DISCUSSION
In general, we found significantly negative effects of N addition on
total microbial biomass. Further analysis revealed that total microbial
biomass displayed different responses to various N types. As an
important component of amino acids,
NH4+-N is the main N source for soil
microorganisms (Guo et al. 2011; Du et al. 2014). Its
addition may significantly accelerate the metabolism of soil
microorganisms. In contrast to our hypothesis,
NH4+-N addition led to a rather
significant microbial biomass decline than other N types across all
ecosystems. This might be because the absorbed
NH4+-N would be replaced by
H+ in soils, which significantly decrease soil pH
(Hong et al. 2019) and eventually induces serious soil microbial
biomass decline. Of all N types,
NH4+-N addition induced the greatest
soil pH decline and displayed the most negative effect on microbial
biomass especially for
(NH4)2SO4 (-21.3 %,P < 0.01, Supplementary Online Material 8 ). In
addition, NH4+-N additions may
aggravate nitrification in soils. The nitrification process may be toxic
to microorganisms, and then lead to a decrease in the microbial biomass
(Zhang et al. 2017). NO3−-N
addition also significantly inhibited total microbial biomass but less
than NH4+-N. Firstly, the excessive
addition of NO3−-N increased the
activities of nitrate reductase and the accumulation of nitrite, which
might be harmful to soil microorganisms (Luan et al. 2019).
Secondly, as a type of anion, NO3−-N
is more mobile and subjected to leaching (Currey et al. 2010).
The direct negative effect of NO3−-N
on microorganisms may be less obvious than
NH4+-N. The significantly negative
relationship between microbial biomass decrease and MAP supports our
hypothesis (Supplementary Online Material 10) . Thirdly, the
ability of soil microorganisms to absorb
NO3−-N may be lower (Fu & Shen 2017).
Most soil microorganisms cannot immobilize
NO3−-N under N addition (Brenneret al. 2005). Finally, it induced less soil acidification
(Supplementary Online Material 8 ), which may reduce the
negative effect of NO3−-N on soil
microorganisms.
As the most used fertilizer in field experiment,
NH4NO3 addition significantly decreased
microbial biomass as well, but less than single
NH4+-N and
NO3−-N. The reason may be its less
acidification than single NH4+-N and
less nitrite accumulation than single
NO3−-N under the same dose. Based on
the results, we hypothesized that N addition with extremely massive
NH4+-N or
NO3−-N may destroy the original
balance of NH4+-N to
NO3−-N in microorganisms and finally
cause potential toxic effects on them. If
NH4NO3 was fertilized in the area with
extremely higher or lower background level of
NH4+-N/NO3−-N
ratio, the results might mislead the estimation of the effect of
elevated natural N deposition on the ecosystem.
CO(NH2)2 is a type of organic N and
commonly used as fertilizers in agro-ecosystems. It was also used in
some field experiments (Li et al. 2017; Zong et al. 2019).
In this work, CO(NH2)2 addition induced
the smallest microbial biomass decrease. Microbial biomass even
accelerated after CO(NH2)2 addition in
some reports (Thirukkumaran & Parkinson 2000; Guo et al. 2011;
Du et al. 2014). It might be because of the slowed soil
acidification, as NH3 was released when
CO(NH2)2 was hydrolyzed in soil (Guoet al. 2011), its less soil pH decrease compared with other N
types supported our analysis (Supplementary Online Material 8 ).
Previous work has shown that when other nutrients were added in addition
to N fertilizer, the effect sizes might change (Wang et al.2018a). Among all N types, actually, total microbial biomass only
increased after Ca(NO3)2 addition (+3.66
%). This may be attributable to the alleviation of
Ca2+ limitation (Treseder 2008), which microorganisms
could more tolerant to greater N. Additionally, the base cations
Ca2+ and K+ played critical roles in
buffering against N-induced soil acidification, especially at the early
stage (Tian & Niu 2015). Contrarily, total microbial biomass decreased
the most after (NH4)3PO4addition (-45.9 %, P < 0.001). However, the data were
extracted from only one observation, the result was less reliable.
Although KNO3, Ca(NO3)2,
(NH4)3PO4 or
(NH4)2SO4 were sometimes
used as fertilizer in some field experiment, the added
K+, Ca2+,
PO43− or
SO42− with N fertilizers may mask,
offset and even mislead the direct effects of N on soil microorganisms.
Thus, they were not recommended as N fertilizers in field experiment.
Contrarily, NH4Cl and NaNO3 were
suggested to be selected as NH4+-N and
NO3−-N, as Cl− and
Na+ was relative abundant in soil and microbial
metabolism was less limited by them.
Fungi and bacteria are the main constituents of soil microbial
community. The biomass of both fungi and bacteria significantly
decreased after N addition. Compared with fungal biomass, bacterial
biomass less decreased, which indicated greater sensitivity of fungi to
N addition (Zhang et al. 2017). In line with previous results,
NH4+-N addition induced significant
and dramatical decline in fungal and bacterial biomass compared with
other N types, which also meant its serious negative impact on soil
microorganisms. A higher fungi to bacteria (F/B) ratio indicates
stronger soil ecosystem buffering capacity, better sustainability of
soils and higher microbial metabolic efficiency (Chen et al.2018). The decreased F/B ratio after N addition in this work indicated
less sustainability of soil ecosystem. Furthermore, the ratio was
sensitive to N type. NH4+-N addition
led to significant and the largest F/B ratio decline than other N types,
especially for
(NH4)2SO4, which also
indicated its serious negative impact on the function and sustainability
of soils. Compared with NH4NO3,
CO(NH2)2 addition resulted in a more
significant F/B ratio decline, which meant soil microorganisms may be
more sensitive to CO(NH2)2 (Guo et
al. 2011; Du et al. 2014).
In soil microbial community, both actinomycete and saprophytic fungal
biomass were less affected by N addition. Actinomycete is thought to be
largely unaffected by soil pH (Rousk et al. 2010), the influence
of acidification caused by N addition might be less obvious. The
insignificant relationship between actinomycete biomass and soil ΔpH
supported our hypothesis (P = 0.439, Supplementary Online
Material 9 ). Soil saprophytic fungi are primarily C limited (Bonneret al. 2019), the direct effects of different N types on them
might be similar. Although no statistically significant differences were
shown among different N types for them,
NH4+-N addition resulted in the most
serious biomass decline as well. The biomass of AM fungi declined the
most in all types of microorganisms. When N was added, plant roots might
assimilate these available N compounds directly and might not have to
absorb them via mycorrhizal symbionts. It would finally lead to AM
fungal biomass decline (Kong et al. 2018). Interestingly, the
effect of CO(NH2)2 addition on AM fungal
biomass was similar to NH4+-N. When
CO(NH2)2 was fertilized, part of it will
be directly absorbed by hyphae and then broken down to
NH4+-N, while others will be
hydrolyzed to NH4+-N in soil and then
absorbed by hyphae (Govindarajulu et al. 2005). Thus,
CO(NH2)2 displayed a similar effect as
NH4+-N. Although
NO3−-N can also be taken up by AM
fungal hyphae, it will be converted to
NH4+-N (Hodge 2017). However, it is a
high-energy demand process (Wang et al. 2015) and more AM fungi
would be needed to translocate these massive
NO3−-N.
G+ and G− bacterial biomass were
less impacted by N addition. However, N addition induced a increased
G+/G− ratio, especially after
CO(NH2)2 addition. A possible
explanation is the reduced belowground C-allocation by the trees with
increasing N loading (Wang et al. 2015). Additionally,
G+ bacteria are more stress-tolerant than
G− bacteria (Wang et al. 2018b). In soils,
higher G+/G−ratio indicates greater
soil organic carbon accumulation (Zhang et al. 2015). The
greatest G+/G− ratio increase after
CO(NH2)2 addition not only indicated a
lower quality substrate but also an acclimation of microorganisms to
changes in substrate and nutrient availability. Although
NH4+-N addition induced significant
G+ and G− bacterial biomass and
G+/G− ratio decline, they were
derived from only one observation. We therefore do not have strong
evidence to conclude that NH4+-N
addition significantly affects G+ and
G− bacterial biomass.
The impacts of N addition on soil microbial biomass may also depend on
the ecological factors, such as MAT, MAP, ecosystem type, N addition
rate and application duration (Treseder 2008; Fu & Shen 2017; Wanget al. 2018a; You et al. 2018; Zhang et al. 2018;
Jia et al. 2020). Because of low background levels of N
deposition (Wardle et al. 2013), microorganisms of tundra
ecosystem is the most sensitive to N and finally exhibit the most severe
biomass decline when N was added. Compared with forest soils, microbial
biomass of grassland soils decreased greater, which indicated its more
sensitivity to N addition. It may be because of the higher C/N ratios
and long lifetime of C-cycle of forest soils (Townsend et al.1996). Similarly, microbial biomass of coniferous forest were more
sensitive to N deposition than that of broadleaved forest for all N
types. For all types of ecosystem,
NH4+-N induced to the greatest biomass
decline and CO(NH2)2 induced to the
smallest decline, which indicated the significant effects of different N
on microbial biomass were consistent and commonly widespread across all
ecosystem.
Although globally wide ranges of variations in MAT, the responses of
microbial biomass to N addition did not change spatially. However, field
experiment sites with higher precipitation may lead to less significant
soil microbial biomass decline, especially for
NO3−-N (Supplementary Online
Material 10 ). It may be due to its greater leaching than other N types.
Some reports revealed that the responses of ecosystem to N deposition
might be linear or nonlinear, such as linear (Gu et al. 2019) or
exponentially increasing N2O emissions (Shcherbaket al. 2014) and curve changing of soil respiration (Penget al. 2017) to N. Our analysis showed that the general trend of
soil microbial biomass to N addition is linear. In consistent to some
reports (Humbert et al. 2016), soil microbial biomass decline was
significantly correlated with the accumulated N amount (N addition rate
× application duration). It indicated that N addition with great rate
and short duration will lead to similar microbial biomass changes as low
amount for a long term, and the declining trends were consistent among
all ecotypes. Among these N types,
NH4+-N addition revealed the most
serious soil microbial biomass decline, which indicated its more serious
negative effect on soil microorganisms than other N types.
Actually, the deposited N from the atmosphere is mixed with different N
types and the percentages of NH4+-N,
NO3−-N and
CO(NH2)2 are significantly different in
the world scale. In this work, soil microbial biomass significantly
increased (+19.8 %) when mixed N
(NH4NO3+CO(NH2)2)
was fertilized. However, the data were extracted from only two articles.
Less data were not enough to support the conclusion that mixed N
addition significantly accelerated soil microbial biomass. Whether mixed
N fertilization will display significantly different effects on the soil
microorganisms from single N type should be further investigated. All
the results suggested that when we estimate the effects of N deposition
on ecosystem, the type of N fertilizer should be concerned, rather than
only focus on the total N deposition amount. In addition, the natural
deposited N components of sample site should also be concerned and
investigated before experiment.
ACKNOWLEDGEMENTS This work was supported by Foundation of
Talent Training Project of Hebei Province, China (A201901041) and
Natural Science Foundation of Hebei Province, China (C2016417004).