Geoderma 329 (2018) 61–64
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Nitrogen addition aggravates microbial carbon limitation: Evidence from ecoenzymatic stoichiometry
T
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Hao Chen, Dejun Li , Jie Zhao, Wei Zhang, Kongcao Xiao, Kelin Wang Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, Hunan, China Huanjiang Observation and Research Station for Karst Ecosystems, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Huangjiang 547100, Guangxi, China
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Jan Willem Van Groenigen
Nitrogen (N) deposition may change carbon (C) or nutrient limitation of soil microbes, but whether such change can be reflected by ecoenzymatic stoichiometry has not been well studied. Here, we synthesized data from 36 published studies to evaluate the responses of ecoenzymatic stoichiometry to N addition. Results showed that N addition did not change the enzyme ratio (1:1:1) of C, N, and phosphorus (P) acquisition, and also did not change vector angle of ecoenzymatic stoichiometry which is an indicator of microbial P limitation. However, N addition increased two indicators of C-limitation significantly (i.e., activity of β-D-glucosidase and vector length), both of which indicated that N addition aggravated microbial C-limitation. The aggravated C-limitation may be partly due to the reduced decomposition of recalcitrant organic C. Our study extends our understanding of the effects of N deposition on C cycling from a new perspective.
Keywords: N deposition Ecoenzymatic stoichiometry C limitation Nutrient limitation
1. Introduction The growth and activity of soil microbes is often limited or colimited by resource (such as carbon (C), nitrogen (N) or phosphorus (P)) in terrestrial ecosystems. This is referred to microbial resources limitation (Ekblad and Nordgren, 2002; Hill et al., 2014). However, over the past few decades, the pattern of microbial resource limitation has been changing slowly due to elevated deposition of atmospheric N (Galloway et al., 2008). Given the N saturation hypothesis, elevated input of N may reduce the microbial requirements for additional N in all ecosystems, therefore, C or P limitation of soil microbes are expected to be motivated or aggravated (Aber et al., 1989; Gress et al., 2007; Kopáček et al., 2013). Due to the importance of soil microbes in soil biogeochemical cycles, understanding how microbial resource limitation changes after N limitation is removed will largely improve our prediction of future composition and dynamics of ecosystems in response to long-term N deposition. However, most of previous studies focused on the nutrient limitation of plants (Finzi, 2009; Peñuelas et al., 2015), how microbial resource limitation changes after N addition has long been neglected (Gress et al., 2007). The one of the reasons for the lack of studies may be due to the limitation of methods for quantifying microbial resource limitation. Traditional methods generally measure the effects of substrate addition on microbial biomass or respiration as a proxy for
microbial resource limitation (Stotzky and Norman, 1961). However, these methods are time-consuming and have lower accuracy of the results. Therefore, we almost did not find any studies that discuss the effect of N addition on microbial resource limitation using above methods (Gnankambary et al., 2008). Recently, ecoenzymatic stoichiometry has been suggested to be a useful indicator of the relative resource limitations of microbial assemblages, because soil extracellular enzyme activity (EEA) reflects the microbial cell's response to meet its metabolic resource demands (Sinsabaugh et al., 2009). This method generally needs measuring four extracellular enzymes, i.e., β-D-glucosidase (BG), L-leucine aminopeptidase (LAP), β-N-acetylglucosaminidase (NAG), and acid/alkaline phosphatase (AP), which are assumed to be proxy indicators of overall C, N, and P acquisition (Sinsabaugh, 1994) (Table 1). Within this context, Sinsabaugh et al. (2009) initiated that there exists a functional enzymatic ratio with 1:1:1 for the microbial C, N, and P acquisition. Sinsabaugh et al. (2008) suggested that the relative activities of BG/AP and BG/(NAG + LAP) reflect the relative demands in acquiring C vs. P and C vs. N, respectively. Moorhead et al. (2016) proposed that calculating the vector length and angle of ecoenzymatic stoichiometry can reflect relative C vs. nutrient limitation and relative P vs. N limitation of soil microbes. In this sense, studying the response of ecoenzymatic stoichiometry to N addition may provide a new perspective for understanding how
⁎ Corresponding author at: Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, Hunan, China. E-mail address:
[email protected] (D. Li).
https://doi.org/10.1016/j.geoderma.2018.05.019 Received 24 January 2018; Received in revised form 12 April 2018; Accepted 16 May 2018 0016-7061/ © 2018 Elsevier B.V. All rights reserved.
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2007). The regression slopes of EEAs were used to reflect the pattern of resource allocation by soil microbes (Sinsabaugh et al., 2009). The regression intercepts were used to reflect the microbial C, N or P limitation, basing on the relative extent of the changes in C, N, and P acquisition enzymes. In the current study, for example, the higher intercept for the C vs. N acquisition enzymes, which results from more increase in C-acquisition enzyme, suggests an aggravated C limitation. Resource limitation was measured using vector analysis (length, L; angle, A) of ecoenzymatic stoichiometry (Moorhead et al., 2013).
Table 1 Names, Abbr. (abbreviations), functions and groups of soil enzymes studied. Group
Function
Enzyme name
Abbr.
C acquisition
Hydrolysis of cellobioside Oxidation lignin Hydrolysis of protein Hydrolysis of chitin Hydrolysis of organic P
β-D-Glucosidase Polyphenol oxidase L-Leucine aminopeptidase β-N-Acetylglucosaminidase Acid phosphatase
BG POX LAP NAG AP
N acquisition P acquisition
Vector L =
microbial resource limitation changes in the context of global N deposition. Over the past few decades, a large numbers of studies have reported the effects of N addition on soil EEAs (Chen et al., 2016; Jian et al., 2016). These studies showed that the responses of C, N and P acquisition enzymes to N addition were different. For example, recent meta-analysis studies have reported that N addition increased activities of C-acquisition hydrolases (Chen et al., 2016), and the P-acquisition enzyme (Marklein and Houlton, 2012). By contrast, other meta-analysis studies showed that N addition have minor or negative effects on Nacquisition enzyme (Chen et al., 2018; Jian et al., 2016). Due to these differential responses, it is reasonable to believe that N addition may change ecoenzymatic stoichiometry and thus microbial resource limitation. To our knowledge, however, there are very few studies that have assessed the response of ecoenzymatic stoichiometry to N addition (Wang et al., 2015). In addition, no study synthesized the data of enzyme activity that was measured in previous N-addition experiments to show the general pattern for the response of ecoenzymatic stoichiometry to N addition. To fill this gap, we calculated ecoenzymatic stoichiometry in this study, by summarizing enzymatic data from published studies with N-addition experiments. Our results will extend our understanding regarding the effects of N deposition on biogeochemical cycle in terrestrial ecosystems.
(lnBG/ln[NAG + LAP ])2 + (lnBG/lnAP)2
(1)
Vector A = Degrees(ATAN 2((ln BG /lnAP), (lnBG/ln[NAG + LAP ]))) (2) Relatively longer vector L indicated greater C-limitation; the vector A < 45° and > 45° indicated relative degrees of N- and P-limitation, respectively. To compare whether the regression slopes and intercepts of EEAs were different between control samples and those with N-addition, we used an analysis of covariance. To compare the differences in other variables (including vector A and L, fungal and bacterial biomass, fungi/bacteria, enzyme activities and their ratios) between control and N-addition, we performed a paired-samples t-test. 3. Results Regression slopes for the Ln(BG) vs. Ln (NAG + LAP) and Ln (BG) vs. Ln (AP) were 1.02 and 0.96, respectively, across the controls (Fig. 1a and Fig. 1b), and were 1.00 and 0.96, respectively, across the N-addition treatment (Fig. 1c and Fig. 1d). Analysis of covariance showed that the ratios had no significant difference between control and N-addition treatment (P > 0.01). There was also no significant difference between the control and N-addition samples in the regression intercepts for N and P enzyme activity (Fig. 1b and d). However, the regression intercepts for the C and N enzyme activity increased significantly from −8.4 in the control to −3.4 in the samples with added N (Fig. 1a and Fig. 1c, P < 0.05). Nitrogen addition significantly increased the Ln (BG), Ln (AP), Ln BG: Ln (NAG + LAP), and Ln (bacterial biomass), and significantly decreased Ln (POX), Ln (fungal biomass), and Fungi/Bacteria, but did not change Ln (NAG + LAP), Ln BG: Ln AP, and Ln (NAG + LAP): Ln AP (Table 2). The mean vector A was greater than 45° across all observations in controls. Nitrogen addition significantly increased vector L, but did not change vector A (Table 2).
2. Material and methods 2.1. Data compilation Peer-reviewed publications (1990–2016) that reported the responses of soil EEAs to N addition were selected by searching Web of Science and Google Scholar. The following criteria were applied to select studies: (1) studies that measured simultaneously the enzyme activities of BG, LAP, NAG, AP, and polyphenol oxidase (POX) were selected (Table 1); (2) N treatment and control plots were deployed under the same climate, soil and vegetation conditions to avoid confounding factors; (3) studies conducted along N deposition gradients were excluded due to possible effects of factors other than N; (4) studies using organic materials, such as manure or straw, as N addition sources, were excluded; (5) we only extracted data measured in organic horizon and top layer of mineral soil if multiple soil horizons were reported; and (6) for studies with multiple factors being manipulated (e.g. a full factor design with four treatments: control, P addition, N addition, and combined N and P addition), we only extracted data from control and N addition treatments. The raw data were either obtained from tables or extracted by digitizing graphs using the GetData Graph Digitizer (version 2.24, Russian Federation). In total, 419 observations from 36 studies were involved (Table S1). In addition, data on fungal and bacterial biomass were also extracted when these data were reported along with soil enzymes (Table S1).
4. Discussion We found that the mean enzyme ratio of C, N, and P acquisition was close to 1:1:1 in the controls (Fig. 1). This ratio was in agreement with a recent meta-analysis at a global scale (Sinsabaugh et al., 2009) and several studies at regional scales (Tapia-Torres et al., 2015; Waring et al., 2014). In addition, this 1:1:1 ratio was not altered by N addition (Fig. 1). The no-change in this 1:1:1 ratio was not surprising, because it was demonstrated partly by the results of the controls, which were from different habitats with various N deposition rates. Our results confirmed the view that the microbes may have shared a common functional stoichiometry in relation to organic nutrient acquisition (Sinsabaugh et al., 2009), and it also suggested that this functional stoichiometry was not altered by the changed deposition of N. Our results demonstrated that addition of N aggravated microbial C limitation. There were two pieces of evidence for this. First, the regression intercept for the C and N enzyme activity increased significantly under N-addition treatment. The increased intercept resulted from the increased Ln BG: Ln (NAG + LAP), which contributed to the significant increase in BG activity and the no-change in (NAG + LAP) activity (Table 2). Due to the more increase in BG activity after N, this
2.2. Data analysis Data of enzyme activity was loge-transformed before analysis for fitting the conventions of stoichiometric analyses and for normalizing variance (Sinsabaugh and Shah, 2012). After that, the ratios between EEAs were calculated. The relationships between EEAs were analyzed with type II regression using SMART (R Development Core Team, 62
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Fig. 1. Organic nitrogen acquisition activity and organic phosphorus acquisition activity in relation to organic carbon acquisition activity in soils. (a)–(b): the relationships under control treatment; (c)–(d): the relationships under N addition treatment.
organic matter following N addition (Prescott, 2010). This has been found in many previous studies, which suggested that N increased recalcitrant organic matter (alkali insoluble fraction, or humin) through condensation reactions between mineral N and organic matter (Cusack et al., 2010; Sollins et al., 1996). The second mechanism is the lower activity of lignin-degrading enzyme following N addition (Carreiro et al., 2000). This was supported by our result with lower POX activity after N input (Table 2). Many studies have found the reduced activity of lignin-degrading enzyme after N addition (Carreiro et al., 2000; Waldrop et al., 2004), and they generally attribute the reasons to the reduced fungal biomass and thus the fungi: bacteria ratio (Frey et al., 2004), because lignin-degrading enzyme are produced by a small group of fungi which is very sensitivity to the N availability (Carreiro et al., 2000; Frey et al., 2004). In this study, we also found a significant decrease in the fungi: bacteria ratio due to the increased bacterial biomass and decreased fungal biomass (Table 2). This greatly supports the above explanation. The result of vector angle (greater than 45° across all observations in controls) indicated that microbial P limitation is widespread in terrestrial ecosystems. The higher vector angles in this study were consistent with other studies that also calculated this variable across global data (Moorhead et al., 2016). However, this result was interesting, because it rejected our general belief that N is the major limitation in terrestrial ecosystems (LeBauer and Treseder, 2008). Nevertheless, a recent study also suggested that P limitation is as important in terrestrial ecosystems as N limitation (Elser et al., 2007), which partly supports our result. Moreover, vector angles were not significantly different between the control and N-addition treatment (Table 2), which suggested that addition of N did not aggravate microbial P-limitation. In support of this, several studies also found that N addition did not cause P limitation, but led to calcium or magnesium limitation (Crowley et al., 2012). The nochange in P limitation that followed N addition may have been due to the accelerated P cycling rates which caused by the increase in soil phosphatase activity (Marklein and Houlton, 2012). In summary, this study conducted a meta-analysis regarding the effects of N addition on microbial resource limitation by using
Table 2 Results of paired-samples t-test for the effects of N addition on soil ecoenzymatic stoichiometry. Value is mean ± standard error. The bold numbers denote significant difference (P < 0.05) between control and N addition.
Ln (BG) Ln (POX) Ln (NAG + LAP) Ln (AP) Ln (BG): Ln (AP) Ln (BG): Ln (NAG + LAP) Ln (NAG + LAP): Ln (AP) Vector L (unitless) Vector A (degree) Ln (Fungal biomass) Ln (Bacterial biomass) Fungi/Bacteria
Control
N addition
P value
7.62 (0.27) 7.57(0.12) 6.40(0.51) 8.49(0.27) 0.87 (0.01) 0.81 (0.07) 0.88 (0.06) 1.18 (0.06) 50.03 (1.86) 1.56(0.48) 2.05 (0.38) 0.83 (0.18)
7.75(0.27) 7.30(0.12) 6.40 (0.51) 8.62 (0.27) 0.87 (0.01) 0.87 (0.06) 0.89 (0.06) 1.23 (0.06) 49.77 (1.86) 1.50 (0.46) 2.06 (0.39) 0.73 (0.15)
0.000 0.000 0.989 0.010 0.931 0.039 0.597 0.028 0.674 0.000 0.045 0.000
result indicated that more C was needed by microbes compared to N. Second, vector length, which is a direct indicator of C limitation, increased significantly from 1.18 in the control to 1.23 in the N-addition samples (Table 2). Aggregated microbial C limitation that was induced by added N has been predicted in the previous studies (Kopáček et al., 2013), but evidence has been rare. To our knowledge, this is the first study that demonstrated aggravated microbial C limitation followed addition of N using evidence from enzymatic stoichiometry. Previous studies have proposed several potential mechanisms, including the increased C loss and decreased C input (Kopáček et al., 2013). In the current study, however, the reasons for the aggravated Climitation needs further addressed due to the limited data. Here we suspect that one of the reasons for our results may be the decline in the decomposition of recalcitrant organic C, which is one way of the C inputs. It has been found commonly that N addition retarded mineralization of old soil organic matter, especially in the later stage of decomposition (Hagedorn et al., 2003; Knorr et al., 2005). At this stage, there are two mechanisms that are often proposed to cause the slower SOM decomposition. The first mechanism is the reducing quality of 63
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ecoenzymatic stoichiometry as indicators. There were two main findings. Firstly, we found that N addition aggravated microbial C limitation, which was partly due to the reduced recalcitrant C decomposition. Secondly, we found that N addition did not affect soil microbial Plimitation, which may be due to the accelerated P cycling rate. These findings provide important knowledge for understanding the effects of N deposition on C, N and P cycling, which is useful for predicting the changes of terrestrial ecosystem function and its feedback to global climate under the context of elevated N deposition. However, considering that the data for our calculation were still limited, we highlight that further studies based on a larger dataset and at a larger scale are needed to assess the generality of our results. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.geoderma.2018.05.019.
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Acknowledgements This work was funded by the National Key Research and Development Program of China (2016YFC0502404), the National Science Foundation of Guangxi Province of China (2017GXNSFAA198038), the National Natural Science Foundation of China (31500405), the Chinese Academy of Sciences through its OneHundred Talent Program to Dejun Li (Y523101030), the Chinese Academy of Sciences through its “Light of West China” Program to Hao Chen, and the Youth Innovation Team Project of ISA, CAS (2017QNCXTD_ZJ). References Aber, J.D., Nadelhoffer, K.J., Steudler, P., Melillo, J.M., 1989. Nitrogen saturation in northern forest ecosystems. Bioscience 39, 378–386. Carreiro, M.M., Sinsabaugh, R.L., Repert, D.A., Parkhurst, D.F., 2000. Microbial enzyme shifts explain litter decay responses to simulated nitrogen deposition. Ecology 81, 2359–2365. Chen, J., Luo, Y.Q., Li, J.W., Zhou, X.H., Cao, J.J., Wang, R.W., Wang, Y.Q., Shelton, S., Jin, Z., Walker, L.M., Feng, Z.Z., Niu, S.S., Feng, W.T., Jian, S.Y., Zhou, L.Y., 2016. Co-stimulation of soil glycosidase activity and soil respiration by nitrogen addition. Glob. Chang. Biol. 23, 1328–1337. Chen, H., Li, D., Zhao, J., Xiao, K., Wang, K., 2018. Effects of nitrogen addition on activities of soil nitrogen acquisition enzymes: a meta-analysis. Agric. Ecosyst. Environ. 252, 126–131. Crowley, K., McNeil, B., Lovett, G., Canham, C., Driscoll, C., Rustad, L., Denny, E., Hallett, R., Arthur, M., Boggs, J., 2012. Do nutrient limitation patterns shift from nitrogen toward phosphorus with increasing nitrogen deposition across the northeastern United States? Ecosystems 15, 940–957. Cusack, D., Silver, W., Torn, M., McDowell, W., 2010. Effects of nitrogen additions on above- and belowground carbon dynamics in two tropical forests. Biogeochemistry 104, 203–225. Ekblad, A., Nordgren, A., 2002. Is growth of soil microorganisms in boreal forests limited by carbon or nitrogen availability? Plant Soil 242, 115–122. Elser, J.J., Bracken, M.E.S., Cleland, E.E., Gruner, D.S., Harpole, W.S., Hillebrand, H., Ngai, J.T., Seabloom, E.W., Shurin, J.B., Smith, J.E., 2007. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol. Lett. 10, 1135–1142. Finzi, A.C., 2009. Decades of atmospheric deposition have not resulted in widespread phosphorus limitation or saturation of tree demand for nitrogen in southern New England. Biogeochemistry 92, 217–229. Frey, S.D., Knorr, M., Parrent, J.L., Simpson, R.T., 2004. Chronic nitrogen enrichment affects the structure and function of the soil microbial community in temperate hardwood and pine forests. For. Ecol. Manag. 196, 159–171.
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