Impact of long- and short-term nutrient input on microbial nitrogen cycling communities
Authors:   , Mitsuta Akari 
Published: December 2024

Microorganisms are vital to the soil nitrogen (N) cycle, driving key processes such as N-fixation, nitrification, and denitrification. While these processes support plant productivity, they can also contribute to environmental issues such as N2O emissions. Over the last decades, fertilizer use has increased to meet food demands, but its long-term effects on microbial N-cycling functions remain poorly understood. This thesis investigates how chemical fertilization affects microbial N-cycling functions and explores the roles of soil pH and microbial diversity in these processes. In Chapters 2 and 3, I examined the gene abundances associated with N-cycling (amoA, nirS, nirK, nosZ, and nifH) using soil samples from the Ossekampen grassland, a long-term fertilization experiment in the Netherlands. This grassland has received consistent applications of fertilizers, including P (superphosphate), K (potassium sulfate), N (ammonium nitrate), PK, NPK, PK+N, and lime for 65 years. Soil sampling was conducted at intervals during the grass growing season. Chapter 2 revealed that soil pH is the dominant factor influencing gene abundances related to N-fixation and denitrification, outweighing the direct effects of fertilizer application. Long-term N fertilizer use reduced soil pH, leading to lower abundances of diazotrophs and denitrifiers. However, nitrifiers increased with N fertilizer use, regardless of soil acidification. Notably, N2O emissions were positively correlated with AOA abundance but not with denitrifiers. Our result suggest that fertilizer impacts on microbial N-cycling are mediated indirectly through soil pH changes. Chapter 3 explored the effect of liming on microbial N-cycling genes. Liming increased abundances of nitrifiers, denitrifiers, and diazotrophs without increasing N2O emissions. This was likely due to increased nosZ gene abundance, which encodes the N2O reductase enzyme. Liming also stabilized N-cycling microbial populations against seasonal moisture changes. These findings suggest that mitigating soil acidity through liming enhances microbial N-cycling gene abundances without increasing N2O emissions. In Chapter 4, an incubation experiment examined the effect of soil pH (adjusted to 4.5 or 6.0) and fertilizer treatments (urea, concentrated vinasse, or their combination) on microbial metabolic pathways. Shotgun metagenomics revealed that soil pH had a stronger effect on microbial metabolic pathways (N metabolism, sulfur metabolism, methane metabolism, and carbon fixation) than fertilizer type. High pH enhanced relative gene abundances linked to amino acid synthesis, while low pH increased genes associated with N2O emissions. Additionally, the application of urea at high soil pH led to an increase in genes for denitrification and a decrease in genes related to methanogens compared to the control treatment. These findings suggest an important role of soil pH in shaping microbial functions. Chapter 5 assessed the effect of microbial diversity on AOB and AOA communities under repeated N fertilizer application using a dilution-to-extinction approach. Greater microbial diversity enhanced AOB stability and abundance, while AOA stability was less affected, and its abundance declined with repeated fertilization. Overall, this thesis highlights soil pH as an important factor of microbial N-cycling functions, outweighing fertilizer effects. Additionally, it also sheds light on the role of microbial diversity in maintaining the stability of functional communities under repeated N fertilizer application.