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+/Gratio 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).