Introduction
Ecosystems worldwide are receiving increasing amounts of reactive
nitrogen (N) through anthropogenic activities (Galloway et al. 2008;
Canfield et al. 2010). The most considerable change in the ecosystem’s N
cycle caused by N enrichment is rapid nitrification, defined as the
ammonium conversion via nitrite to nitrate by soil microorganisms.
Nitrification causes much of the reactive N to be lost in the
environment by nitrate leaching and gaseous nitrous oxide production,
leading to soil acidification (Liu et al. 2010; Coskun et al. 2017). In
the last decades, N deposition levels have begun to decline in some
parts of Europe (Sutton et al. 2011; Stevens, 2016). Since 2008, China’s
cleaner technologies and policy frameworks have reduced atmospheric
pollution and N fertilizer input (Liu et al. 2016). However, it is
difficult to assess whether the high nitrification environment caused by
long-term N enrichment can be reversed to a low nitrification
environment after reduction or cessation of nitrogen enrichment due to
the few studies examining the duration of the N-induced impacts on
nitrification after N input cessation (Clark et al. 2009).
Within the N cycle,
ammonia
oxidizers are functionally essential groups of microorganisms that
perform the first and rate-limiting nitrification steps.
Ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) are
considered classical ammonia oxidizers. Many studies in various
ecosystems have examined the increased exposure N inputs effects on AOA
and AOB (Xiao et al. 2020). It is well established that mineral
N-fertilizer application stimulates nitrifier activity and the
subsequent development of high-nitrifying soil environments (Di et al.
2009; Lu et al. 2012; Ouyang et al. 2016). For example, a recent
meta-analysis reported that N addition increased AOA and AOB abundances
by 27% and 326%, respectively (Carey et al. 2016).
Similarly, a comprehensive study
from agricultural soils showed that N fertilization significantly
increased archaeal amoA (31%), bacterial amoA (313%), nirK (53%),
nirS (40%), and nosZ (75%) genes (Ouyang et al. 2018). However, the
discovery of comammox (complete ammonia oxidation) Nitrospira ,
which can independently oxidize ammonia to nitrate challenged this
long-held perception (Daims et al. 2015; van Kessel et al. 2015;
Santoro, 2016). Comammox bacteria are differentiated into two clades (A
and B) based on phylogeny of the ammonia monooxygenase α-subunit gene
(amoA) (Daims et al., 2015; Pjevac et al. 2017). Recently, some studies
have indicated that N input significantly increases comammox abundances,
especially clade A (Li et al. 2019; Wang et al. 2019). Compared to a
good understanding of the response of ammonia oxidizers to nitrogen
addition, the reversibility of N-induced shifts in these groups are
largely unknown.
Soil ammonia availability has been proposed to be a primary driver
controlling the AOA and AOB abundances (Verhamme et al. 2011; Daebeler
et al. 2015). High N accumulation occurs in regions with high mineral
fertilizer application in the ecosystem. The amount of ammonia is
sufficient for the development of microbial communities involved in
nitrification, leading to nitrification becoming a highly significant
process during the N enrichment period. But soil ammonium and nitrate
concentrations were not significantly different from the control plots
in the Minnesota prairie after N cessation (Clark et al. 2009). Similar
results were observed at Tadham Moor (Stevens et al. 2012) and Wardlow
Hay Cop (O’Sullivan et al. 2011) where nutrient additions were
discontinued. These suggest soil nitrate and ammonium concentrations
typically showed recovery signs after the cessation of N additions. In
contrast, soil mineralization and nitrification show weak recovery
because the cessation of nutrient addition did not reduce nitrification
rates (Corre & Lamersdorf, 2004). Similarly, elevated
N2O emissions after seven years of N addition ceassation
were observed at a solling site (Borken et al. 2002). Higher net
nitrification rates combined with negligible amounts of dissolved
inorganic N (DIN) suggest that soil N retained in another form could
influence internal cycling many years after N inputs cessation. The lack
of recovery in soil processes is potentially related to changes in the
soil microbial community. Consequently, long-term impacts on ammonia
oxidizer communities are plausible.
Ammonia oxidizers played a key role in N cycling changes. The long-term
retention of added N and its impacts on ammonia oxidizers dictate the
irreversibility of N-induced changes. On the other hand, the microbial
community legacy effects may also contribute to durable changes in N
cycling due to N addition (Johnson, 1993; Bradley et al. 2006). We
hypothesized that both retention and ammonia oxidizers properties
influenced the reversibility of N-induced shifts in the ecosystem
function. Here, we performed a 15 years long nutrient addition
experiment (2000–2014) followed by a 7 years cessation of nutrient
inputs (2015–2021) in the alpine meadow ecosystem to examine the
effects of nutrient inputs on soil ammonia oxidizers. Our objective was
to compare the effects of nitrogen addition and cessation effects on
soil ammonia oxidation and nitrification and the underlying mechanisms
during these two periods.