Studies have reported increases of 25 % in wet atmospheric nitrogen (N) deposition over the past decade (Jia et al., 2014), which has resulted in a range of problems in forest ecosystems, such as induced soil acidification, aggravation of cation and nitrate leaching, and decreased microbial biomass (Liu et al., 2011; Huang et al., 2014; Gao et al., 2015; Liu et al., 2013). While wet atmospheric N deposition is mostly comprised of ammonium, nitrate deposition has increased over recent years, so that the ratio of ammonium to nitrate has decreased from 5 to 2 (Liu et al., 2013). It is therefore important to study the individual influences of these two forms of N on soil microorganisms to support improved predictions of C, N, and P cycling under increased nitrate deposition.

Soil microorganisms supply nutrients to forests by producing enzymes that catalyze the degradation of soil organic matter, and they drive carbon (C), nitrogen (N), and phosphorus (P) cycling, with consequences for forest productivity and sustainability (Heijden et al., 2008). The soil microbial biomass of different communities may be quantified by phospholipid fatty acid (PLFA) biomarkers. Even though the PLFA signature method is not as advanced as genomic technology, it has been used extensively with good results to analyze the biomass and structures of microbial communities (Frostegård et al., 2011). Bacteria, including Gram-positive (G+) and negative (G-) bacteria, generally degrade labile compounds by excreting hydrolase, while fungi, including arbuscular mycorrhizal fungi (AMF) and saprophytes (SAP), are responsible for degrading recalcitrant compounds by secreting oxidase (Burns et al., 2013; Sinsabaugh et al., 2010; Willers et al., 2015).

To date, most studies have considered the influence of organic N on microbial communities (Guo et al., 2010; Hobbie et al., 2012) and few studies have reported how ammonium and nitrate individually influence microbial communities in forest soils. Positively charged ammonium is more easily absorbed by negatively charged soil colloids than nitrate, meaning that ammonium is more available to microorganisms than nitrate. In our previous study, we showed that ammonium promoted the activities of β-1,4-glucosidase (βG) and β-1,4-N-acetylglucosaminidase (NAG) in soil aggregates were strongly than nitrate (Yan et al., 2017). However, the process of nitrification, i.e., where ammonium is rapidly transformed to nitrate when it enters soil, may sterilize microorganisms in the soil (Dail et al., 2001). Ammonium and nitrate have different effects on the microbial decomposition rate and microbial respiration of soil organic matter. For example, substrate respiration in peatlands increased when ammonium was added, but did not change when nitrate was added (Currey et al., 2010). Nitrate additions strongly promoted the decomposition rates of soil organic matter of fir plantations in the early incubation phase (0–15 days; Zhang et al., 2012). However, from a laboratory incubation experiment, Ramirez et al. (2010) showed that nitrate and ammonium had similar inhibitory effects on soil microbial respiration.

It is well known that microorganisms and enzymes are sensitive to soil pH. Tian and Niu (2015), from their meta-analysis of soil acidification caused by N additions, suggested that ammonium nitrate (NH4NO3) contributed more to soil acidification than ammonium. Further, most studies have not separated the individual effects of additions of different nitrogen forms on PLFAs and microbial biomass carbon (MBC) in forest ecosystems. From their meta-analysis, Treseder et al. (2008) reported that N additions caused MBC to decrease by 15 %, and that fungi were more sensitive to N additions than other microbial communities. The responses of microbial biomass to N additions may be influenced by a wide range of factors, including forest type and geographical location. For example, in temperate regions, the total PLFA contents decreased in American beech (Fagus grandifolia Ehrh) and yellow birch (Betula alleghaniensis Britton), but increased in eastern hemlock (Tsuga Canadensis (L.) Carr) and red oak (Quercus rubra (L.) Britton) forests when NH4NO3 was added, with variable responses from bacteria and fungi (Weand et al., 2010). In subtropical forests, NH4NO3 additions resulted in an increase in total PLFA contents in a Chinese fir forest (Dong et al., 2015), a decrease in soil MBC contents in an evergreen broadleaved forests, but no change in the pine broadleaved mixed forest (Wang et al., 2008).

Soil enzymes catalyze the decomposition of soil organic matter (Burns et al., 2013). Enzymes involved in labile C breakdown that can decompose starch, cellulose, and hemicellulose include α-1,4-glucosidase (αG), cellobiohydrolase (CBH), β-1,4-xylosidase (βX), and βG. NAG, a nitrogen-degradation enzyme, can decompose oligosaccharides. Acid phosphatase (AP), a phosphorus-degradation enzyme, can decompose chitin lipophosphoglycan (Stone et al., 2014). Recalcitrant C-degradation enzymes that can decompose lignin, and aromatic and phenolic compounds including peroxidase and phenol oxidase (Sinsabaugh et al., 2010). When added to peatland, Currey et al. (2010) found that ammonium and nitrate had different effects on carbon- and phosphorus-enzyme activities (CBH and AP) but had similar effects on polyphenol oxidase (PPO) activities, while Tian et al. (2014) found that the effects of ammonium and nitrate were not significantly different when added to an alpine meadow. To date, few studies have reported how ammonium and nitrate additions individually influence soil enzyme activities in forest ecosystems.

Microorganisms will allocate energy to the relatively absent resources so that N additions will cause C- and P-acquisition enzymes to increase, and N-acquisition enzymes to decrease (Burns et al., 2013). It has been reported that, when inorganic N forms were not considered, N additions caused C-degradation enzymes (αG, βG, CBH, and βX) and P-degradation enzymes (AP) to increase and restricted oxidase (PPO and PER), but they did not inhibit N-degradation enzymes (NAG) (Jian et al., 2016; Marklein and Houlton, 2012), which suggests that the allocation of enzyme activities does not always correspond exactly with the economic theory.

The responses of enzyme activities to N additions are influenced by a range of factors including environmental conditions, plant types, and N background values. For example, in temperate regions, the soil activities of βG, CBH, NAG, and PPO increased in a dogwood forest, decreased in an oak forest, and did not change in a maple forest when NH4NO3 was added (Sinsabaugh et al., 2002). The AP activities increased in dogwood and maple forests, but were invariant in an oak forest after NH4NO3 additions (Sinsabaugh et al., 2002). However, in acidified temperate regions, the soil βG activities increased in a maple forest, but the soil βG, NAG, and AP activities did not change in yellow birch, oak, hemlock, and beech forests when NH4NO3 was added (Weand et al., 2010). In subtropical and tropical forests, the βG, NAG, and AP activities increased, and oxidase (PPO and PER) activities decreased after NH4NO3 additions (Dong et al., 2015; Guo et al., 2011; Cusack et al., 2011). To date, we are still not sure whether ammonium and nitrate additions have different effects on the soil microbial biomass of different communities and on enzyme activities. To support improved predictions of the effects of elevated N deposition on C, N, and P cycling in soil, we therefore need to evaluate the individual effects of ammonium and nitrate additions on the soil microbial biomass of different communities and enzyme activities.

The N-rich subtropical soils in southern China have experienced increased nitrate deposition in the recent past. To facilitate an exploration of the different effects of ammonium and nitrate additions on soil microbial communities and enzyme activities, we established a long-term ammonium and nitrate trial in a slash pine (Pinus elliottii) plantation in a subtropical area. We hypothesized that (1) ammonium would have stronger inhibitory effects on total PLFA, fungi PLFA contents, and enzyme activities than nitrate because of its strong negative effect on soil pH; (2) that ammonium and nitrate additions would result in increased C- and P-hydrolase activities, and decreased N-hydrolase activities in line with the economic theory; and (3) that oxidase activities would be restricted due to their inhibitory effects on fungi.

Our results agree with our first hypothesis and show that the inhibition effects on soil PLFA contents of bacteria, fungi, and actinomycetes across the three sampling events or seasons were stronger when ammonium was added than when nitrate was added (Figs. 3b and 4a, b, Table 1). Results from RDA suggest that acidification because of the ammonium additions triggered the decrease in the microbial biomarkers-PLFA contents (Fig. 6b). Soil microbial biomass may be inhibited by resource availability and acidification (Sinsabaugh et al., 2014; Moorhead et al., 2006). However, C and N availability either increased or stayed the same over the three sampling events when ammonium and nitrate were added (Figs. 1b and 2a, b). Ammonium additions may aggravate nitrification in subtropical soils (Tang et al., 2016), and nitrification may be toxic to microorganisms (Dail et al., 2001), which may then lead to a decrease in the microbial PLFA contents.

The soil pH did not change when nitrate was added (Fig. 1a), which may explain why nitrate had weaker inhibition effects on PLFA biomarker contents than ammonium. Nitrate additions may inhibit the PLFA biomarker contents because of accelerated leaching of Ca2+ and Mg2+ (Qian et al., 2007), increases in the soil osmotic potential, and activation of Al3+ absorbed by soil colloids (Treseder et al., 2008). The PER activity was lower when ammonium and nitrate were added (Table 2), which may eventually result in polyphenol accumulation in soil. Accumulated polyphenol may be toxic to microorganisms (Sinsabaugh et al., 2010) and may have contributed to the decrease in the contents of the PLFA biomarkers. Moreover, the higher soil DOC concentrations observed in the nitrate-addition treatments (Fig. 2a) may be attributed to changes in the diversity of the composition of saprophytic bacteria (Freedman and Zak, 2014; Freedman et al., 2016).

In our study, the fungi / bacteria ratios were lower in the ammonium and nitrate treatments than in the control, which suggests that fungi were more sensitive to N additions than bacteria. In an earlier study, we found that the fine-root biomass decreased after N additions (Kou et al., 2015), which suggests that N might upset the symbiosis between AMF and plants, thereby restricting the AMF-PLFA contents.

Our study showed that the absolute activities of C, N, and P hydrolases and oxidase were inhibited by ammonium and nitrate in the three seasons (Table 2). This agrees with our second and third hypothesis, i.e., that N additions caused the absolute activities of the N-acquisition enzyme (NAG) to decrease, in line with the microbial economic theory, and that N additions reduced the absolute activities of the oxidase by decreasing the PLFA contents of fungi. However, we did not expect the C- or P-acquisition enzymes to decrease. As a main producer of soil enzymes, the microbial biomass would decrease in response to ammonium and nitrate additions, resulting in lower absolute enzyme activities in the treated plots than in untreated plots (Allison et al., 2005).

The ratios of C or P to N acquisition enzyme activities were higher in the ammonium and nitrate treatments than in the control plots, and the N-acquisition enzyme activities per unit of microbial biomass were lower in the ammonium and nitrate treatments than in the control (Fig. 5a), indicating that microorganisms secreted enzymes in line with the economic theory. Measured absolute enzyme activities were positively correlated with soil pH and ammonium contents, and negatively correlated with nitrate contents (Fig. 6a). The inhibitory effects of N on the soil absolute enzyme activities may be more closely related to abiotic factors, i.e., soil pH and nitrification, than biotic factors (Kivlin et al., 2016).

We also found that ammonium and nitrate additions inhibited AP activities (Table 2). However, P-acquisition enzyme activities per unit of microbial biomass increased in the ammonium treatments (Fig. 5b). Li et al. (2016) reported that N applications aggravated the P limitations on biomass production. In line with the microbial economic theory, when the P availability was low, the activities of P-acquisition enzymes were higher. The decreased AP activities that resulted from ammonium additions may be more strongly related to abiotic inhibition caused by the ammonium, such as acidification, aggravated nitrification, and leaching of cations and nitrate, than biotic inhibition.

The N treatments also varied significantly on a seasonal basis and there were interaction effects between N treatments and seasons on the contents of some PLFA biomarkers and enzyme activities (Table 2). Climate conditions, plant growth, the amount of litter returned, and plant–microorganism competitive relationship varied across the three seasons. The temperature ranged from 13.5 to 27.6 ∘C, and precipitation ranged from 88.2 to 176.6 mm, across the three seasons (Fig. S1), and did not limit the growth of microorganisms. The positive relationships between PLFA biomarker contents and soil moisture contents indicate that soil moisture had a strong influence on soil microbial community biomass. There may be interaction effects between plant growth, the mass and quality of litter, plant–microbe competition, and soil nutrient dynamics. For example, compared with the control plots, the soil DOC contents were lower, and soil nitrate contents stayed the same in June (the growing season) in the ammonium treatment, but the soil DOC and nitrate contents were higher in the ammonium and nitrate treatments in March and October (non-growing season, Fig. 2a). This indicates that there was stronger competition between plants and microbes for available C and N in June than in March and October, and that there were interaction effects between plants and microbes on soil C and N availability. This might explain the interaction effects between N additions and seasons on the activities of C and N-acquisition enzymes. The effects of interactions between N additions and season on the AMF PLFA contents, along with available C and N dynamics, may result from plant growth as plant–AMF symbiotic systems may be influenced by fine-root biomass.

The results showed that soil bacteria, fungi, and actinomycetes–PLFA biomarker contents decreased after ammonium and nitrate additions. Ammonium inhibited the biomass of different soil microbial communities except SAP more strongly than nitrate, perhaps because of acidification caused by ammonium. The microbial communities were dominated by G+ and bacteria after ammonium additions, and were dominated by bacteria under nitrate additions.

The absolute activities of C-, N-, and P-acquisition hydrolases and oxidase decreased after additions of ammonium and nitrate, and nitrate had a stronger inhibition effects on P-acquisition absolute enzyme activities than ammonium. However, ammonium improved the P demand per unit of microbial biomass. C- and P-acquisition absolute enzyme activities were higher than N-acquisition absolute enzyme activities under ammonium and nitrate additions. Because of the positive correlation between the measured absolute enzyme activities and soil pH, the decreases in the absolute hydrolase and oxidase activities reflected abiotic restrictions, i.e., acidification and nitrification caused by ammonium additions, rather than biotic restrictions.

Ammonium and nitrate additions had a range of effects on soil microbial communities and the activities of specific enzymes. Our results show that the effects of ammonium and nitrate need to be discussed separately to provide the information that we need to predict the effects of elevated N deposition on soil microbial biomass and enzyme activities.