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Proximal and distal control by pH of denitrification rate in a pasture soil
Agriculture, Ecosystems and Environment 141 (2011) 230–233
Contents lists available at ScienceDirect
Agriculture, Ecosystems and Environment journal homepage: www.elsevier.com/locate/agee
Short communication
Proximal and distal control by pH of denitrification rate in a pasture soil ∗ ˇ ˇ Jiˇrí Cuhel , Miloslav Simek ˇ Biology Centre of the ASCR, v. v. i. – Institute of Soil Biology, and University of South Bohemia – Faculty of Science, Na Sádkách 7, 370 05 Ceské Budˇejovice, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 May 2010 Received in revised form 9 February 2011 Accepted 11 February 2011 Available online 5 March 2011 Keywords: Denitrification pH Nitrous oxide Dinitrogen Denitrifying enzyme activity Soil
a b s t r a c t Soil pH can influence denitrification both proximally and distally. Proximal control by pH involves direct changes in denitrification reductase activity while distal control by pH involves changes in the denitrifier community, which is a key component affecting the denitrification rate. The current study separated the proximal and distal control by pH of the denitrification rate and of the relative proportion of two denitrification gas products (N2 O and N2 ). The potential denitrifying enzyme activity (DEA) was measured in the presence or absence of acetylene in three pasture soils differing in pH management in the field. The pH of these soils was further manipulated just before DEA measurement to determine the effect of short-term changes in pH. DEA was driven by the pH management in the field rather than by current pH resulting from short-term changes in pH. However, the N2 O/(N2 O + N2 ) ratio was driven by the effects of the current pH value on the kinetics of N2 O production and reduction. The data suggest that even if the pH-induced changes in the structure of denitrifying community can control the absolute denitrification rate (distal control by pH), the community does not influence the proportion of denitrification products, which is regulated solely by the proximal control by pH. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In conjunction with nitrification, denitrification is the main soil process responsible for emissions of nitrous oxide (N2 O) (Conrad, 1996), a significant contributor to stratospheric ozone depletion and global warming (Conrad, 1996; Knowles, 1982). Soil pH is a crucial abiotic factor influencing not only the denitrification rate but, even more importantly, the proportion of the two major denˇ itrification products, N2 O and N2 (Simek and Cooper, 2002). In general, activity of denitrification enzymes increases with increasing pH values (up to the pH optimum), while in contrast the N2 O/(N2 O + N2 ) ratio decreases. Wallenstein et al. (2006) defined the effects of environmental parameters (including pH) on the kinetics of denitrification enzymes as “proximal control”. Soil pH, however, also influences the denitrifier community (Enwall et al., 2005; Parkin et al., 1985), whose abundance and/or composition can be important drivers of denitrification activity and molar ratio of denitrification products (Bremer et al., 2009; Cavigelli and Robertson, 2000; Holtan-Hartwig et al., 2000). Wallenstein et al. (2006) defined this effect as “distal control” because it influences the composition and abundance of the denitrifying community over the long term. The denitrifying community, in turn, acts as a transducer through which proximal controls on denitrification are realized. The concept of “proximal and distal control” is very
∗ Corresponding author. Tel.: +420 387 775 767; fax: +420 385 310 133. ˇ E-mail address: [email protected] (J. Cuhel). 0167-8809/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.agee.2011.02.016
useful, because even if both controls stabilize during different time periods (short- and long-term, respectively), in the end they both influence the instantaneous activity of denitrification enzymes. ˇ In our previous study (Cuhel et al., 2010), we showed that a 10-month manipulation of soil pH in an experimental grassland led to changes in the abundance of denitrifiers possessing the nirS gene encoding cytochrome cd1 nitrite reductase (NirS). The previous study also documented a relationship between the abundance of NirS-denitrifiers and denitrifying enzyme activity (DEA). It was not clear, however, whether soil pH influenced denitrification rate directly through the kinetics of the denitrification reactions (proximal pH control) or indirectly through the size of denitrifying community possessing this gene (distal pH control). The objective of the present study was to explore how soil pH influences the denitrification rate and N2 O/(N2 O + N2 ) ratio both proximally (direct pH effect) and distally (indirect pH effect). 2. Materials and methods Soils were sampled at an experimental field in a grassland area in South Bohemia, Czech Republic, which is described in detail in ˇ our previous study (Cuhel et al., 2010). Briefly, the experimental site was established in July 2007 and included 12 plots (each 3 m × 3 m) with three different pH treatments: each of four plots was amended three times (in July 2007, September 2007, and April 2008) with a KOH solution, with an H2 SO4 solution, or with water as described ˇ by Cuhel et al. (2010). Three independent soil samples were taken from each plot (12 samples for each pH treatment) in July 2009.
ˇ J. Cuhel, M. Sˇ imek / Agriculture, Ecosystems and Environment 141 (2011) 230–233 Table 1 Soil pH, moisture, mineral nitrogen (NH4 + , NO3 − ) content, total nitrogen (Ntot ), organic carbon (Corg ), carbon in microbial biomass (Cmic ), and glucose-induced respiration (GIR) in soils from the experimental plots differing in pH treatment. Values are means ± standard deviations (n = 4). Values in a row followed by different letters are significantly different (P < 0.05). ParameterA
A
by the Wilcoxon rank sum test. The statistical analyses were performed using the R package version 2.12.1 (R Development Core Team, 2010). 3. Results and discussion
Soil Acidic
pH (H2 O) Moisture (g H2 O g−1 ) NH4 + (g N g−1 ) NO3 − (g N g−1 ) Ntot (mg N g−1 ) Corg (mg C g−1 ) Cmic (g C g−1 ) GIR (g C g−1 h−1 )
231
5.00a 0.207a 0.82a 2.79a 3.82a 23.1ab 308.3a 10.5a
pH-Natural ± ± ± ± ± ± ± ±
0.02 0.004 0.00 0.16 0.19 1.3 44.6 0.4
6.03b 0.206a 0.55b 13.93b 1.30b 21.0a 397.3a 21.1b
± ± ± ± ± ± ± ±
0.01 0.004 0.08 0.16 0.26 0.6 75.0 1.4
Alkaline 7.07c 0.195b 0.99c 15.06c 2.83ab 24.2b 675.3b 24.5c
± ± ± ± ± ± ± ±
0.04 0.003 0.08 0.28 1.59 1.1 64.0 1.9
ˇ For details on methods, see Cuhel et al. (2010) and references listed ibid.
The samples were passed through a 5-mm mesh sieve, combined to produce one composite sample for each pH treatment, and stored at 4 ◦ C in plastic bags. The soil samples were labelled acidic, pHnatural, and alkaline (Table 1). DEA was measured during the first 7 days after soil sampling, while less sensitive soil characteristics like total N and organic C (Table 1) were analyzed later, in the following 2 weeks. DEA was determined in the soils by the phase I assay of Smith and ˇ Tiedje (1979), which was slightly altered as described in Simek and Hopkins (1999) and in this paper. Soil was placed in 120-ml serum bottles (four replicate bottles per soil treatment and 25 g of fieldmoist soil per bottle) and allowed to equilibrate to 25 ◦ C for 1 h. Then 20 ml of water, H2 SO4 , or KOH solution (see next paragraph) was added to the bottles, and the resulting slurries were vigorously shaken. After 20 min of equilibration, 5 ml of the glucose solution (1000 mg l−1 ) and KNO3 solution (500 mg l−1 ) used for DEA determination was added, the bottles were vigorously shaken again, and pH of the slurries was measured using a combined electrode (SenTix 61, WTW, Germany) and pH meter (526/538 pH Meter, WTW, Germany). Bottles were capped with rubber stoppers and metal holders and were evacuated and flushed four times with 99.99% He. The slurries were then incubated either with or without acetylene (10%, v/v) on an end-to-end shaker at 25 ◦ C for measurement of DEA (N2 O + N2 ) and N2 O production. After 30 and 60 min, the concentration of N2 O in the headspace was quantified using gas ˇ chromatography (for details see Cuhel et al., 2010); then the pH of the slurries was measured again. We first determined DEA in the three soils (acidic, pH-natural, and alkaline) sampled in the field without any additional pH manipulation. We then used other portions of the pH-natural soil and added 20 ml of 4.5 mM H2 SO4 or 8.8 mM KOH solutions to the soil slurries just before DEA measurement to shift their original pH values (6.03) to those of the acidic (pH 5.00) or alkaline (pH 7.07) soils, respectively. Finally, we used the acidic and alkaline soils and shifted their pH values to that of the pH-natural soil just before DEA measurement by adding 20 ml of 9.4 mM KOH or 2.7 mM H2 SO4 solutions to the soil slurries, respectively. Concentrations of H2 SO4 and KOH solutions necessary to change pH to target values were determined in preliminary experiments. Results of pH determination before and after DEA measurement showed that adjusted pH values fluctuated less than ±0.2 pH units during 1 h of incubation. Therefore, we calculated the adjusted pH values as the average pH before and after DEA measurement. The relationships between pH and DEA or the N2 O/(N2 O + N2 ) ratio were evaluated by calculating Spearman’s rank correlation coefficients, and the differences between the effects of long-term (field manipulation) and short-term (laboratory manipulation) changes in pH on DEA (or the N2 O/(N2 O + N2 ) ratio) were analyzed
Analysis of DEA in the soils with different field pH management resulted in the ˇ previously described pattern (Simek and Cooper, 2002): in agreement with our preˇ vious findings (Cuhel et al., 2010), DEA was highest in the alkaline soil and lowest in the acidic soil (Fig. 1A) but the N2 O/(N2 O + N2 ) ratio was highest in the acidic soil and lowest in the alkaline soil (Fig. 1D). Although DEA of the pH-natural soil did not change if the soil pH was decreased by addition of H2 SO4 just before DEA determination (Fig. 1B), DEA of the pH-natural soil did increase if the soil pH was increased by the addition of KOH just before DEA determination. The difference between the alkaline and pH-natural soil, however, was greater than the difference between the pH-natural soil with and without the addition of KOH (Fig. 1A vs. B). In contrast, pH manipulations of the pH-natural soil shifted the N2 O/(N2 O + N2 ) ratios (Fig. 1E) to those found in the soils differing in pH (Fig. 1D); we did not find any difference in the N2 O/(N2 O + N2 ) ratio between acidic soil and pH-natural soil adjusted to an acidic pH or between alkaline soil and pH-natural soil adjusted to an alkaline pH (compare Fig. 1D and E). These results are also supported by DEA measurements (Fig. 1C) and N2 O/(N2 O + N2 ) ratios (Fig. 1F) of acidic and alkaline soils whose pH values were adjusted to that of pH-natural soil just before DEA determination (after adjustment, the pH of the acidic and alkaline soil was 5.95 and 6.08, respectively). While pH adjustment did not alter the DEA of the acidic and alkaline soils (compare Fig. 1A and C), the N2 O/(N2 O + N2 ) ratios were shifted to values typical of the pH-natural soil (Fig. 1F). The weak correlation between pH and DEA (Spearman’s rank correlation coefficient r = 0.643; P = 0.069) indicated that DEA was dependent not only on the current pH value but also on the “pH history” of the studied soils. Further, the Wilcoxon test revealed that the long-term pH effect was more important for DEA than the shortterm pH effect at a risk of 6.7% (P = 0.067), which could be considered significant considering the low number of measures. On the other hand, the negative correlation between pH and the N2 O/(N2 O + N2 ) ratio (Spearman’s rank correlation coefficient r = −0.964; P = 0.001) showed that the current pH value was extremely important for relative N2 O production. This finding was also supported by the Wilcoxon test (P = 0.400), indicating that the pH shift achieved in the laboratory on a short-term basis did not influence the relative N2 O production more than the pH shift achieved by the field adjustment. Although hundreds of papers published in the last three decades have used DEA measurements for estimating the denitrification rate, there are still some doubts about the relationship between the DEA data and actual denitrification, as recently discussed, e.g., by Oehler et al. (2010). DEA estimates the process of denitrification by incubation under optimal laboratory conditions and is believed to represent the size of the denitrifying enzyme pool present in the soil sample at the time of measurement (Smith and Tiedje, 1979). We found it advantageous to use DEA in a present study because it is a standardized technique in which environmental factors (other than pH) are invariable and strictly defined and could not overshadow the effect of pH. As noted, however, DEA measures potential rather than actual denitrification, and this limitation should be recognized when considering the data reported in this paper. Our present results clearly indicate that DEA (overall N2 O and N2 production) was more affected by the relatively long-term pH management in the field (2 years in this case), which led to the changes in the abundance of denitrifiers possessing ˇ et al., 2010), the nirS gene encoding cytochrome cd1 nitrite reductase (NirS) (Cuhel than by short-term changes in pH. We expect that, in addition to the changes of ˇ denitrifier abundance (Cuhel et al., 2010) and of other soil parameters (Table 1), the pH adjustment in the field also changed the composition of the denitrifying community for the following reasons: soil pH is an important factor driving bacterial community composition (Fierer and Jackson, 2006); 2 years of liming was shown to be sufficient to change the structure of bacterial community in grassland soils (Gray et al., 2003); and the ability to denitrify has been identified in a very diverse group of phylogenetically unrelated bacteria (Zumft, 1997). It is evident that DEA as a measure of the denitrification rate in the present study was controlled by pH indirectly, i.e., denitrification was evidently more affected by the size and composition of denitrifying community than by the current or direct pH effect, even if we did not analyze the composition of denitrifier community in the experimental soils and cannot separate the effects of the community abundance vs. those of community composition. The direct pH effect can also substantially conˇ trol denitrification rate as shown by Simek et al. (2002), but the direct effect of pH in the current study was probably limited by the relatively narrow range of pH values. The pH range did, however, support a substantial indirect effect of pH on DEA. On the other hand, the relative production of N2 O and N2 , here expressed as the N2 O/(N2 O + N2 ) ratio, was affected by the current pH value of the soil slurries during measurement rather than by the long-term pH value. Thus, the kinetics of N2 O production and reduction were controlled exclusively by the direct pH effect. When pH is adjusted just before DEA determination, it is unlikely that the denitrifying community can change its abundance or composition, and induction of new
232
ˇ J. Cuhel, M. Sˇ imek / Agriculture, Ecosystems and Environment 141 (2011) 230–233
Fig. 1. Denitrifying enzyme activity (DEA) (separated into N2 O and N2 production) (A–C) and relative N2 O production expressed as the N2 O/(N2 O + N2 ) molar ratio (D–F) of soils with different pH values. Results are from the acidic, pH-natural, and alkaline soils sampled in the field without any additional pH manipulations (A and D); from the pH-natural soil adjusted to the pH values of the acidic and alkaline soils (B and E); and from the acidic and alkaline soils adjusted to the pH values of the pH-natural soil (C and F). Under the labels of pH treatment on the X-axis, the pH values of the three soils and shifts in pHs are presented. Values are means ± standard deviations (n = 4).
denitrification reductases is also negligible (Smith and Tiedje, 1979), because the soil slurries are incubated for only 1 h. In recent years, there has been a strong effort to find a linkage between the size or composition of the denitrifying bacterial community and denitrification rate and especially relative N2 O production (Cavigelli and Robertson, 2000; Chèneby et al., 1998; Holtan-Hartwig et al., 2000; Philippot et al., 2009; Rich and Myrold, 2004). Chèneby et al. (1998) found significant differences in denitrifier community composition for two agricultural soils with contrasting N2 O molar ratios. However, even if the two soils in their study contained different denitrifying strains (either with or without the ability to reduce N2 O to N2 ), the N2 O molar ratio of the soils (0.92 and 0.03) was driven by soil pH (5.2 and 8.1, respectively). Philippot et al. (2009) found a correlation between the N2 O/(N2 O + N2 ) ratio and the proportion of bacte-
ria genetically capable of performing N2 O reduction relative to the total bacteria in pasture soils (r = −0.530; P < 0.01). Nevertheless, the N2 O/(N2 O + N2 ) ratio was also negatively correlated with soil pH (r = −0.706; P < 0.01), and it is possible that there was no causal relationship between the relative N2 O production and the relative abundance of N2 O-reducing bacteria. Our results suggest that even if soil pH can control the abundance or composition of the denitrifying community (distal control by pH), the community does not influence the proportion of denitrification products, which is controlled by the current pH value (proximal control by pH), not excluding other possible controllers. In agreement with our results, Liu et al. (2010) recently reported that the dependency of the ratio of the denitrification products on soil pH is a post-transcriptional phenomenon, because the relative N2 O production from soils differing in pH in their study did not correspond to the relative abundances or to the
ˇ J. Cuhel, M. Sˇ imek / Agriculture, Ecosystems and Environment 141 (2011) 230–233 relative transcription rates of the denitrification genes nosZ (encoding N2 O reductase) and nirS. Liu et al. suggested that pH affects the translation, protein assembly, or the activity of N2 O reductase. In the present study, we demonstrated only the direct influence of pH on the activity of N2 O reductase because we measured denitrification activity during the phase I of denitrification, when induction of new denitrification reductases does not occur (Smith and Tiedje, 1979). In conclusion, we were able to separate the proximal and distal controls by pH of the denitrifying enzyme activity. The absolute denitrification rate was largely under distal control by pH (i.e., it was influenced by changes in the size and composition of the denitrifying community as affected by long-term pH management) rather than under proximal control by pH. The relative N2 O production, however, was solely under proximal control by pH (i.e., it resulted from the direct influence of pH on the kinetics of N2 O production and reduction). These results increase our understanding of the control and regulation of the denitrification process in general and of the effects of pH on the activity of denitrification enzymes in particular.
Acknowledgements ˇ Linda Jíˇsová and Veronika Slajchrtová are gratefully acknowledged for laboratory analyses, and Vlastimil and Marcela Kamír are thanked for allowing access to the experimental field. This study was supported by the Grant Agency of the Academy of Sciences of the Czech Republic (IAA600660605); Grant Agency of the University of South Bohemia (GAJU 142/2010/P); the Ministry of Education, Youth and Sports of the Czech Republic (LC 06066); and by the research intentions of the Biology Centre of the AS CR, v. v. i. (AV0Z60660521) and of the Faculty of Science, University of South Bohemia (MSM 6007665801). References Bremer, C., Braker, G., Matthies, D., Beierkuhnlein, C., Conrad, R., 2009. Plant presence and species combination, but not diversity, influence denitrifier activity and composition of nirK-type denitrifier communities in grassland soil. FEMS Microbiol. Ecol. 70, 377–387. Cavigelli, M.A., Robertson, G.P., 2000. The functional significance of denitrifier community composition in a terrestrial ecosystem. Ecology 81, 1402–1414. Chèneby, D., Hartmann, A., Hénault, C., Topp, E., Germon, J.C., 1998. Diversity of denitrifying microflora and ability to reduce N2 O in two soils. Biol. Fert. Soils 28, 19–26. Conrad, R., 1996. Soil microorganisms as controllers of atmospheric trace gases (H2 , CO, CH4 , OCS, N2 O, and NO). Microbiol. Rev. 60, 609–640.
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ˇ ˇ Cuhel, J., Simek, M., Laughlin, R.J., Bru, D., Chèneby, D., Watson, C.J., Philippot, L., 2010. Insights into the effect of soil pH on N2 O and N2 emissions, and denitrifier community size and activity. Appl. Environ. Microbiol. 76, 1870–1878. Enwall, K., Philippot, L., Hallin, S., 2005. Activity and composition of the denitrifying bacterial community respond differently to long-term fertilization. Appl. Environ. Microbiol. 71, 8335–8343. Fierer, N., Jackson, R.B., 2006. The diversity and biogeography of soil bacterial communities. Proc. Natl. Acad. Sci. U. S. A. 103, 626–631. Gray, N.D., Hastings, R.C., Sheppard, S.K., Loughnane, P., Lloyd, D., McCarthy, A.J., Head, I.M., 2003. Effects of soil improvement treatments on bacterial community structure and soil processes in an upland grassland soil. FEMS Microbiol. Ecol. 46, 11–22. Holtan-Hartwig, L., Dörsch, P., Bakken, L.R., 2000. Comparison of denitrifying communities in organic soils: kinetics of NO3 − and N2 O reduction. Soil Biol. Biochem. 32, 833–843. Knowles, R., 1982. Denitrification. Microbiol. Rev. 46, 43–70. Liu, B., Mørkved, P.T., Frostegård, Å., Bakken, L.R., 2010. Denitrification gene pools, transcription and kinetics of NO, N2 O and N2 production as affected by soil pH. FEMS Microbiol. Ecol. 72, 407–417. Oehler, F., Rutherford, J.C., Coco, G., 2010. The use of machine learning algorithms to design a generalized simplified denitrification model. Biogeosciences 7, 3311–3332. Parkin, T., Sexstone, A.J., Tiedje, J.M., 1985. Adaption of denitrifying populations to low soil pH. Appl. Environ. Microbiol. 49, 1053–1056. ˇ ˇ Philippot, L., Cuhel, J., Saby, N.P.A., Chèneby, D., Chronáková, A., Bru, D., Arrouays, ˇ D., Martin-Laurent, F., Simek, M., 2009. Mapping field-scale spatial patterns of size and activity of the denitrifier community. Environ. Microbiol. 11, 1518–1526. R Development Core Team, 2010. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, ISBN 3900051-07-0, http://www.R-project.org. Rich, J.J., Myrold, D.D., 2004. Community composition and activities of denitrifying bacteria from adjacent agricultural soil, riparian soil, and creek sediment in Oregon, USA. Soil Biol. Biochem. 36, 1431–1441. ˇ Simek, M., Cooper, J.E., 2002. The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years. Eur. J. Soil Sci. 53, 345–354. ˇ Simek, M., Hopkins, D.W., 1999. Regulation of potential denitrification by soil pH in long-term fertilized arable soil. Biol. Fert. Soils 30, 41–47. ˇSimek, M., Jíˇsová, L., Hopkins, D.W., 2002. What is the so-called optimum pH for denitrification in soil? Soil Biol. Biochem. 34, 1227–1234. Smith, M.S., Tiedje, J.M., 1979. Phases of denitrification following oxygen depletion in soil. Soil Biol. Biochem. 11, 261–267. Wallenstein, M.D., Myrold, D.D., Firestone, M., Voytek, M., 2006. Environmental controls on denitrifying communities and denitrification rates: insights from molecular methods. Ecol. Appl. 16, 2143–2152. Zumft, W.G., 1997. Cell biology and molecular basis of denitrification. Microbiol. Mol. Biol. Rev. 61, 533–552.
Contents lists available at ScienceDirect
Agriculture, Ecosystems and Environment journal homepage: www.elsevier.com/locate/agee
Short communication
Proximal and distal control by pH of denitrification rate in a pasture soil ∗ ˇ ˇ Jiˇrí Cuhel , Miloslav Simek ˇ Biology Centre of the ASCR, v. v. i. – Institute of Soil Biology, and University of South Bohemia – Faculty of Science, Na Sádkách 7, 370 05 Ceské Budˇejovice, Czech Republic
a r t i c l e
i n f o
Article history: Received 19 May 2010 Received in revised form 9 February 2011 Accepted 11 February 2011 Available online 5 March 2011 Keywords: Denitrification pH Nitrous oxide Dinitrogen Denitrifying enzyme activity Soil
a b s t r a c t Soil pH can influence denitrification both proximally and distally. Proximal control by pH involves direct changes in denitrification reductase activity while distal control by pH involves changes in the denitrifier community, which is a key component affecting the denitrification rate. The current study separated the proximal and distal control by pH of the denitrification rate and of the relative proportion of two denitrification gas products (N2 O and N2 ). The potential denitrifying enzyme activity (DEA) was measured in the presence or absence of acetylene in three pasture soils differing in pH management in the field. The pH of these soils was further manipulated just before DEA measurement to determine the effect of short-term changes in pH. DEA was driven by the pH management in the field rather than by current pH resulting from short-term changes in pH. However, the N2 O/(N2 O + N2 ) ratio was driven by the effects of the current pH value on the kinetics of N2 O production and reduction. The data suggest that even if the pH-induced changes in the structure of denitrifying community can control the absolute denitrification rate (distal control by pH), the community does not influence the proportion of denitrification products, which is regulated solely by the proximal control by pH. © 2011 Elsevier B.V. All rights reserved.
1. Introduction In conjunction with nitrification, denitrification is the main soil process responsible for emissions of nitrous oxide (N2 O) (Conrad, 1996), a significant contributor to stratospheric ozone depletion and global warming (Conrad, 1996; Knowles, 1982). Soil pH is a crucial abiotic factor influencing not only the denitrification rate but, even more importantly, the proportion of the two major denˇ itrification products, N2 O and N2 (Simek and Cooper, 2002). In general, activity of denitrification enzymes increases with increasing pH values (up to the pH optimum), while in contrast the N2 O/(N2 O + N2 ) ratio decreases. Wallenstein et al. (2006) defined the effects of environmental parameters (including pH) on the kinetics of denitrification enzymes as “proximal control”. Soil pH, however, also influences the denitrifier community (Enwall et al., 2005; Parkin et al., 1985), whose abundance and/or composition can be important drivers of denitrification activity and molar ratio of denitrification products (Bremer et al., 2009; Cavigelli and Robertson, 2000; Holtan-Hartwig et al., 2000). Wallenstein et al. (2006) defined this effect as “distal control” because it influences the composition and abundance of the denitrifying community over the long term. The denitrifying community, in turn, acts as a transducer through which proximal controls on denitrification are realized. The concept of “proximal and distal control” is very
∗ Corresponding author. Tel.: +420 387 775 767; fax: +420 385 310 133. ˇ E-mail address: [email protected] (J. Cuhel). 0167-8809/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.agee.2011.02.016
useful, because even if both controls stabilize during different time periods (short- and long-term, respectively), in the end they both influence the instantaneous activity of denitrification enzymes. ˇ In our previous study (Cuhel et al., 2010), we showed that a 10-month manipulation of soil pH in an experimental grassland led to changes in the abundance of denitrifiers possessing the nirS gene encoding cytochrome cd1 nitrite reductase (NirS). The previous study also documented a relationship between the abundance of NirS-denitrifiers and denitrifying enzyme activity (DEA). It was not clear, however, whether soil pH influenced denitrification rate directly through the kinetics of the denitrification reactions (proximal pH control) or indirectly through the size of denitrifying community possessing this gene (distal pH control). The objective of the present study was to explore how soil pH influences the denitrification rate and N2 O/(N2 O + N2 ) ratio both proximally (direct pH effect) and distally (indirect pH effect). 2. Materials and methods Soils were sampled at an experimental field in a grassland area in South Bohemia, Czech Republic, which is described in detail in ˇ our previous study (Cuhel et al., 2010). Briefly, the experimental site was established in July 2007 and included 12 plots (each 3 m × 3 m) with three different pH treatments: each of four plots was amended three times (in July 2007, September 2007, and April 2008) with a KOH solution, with an H2 SO4 solution, or with water as described ˇ by Cuhel et al. (2010). Three independent soil samples were taken from each plot (12 samples for each pH treatment) in July 2009.
ˇ J. Cuhel, M. Sˇ imek / Agriculture, Ecosystems and Environment 141 (2011) 230–233 Table 1 Soil pH, moisture, mineral nitrogen (NH4 + , NO3 − ) content, total nitrogen (Ntot ), organic carbon (Corg ), carbon in microbial biomass (Cmic ), and glucose-induced respiration (GIR) in soils from the experimental plots differing in pH treatment. Values are means ± standard deviations (n = 4). Values in a row followed by different letters are significantly different (P < 0.05). ParameterA
A
by the Wilcoxon rank sum test. The statistical analyses were performed using the R package version 2.12.1 (R Development Core Team, 2010). 3. Results and discussion
Soil Acidic
pH (H2 O) Moisture (g H2 O g−1 ) NH4 + (g N g−1 ) NO3 − (g N g−1 ) Ntot (mg N g−1 ) Corg (mg C g−1 ) Cmic (g C g−1 ) GIR (g C g−1 h−1 )
231
5.00a 0.207a 0.82a 2.79a 3.82a 23.1ab 308.3a 10.5a
pH-Natural ± ± ± ± ± ± ± ±
0.02 0.004 0.00 0.16 0.19 1.3 44.6 0.4
6.03b 0.206a 0.55b 13.93b 1.30b 21.0a 397.3a 21.1b
± ± ± ± ± ± ± ±
0.01 0.004 0.08 0.16 0.26 0.6 75.0 1.4
Alkaline 7.07c 0.195b 0.99c 15.06c 2.83ab 24.2b 675.3b 24.5c
± ± ± ± ± ± ± ±
0.04 0.003 0.08 0.28 1.59 1.1 64.0 1.9
ˇ For details on methods, see Cuhel et al. (2010) and references listed ibid.
The samples were passed through a 5-mm mesh sieve, combined to produce one composite sample for each pH treatment, and stored at 4 ◦ C in plastic bags. The soil samples were labelled acidic, pHnatural, and alkaline (Table 1). DEA was measured during the first 7 days after soil sampling, while less sensitive soil characteristics like total N and organic C (Table 1) were analyzed later, in the following 2 weeks. DEA was determined in the soils by the phase I assay of Smith and ˇ Tiedje (1979), which was slightly altered as described in Simek and Hopkins (1999) and in this paper. Soil was placed in 120-ml serum bottles (four replicate bottles per soil treatment and 25 g of fieldmoist soil per bottle) and allowed to equilibrate to 25 ◦ C for 1 h. Then 20 ml of water, H2 SO4 , or KOH solution (see next paragraph) was added to the bottles, and the resulting slurries were vigorously shaken. After 20 min of equilibration, 5 ml of the glucose solution (1000 mg l−1 ) and KNO3 solution (500 mg l−1 ) used for DEA determination was added, the bottles were vigorously shaken again, and pH of the slurries was measured using a combined electrode (SenTix 61, WTW, Germany) and pH meter (526/538 pH Meter, WTW, Germany). Bottles were capped with rubber stoppers and metal holders and were evacuated and flushed four times with 99.99% He. The slurries were then incubated either with or without acetylene (10%, v/v) on an end-to-end shaker at 25 ◦ C for measurement of DEA (N2 O + N2 ) and N2 O production. After 30 and 60 min, the concentration of N2 O in the headspace was quantified using gas ˇ chromatography (for details see Cuhel et al., 2010); then the pH of the slurries was measured again. We first determined DEA in the three soils (acidic, pH-natural, and alkaline) sampled in the field without any additional pH manipulation. We then used other portions of the pH-natural soil and added 20 ml of 4.5 mM H2 SO4 or 8.8 mM KOH solutions to the soil slurries just before DEA measurement to shift their original pH values (6.03) to those of the acidic (pH 5.00) or alkaline (pH 7.07) soils, respectively. Finally, we used the acidic and alkaline soils and shifted their pH values to that of the pH-natural soil just before DEA measurement by adding 20 ml of 9.4 mM KOH or 2.7 mM H2 SO4 solutions to the soil slurries, respectively. Concentrations of H2 SO4 and KOH solutions necessary to change pH to target values were determined in preliminary experiments. Results of pH determination before and after DEA measurement showed that adjusted pH values fluctuated less than ±0.2 pH units during 1 h of incubation. Therefore, we calculated the adjusted pH values as the average pH before and after DEA measurement. The relationships between pH and DEA or the N2 O/(N2 O + N2 ) ratio were evaluated by calculating Spearman’s rank correlation coefficients, and the differences between the effects of long-term (field manipulation) and short-term (laboratory manipulation) changes in pH on DEA (or the N2 O/(N2 O + N2 ) ratio) were analyzed
Analysis of DEA in the soils with different field pH management resulted in the ˇ previously described pattern (Simek and Cooper, 2002): in agreement with our preˇ vious findings (Cuhel et al., 2010), DEA was highest in the alkaline soil and lowest in the acidic soil (Fig. 1A) but the N2 O/(N2 O + N2 ) ratio was highest in the acidic soil and lowest in the alkaline soil (Fig. 1D). Although DEA of the pH-natural soil did not change if the soil pH was decreased by addition of H2 SO4 just before DEA determination (Fig. 1B), DEA of the pH-natural soil did increase if the soil pH was increased by the addition of KOH just before DEA determination. The difference between the alkaline and pH-natural soil, however, was greater than the difference between the pH-natural soil with and without the addition of KOH (Fig. 1A vs. B). In contrast, pH manipulations of the pH-natural soil shifted the N2 O/(N2 O + N2 ) ratios (Fig. 1E) to those found in the soils differing in pH (Fig. 1D); we did not find any difference in the N2 O/(N2 O + N2 ) ratio between acidic soil and pH-natural soil adjusted to an acidic pH or between alkaline soil and pH-natural soil adjusted to an alkaline pH (compare Fig. 1D and E). These results are also supported by DEA measurements (Fig. 1C) and N2 O/(N2 O + N2 ) ratios (Fig. 1F) of acidic and alkaline soils whose pH values were adjusted to that of pH-natural soil just before DEA determination (after adjustment, the pH of the acidic and alkaline soil was 5.95 and 6.08, respectively). While pH adjustment did not alter the DEA of the acidic and alkaline soils (compare Fig. 1A and C), the N2 O/(N2 O + N2 ) ratios were shifted to values typical of the pH-natural soil (Fig. 1F). The weak correlation between pH and DEA (Spearman’s rank correlation coefficient r = 0.643; P = 0.069) indicated that DEA was dependent not only on the current pH value but also on the “pH history” of the studied soils. Further, the Wilcoxon test revealed that the long-term pH effect was more important for DEA than the shortterm pH effect at a risk of 6.7% (P = 0.067), which could be considered significant considering the low number of measures. On the other hand, the negative correlation between pH and the N2 O/(N2 O + N2 ) ratio (Spearman’s rank correlation coefficient r = −0.964; P = 0.001) showed that the current pH value was extremely important for relative N2 O production. This finding was also supported by the Wilcoxon test (P = 0.400), indicating that the pH shift achieved in the laboratory on a short-term basis did not influence the relative N2 O production more than the pH shift achieved by the field adjustment. Although hundreds of papers published in the last three decades have used DEA measurements for estimating the denitrification rate, there are still some doubts about the relationship between the DEA data and actual denitrification, as recently discussed, e.g., by Oehler et al. (2010). DEA estimates the process of denitrification by incubation under optimal laboratory conditions and is believed to represent the size of the denitrifying enzyme pool present in the soil sample at the time of measurement (Smith and Tiedje, 1979). We found it advantageous to use DEA in a present study because it is a standardized technique in which environmental factors (other than pH) are invariable and strictly defined and could not overshadow the effect of pH. As noted, however, DEA measures potential rather than actual denitrification, and this limitation should be recognized when considering the data reported in this paper. Our present results clearly indicate that DEA (overall N2 O and N2 production) was more affected by the relatively long-term pH management in the field (2 years in this case), which led to the changes in the abundance of denitrifiers possessing ˇ et al., 2010), the nirS gene encoding cytochrome cd1 nitrite reductase (NirS) (Cuhel than by short-term changes in pH. We expect that, in addition to the changes of ˇ denitrifier abundance (Cuhel et al., 2010) and of other soil parameters (Table 1), the pH adjustment in the field also changed the composition of the denitrifying community for the following reasons: soil pH is an important factor driving bacterial community composition (Fierer and Jackson, 2006); 2 years of liming was shown to be sufficient to change the structure of bacterial community in grassland soils (Gray et al., 2003); and the ability to denitrify has been identified in a very diverse group of phylogenetically unrelated bacteria (Zumft, 1997). It is evident that DEA as a measure of the denitrification rate in the present study was controlled by pH indirectly, i.e., denitrification was evidently more affected by the size and composition of denitrifying community than by the current or direct pH effect, even if we did not analyze the composition of denitrifier community in the experimental soils and cannot separate the effects of the community abundance vs. those of community composition. The direct pH effect can also substantially conˇ trol denitrification rate as shown by Simek et al. (2002), but the direct effect of pH in the current study was probably limited by the relatively narrow range of pH values. The pH range did, however, support a substantial indirect effect of pH on DEA. On the other hand, the relative production of N2 O and N2 , here expressed as the N2 O/(N2 O + N2 ) ratio, was affected by the current pH value of the soil slurries during measurement rather than by the long-term pH value. Thus, the kinetics of N2 O production and reduction were controlled exclusively by the direct pH effect. When pH is adjusted just before DEA determination, it is unlikely that the denitrifying community can change its abundance or composition, and induction of new
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Fig. 1. Denitrifying enzyme activity (DEA) (separated into N2 O and N2 production) (A–C) and relative N2 O production expressed as the N2 O/(N2 O + N2 ) molar ratio (D–F) of soils with different pH values. Results are from the acidic, pH-natural, and alkaline soils sampled in the field without any additional pH manipulations (A and D); from the pH-natural soil adjusted to the pH values of the acidic and alkaline soils (B and E); and from the acidic and alkaline soils adjusted to the pH values of the pH-natural soil (C and F). Under the labels of pH treatment on the X-axis, the pH values of the three soils and shifts in pHs are presented. Values are means ± standard deviations (n = 4).
denitrification reductases is also negligible (Smith and Tiedje, 1979), because the soil slurries are incubated for only 1 h. In recent years, there has been a strong effort to find a linkage between the size or composition of the denitrifying bacterial community and denitrification rate and especially relative N2 O production (Cavigelli and Robertson, 2000; Chèneby et al., 1998; Holtan-Hartwig et al., 2000; Philippot et al., 2009; Rich and Myrold, 2004). Chèneby et al. (1998) found significant differences in denitrifier community composition for two agricultural soils with contrasting N2 O molar ratios. However, even if the two soils in their study contained different denitrifying strains (either with or without the ability to reduce N2 O to N2 ), the N2 O molar ratio of the soils (0.92 and 0.03) was driven by soil pH (5.2 and 8.1, respectively). Philippot et al. (2009) found a correlation between the N2 O/(N2 O + N2 ) ratio and the proportion of bacte-
ria genetically capable of performing N2 O reduction relative to the total bacteria in pasture soils (r = −0.530; P < 0.01). Nevertheless, the N2 O/(N2 O + N2 ) ratio was also negatively correlated with soil pH (r = −0.706; P < 0.01), and it is possible that there was no causal relationship between the relative N2 O production and the relative abundance of N2 O-reducing bacteria. Our results suggest that even if soil pH can control the abundance or composition of the denitrifying community (distal control by pH), the community does not influence the proportion of denitrification products, which is controlled by the current pH value (proximal control by pH), not excluding other possible controllers. In agreement with our results, Liu et al. (2010) recently reported that the dependency of the ratio of the denitrification products on soil pH is a post-transcriptional phenomenon, because the relative N2 O production from soils differing in pH in their study did not correspond to the relative abundances or to the
ˇ J. Cuhel, M. Sˇ imek / Agriculture, Ecosystems and Environment 141 (2011) 230–233 relative transcription rates of the denitrification genes nosZ (encoding N2 O reductase) and nirS. Liu et al. suggested that pH affects the translation, protein assembly, or the activity of N2 O reductase. In the present study, we demonstrated only the direct influence of pH on the activity of N2 O reductase because we measured denitrification activity during the phase I of denitrification, when induction of new denitrification reductases does not occur (Smith and Tiedje, 1979). In conclusion, we were able to separate the proximal and distal controls by pH of the denitrifying enzyme activity. The absolute denitrification rate was largely under distal control by pH (i.e., it was influenced by changes in the size and composition of the denitrifying community as affected by long-term pH management) rather than under proximal control by pH. The relative N2 O production, however, was solely under proximal control by pH (i.e., it resulted from the direct influence of pH on the kinetics of N2 O production and reduction). These results increase our understanding of the control and regulation of the denitrification process in general and of the effects of pH on the activity of denitrification enzymes in particular.
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