Fig. 4: Effects of humic substance (HS) addition on extracellular electron transfer (EET)-mediated denitrification.
From: Electron shuttling promotes denitrification and mitigates nitrous oxide emissions in lakes
a Impact of HS addition on maximum power density (MPD) across all groups and the low-MPD (L-MPD) group (two-tailed paired t-test). b Influence of HS addition on denitrification rates (two-tailed paired t-test) and N2O accumulation levels (two-tailed Wilcoxon matched-pairs signed rank test). c Nonlinear regression analysis (one-phase decay exponential model, GraphPad Prism v9.0.0) demonstrating the quantitative relationship between HS concentration and denitrification enhancement ratio (HS-C/Blank). d The ratio of relative expression levels of denitrifying genes, N2O reduction genes, and nos/nir, i.e., (nosZI+nosZII)/(nirS+nirK), in HS_C groups compared to Blank groups (HS_C/Blank). e The ratio of relative expression levels of EET genes (omcB, omcS, pilA) in HS_C groups compared to Blank groups (HS_C/Blank). f Log-transformed relative expression levels of denitrifying genes (nirS and nirK) and N2O reduction genes (nosZI, and nosZII). g Log-transformed relative expression levels of EET genes (omcB, omcS, and pilA). All gene expression levels were normalized against 16S rRNA levels. Statistical comparisons were performed using paired t-tests (two-tailed), with Benjamini-Hochberg false discovery rate (FDR) correction for multiple comparisons (7 total tests across f and g) to control FDR at 0.05. FDR-adjusted p values (q values) are labeled in the figure. Additional statistical information is provided in Supplementary Table S13. Relative expression levels of nirS were compared across all samples, while nirK, nosZI, nosZII, omcB, omcS, and pilA were analyzed specifically in the L-MPD subgroup. All groups contain 8 biological replicates, while the L-MPD group contains 3 biological replicates. Sediments without added HS are labeled as the Blank group, while sediments with added commercial HS are designated as the HS_C group. In violin plots, horizontal bars indicate medians, while dotted lines represent 25th and 75th percentiles. Statistical significance was determined at p < 0.05.
