Long term combined fertilization and soil aggregate size on the denitrification and community of denitrifiers

Highlights

• Higher PDA was detected in combined fertilization than inorganic fertilization.

• PDA and denitrifying gene abundances were significantly increased in NPKS, particularly in its microaggregates.

• nirS- and nirK-type denitrifiers influenced by soil properties were responsible for the promoted denitrification.

• Aggregate size significantly influenced community structure of nirK-type denitrifiers

Abstract

Balanced fertilization strategies that combine the use of organic and inorganic fertilizers have been adopted to increase soil fertility and crop yield and to safeguard environment. However, knowledge on the influence of these fertilization strategies on microbe-mediated nitrogen cycling in the agricultural ecosystems remains limited. We analyzed the denitrifiers with nitrite reductase genes (nirK and nirS) in three soil aggregate classes (large macroaggregates, >2 mm; small macroaggregates, 0.25–2 mm; and microaggregates, <0.25 mm) in response to long-term application of inorganic fertilizers (NPK), NPK plus straw (NPKS), and NPK plus manure (NPKM) in the fluvo-aquic soil of North China Plain. Both fertilization and aggregate size had significant effects on the nirK and nirS gene abundances and soil potential denitrification activity (PDA). PDA in the combined fertilizer-treated soils (NPKS and NPKM treatments) was two times more than that in inorganic NPK-treated soil. PDA dramatically increased in small macroaggregates and microaggregates of NPKS treatment. The nirS gene abundance was significantly higher than that of the nirK gene abundance, and NPKS and NPKM treatments increased the nirS to nirK ratio in bulk soils and in microaggregates compared with NPK treatment and control. Correlation analysis and automatic linear modeling revealed a primary correlation between PDA and abundance of nirS-type denitrifiers. Soil properties, such as SOC, pH and TN, significantly affected the community structure of nirK- and nirS-type denitrifiers while only aggregate size significantly influenced the composition of nirK-type denitrifiers. Taken together, our results suggest that long-term combined fertilization increased the denitrification potential in small macroaggregate and microaggregate classes, and the increased gene abundance and the shift community structures of nirS- and nirK-type denitrifiers were potentially responsible for an increase in the promoted denitrification.

Introduction

Fertilization processes are indispensable management tools for enhancing soil nutrition and crop yields in a majority of agricultural ecosystems, and are known to influence the microbial regulated nitrogen (N) cycling processes (Hallin et al., 2009). However, the intensive use of fertilizers increase soil denitrification, and nitrate is reduced to nitrogen gas, which potentially generates greenhouse gases, such as nitrous oxide (N₂O) (Syakila and Kroeze, 2011; Akiyama et al., 2013). Approximately 60% of the global N₂O emissions originate from agricultural soils (IPCC, 2014).

N₂O is an atmospheric trace gas with relatively long lifetime. Increase in concentration of N₂O contributes to global warming and damages the ozone layer. In recent years, the excessive use of mineral nitrogen (N) fertilizers has increased N₂O threat (Frink et al., 1999; Liu et al., 2013). Therefore, practices with reduced fertilizers and energy inputs have recommended globally (Mäder et al., 2002). The Chinese government has recommended inorganic fertilizer reduction management practices for environment protection particularly in North China Plain, which is a major grain producing region with intensive double-crop wheat/maize rotation where the use and application of chemical N is high (Guo et al., 2010).

Several studies have demonstrated that reduced quantity of inorganic N fertilizers efficiently reduced denitrification and N₂O emission (Ding et al., 2007; Wang et al., 2009). However, long-term reduced inorganic N may reduce soil fertility and grain production in North China Plain, where calcareous fluvo-aquic soil is predominant. Application of organic manure in combination with reduced quantity of chemical fertilizers is considered effective to maintain crop yield (Zhao et al., 2016; Zhang et al., 2016). When reduced inorganic N combined organic fertilizers, an increase in N₂O emissions was detected. Efforts have been made to reveal the mechanism and their regulatory microorganisms (Jäger et al., 2011; Cui et al., 2016). However, knowledge on microbial denitrification under long-term combined use of organic and reduced inorganic fertilizers is far from complete and is yet to account for biological variation among soil types and the diverse range of farming systems.

Nitrite (NO₂⁻) reduction to nitric oxide (NO) is a key step in denitrification. This conversion is catalyzed by nitrite reductases of denitrifying bacteria. Nitrite reductases are of two types: copper-containing reductase (nirK) and cytochrome cd1-containing reductase (nirS) (Zumft, 1997). Historically, nirK and nirS genes have been widely utilized as molecular markers to investigate the denitrifiers of different terrestrial ecosystems (Hallin and Lindgren, 1999; Chen et al., 2010; Wei et al., 2015) and under different fertilizer sources or types (Brenzinger et al., 2018; Dai et al., 2019). Some researchers have demonstrated that nirS denitrifiers were susceptible to organic fertilizers particularly in upland soils (Tang et al., 2010; Yin et al., 2015; Cui et al., 2016). In contrast, Chen et al. (2010) reported that nirK denitrifiers were responsive to organic fertilization in a paddy soil. Furthermore, nirK gene outnumbered nirS gene in paddy ecosystems (Yoshida et al., 2010; Yuan et al., 2012; Azziz et al., 2017).

Abundance and community structure of denitrifiers depend on soil type (Cavigelli and Robertson, 2000). Studies on nirK- and nirS-denitrifying bacteria under a broad range of soils have implied that the two denitrifiers occupy different ecological niches (Priemé et al., 2002; Braker et al., 2015). Soil fertility management may change the soil environment, which influences the structural and functional differentiation of the microbial community. The two types of denitrifiers and their contribution to potential denitrification may vary depending on long-term combined use of organic and inorganic fertilizers.

Fertilizer management practices can affect soil aggregate distribution. Aggregates are important soil components that provide spatially heterogeneous microclimatic conditions, including water, air, and nutrition, for soil microbes (Mäder et al., 2002; Bach and Hofmockel, 2014). Soil aggregate distribution is directly influenced by fertilization, while soil aggregate size can modify the impacts of fertilization on microbial communities (F.Q. Li et al., 2019). Studies have proven differences in N₂O production in heterogeneous soil aggregate size classes under different fertilization conditions (Uchida et al., 2008; Diba et al., 2011). However, the response of these denitrifying populations to changes in different aggregates size at the microlevel has rarely been investigated.

Here, we investigate the effects of long-term straw or manure application paired with reduced quantity of chemical fertilizer and soil aggregates of different size classes on denitrification and N₂O emission in calcareous fluvo-aquic soil. The abundance and community structures of the denitrifiers within soil aggregates under different fertilizer management practices were analyzed for the following reasons: (1) to understand the effects of straw and manure application on denitrifiers in the aggregates and the possible mechanisms via which denitrification is enhanced or inhibited; (2) to identify the soil physicochemical factors that determine the distribution of nirK and nirS-type denitrifiers in soil aggregates and their correlation with denitrification under different fertilizer management practices; and (3) to compare the aggregate size effects on the abundance, diversity, and community composition of the nirK and nirS-type denitrifiers.