetected. Significant differences among mean values are represented in two ways based on Tukey's test (P < 0.05): (i) differences among treatment means for individual time points are represented by letters adjacent to the time points but only for sampling dates in which significant differences among treatments means exist, and (ii) differences among treatment means averaged across time points are shown by letters in the figure legend.

FIG. 4.

Quantification of nirS_P gene numbers (A) and nirS_P transcripts (B) using qPCR and RT-qPCR, respectively. Treatments consisted of 0 mg C kg⁻¹ dry soil (C0), 1,000 mg glucose-C kg⁻¹ dry soil (G1000), 1,000 mg red clover-C kg⁻¹ dry soil (RC1000), 1,000 mg soybean-C kg⁻¹ dry soil (S1000), and 1,000 mg barley straw-C kg⁻¹ dry soil (B1000). Values are means (n = 6). Error bars are ±1 standard error. Standard curve descriptors and detection levels are as follows: for nirS_P gene numbers, y = −3.50x + 36.01, R² = 0.999, E = 93.4%, and NTC = undetected; for nirS_P transcripts, y = −3.45x + 37.28, R² = 0.993, E = 95.7%, and NTC = undetected. Significant differences among mean values are represented in two ways based on Tukey's test (P < 0.05): (i) differences among treatment means for individual time points are represented by letters adjacent to the time points but only for sampling dates in which significant differences among treatments means exist, and (ii) differences among treatment means averaged across time points are shown by letters in the figure legend.

FIG. 5.

Relationship between denitrification and respiration over the 72-h incubation period. Treatments consisted of 0 mg C kg⁻¹ dry soil (C0), 1,000 mg glucose-C kg⁻¹ dry soil (G1000), 1,000 mg red clover-C kg⁻¹ dry soil (RC1000), 1,000 mg soybean-C kg⁻¹ dry soil (S1000), and 1,000 mg barley straw-C kg⁻¹ dry soil (B1000). The line of best fit indicates the linear relationship described by the following equation: y = 0.342x − 2.27 (R² = 0.90).