Microb Ecol DOI 10.1007/s00248-015-0604-9
SOIL MICROBIOLOGY
Coupling Between and Among Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms Submitted to Elevated CO2 and Nitrogen Supply Marie Simonin 1,2 & Xavier Le Roux 2 & Franck Poly 2 & Catherine Lerondelle 2 & Bruce A. Hungate 3 & Naoise Nunan 1 & Audrey Niboyet 1
Received: 29 July 2014 / Accepted: 23 March 2015 # Springer Science+Business Media New York 2015
Abstract Many studies have assessed the responses of soil microbial functional groups to increases in atmospheric CO2 or N deposition alone and more rarely in combination. However, the effects of elevated CO2 and N on the (de)coupling between different microbial functional groups (e.g., different groups of nitrifiers) have been barely studied, despite potential consequences for ecosystem functioning. Here, we investigated the short-term combined effects of elevated CO2 and N supply on the abundances of the four main microbial groups involved in soil nitrification: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitriteoxidizing bacteria (belonging to the genera Nitrobacter and Nitrospira) in grassland mesocosms. AOB and AOA abundances responded differently to the treatments: N addition increased AOB abundance, but did not alter AOA abundance. Nitrobacter and Nitrospira abundances also showed contrasted responses to the treatments: N addition increased Nitrobacter abundance, but decreased Nitrospira abundance. Our results support the idea of a niche differentiation between Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0604-9) contains supplementary material, which is available to authorized users. * Audrey Niboyet [email protected] 1
Institute of Ecology and Environmental Sciences - Paris, UMR 7618 Université Pierre et Marie Curie/CNRS/AgroParisTech, 78850 Thiverval Grignon, France
2
Microbial Ecology Center, Université de Lyon/Université Lyon 1/CNRS/INRA, UMR CNRS 5557, USC INRA 1364, 69622 Villeurbanne, France
3
Center for Ecosystem Science and Society and Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA
AOB and AOA, and between Nitrobacter and Nitrospira. AOB and Nitrobacter were both promoted at high N and C conditions (and low soil water content for Nitrobacter), while AOA and Nitrospira were favored at low N and C conditions (and high soil water content for Nitrospira). In addition, Nitrobacter abundance was positively correlated to AOB abundance and Nitrospira abundance to AOA abundance. Our results suggest that the couplings between ammonia and nitrite oxidizers are influenced by soil N availability. Multiple environmental changes may thus elicit rapid and contrasted responses between and among the soil ammonia and nitrite oxidizers due to their different ecological requirements. Keywords Global change . Grasslands . Nitrification . Ammonia oxidizers . Nitrite oxidizers . Niche differentiation
Introduction Environmental changes, such as increases in atmospheric carbon dioxide (CO2) and in nitrogen (N) deposition, are expected to alter the functioning of terrestrial ecosystems, partly through modifications of soil microbial community abundance, structure, and activity [1–5]. Previous studies have focused on the effects of increases in atmospheric CO2 or N deposition in isolation, even though atmospheric CO2 and N deposition are increasing simultaneously [6, 7]. As a result, little information is available about the combined effects of elevated CO2 and N addition on soil microbial communities and the ecosystem functions they perform. Some microbial processes, such as organic matter decomposition, are carried out by organisms that span a broad range of phylogenies [8], likely with high functional redundancy and a degree of resistance to environmental changes [9, 10]. Other processes, such as nitrification and some trace gas
M. Simonin et al.
transformations, are performed by organisms with restricted phylogenies [8] and might, therefore, be more sensitive to environmental changes. For instance, several steps of the N cycle are mediated by only a few microbial groups. Among these, nitrification influences the availability of ammonium (NH4+) and nitrate (NO3−) in soils, the two major N sources for plants and microbes [11, 12]. Because the end product of nitrification, NO3−, is mobile in soils and is the substrate for denitrification, nitrification also contributes to N losses from ecosystems [13]. Nitrification is a two-step process: the first step (the oxidation of NH4+ to nitrite, NO2−) is mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) [14], while the second step (the oxidation of NO2− to NO3−) is mediated by nitrite-oxidizing bacteria (NOB), with two main genera of NOB present in soils: Nitrobacter and Nitrospira [15]. The effects of inorganic or organic N additions on soil ammonia oxidizers in grassland ecosystems are well documented; in contrast, very few studies have investigated the effects of elevated CO2 (alone or combined with N additions) [16, 17] (see Table 1). Most of these studies have been conducted in long-term experiments, i.e., for 2 to 10 years under field conditions (Table 1). It is, thus, difficult to know whether the reported effects on soil ammonia oxidizers are progressively induced after several years of treatment or are generated quickly after treatment application. Furthermore, no studies have reported the effects of elevated CO2 and N addition on soil nitrite oxidizers in grassland ecosystems. Most studies have focused solely on the first step of nitrification, as it is considered to be the rate-limiting step [28]. However, NO2− can accumulate in disturbed soil systems [29–31], which indicates that nitrite oxidation may sometimes be limiting for nitrification. Moreover, it has been demonstrated that the two steps of nitrification can respond differently to environmental changes [32]: in a grassland ecosystem exposed to simulated global changes, N additions, and elevated precipitation affected ammonia and nitrite oxidation differently. Elevated CO2 and N additions are expected to have both direct and indirect effects on soil ammonia and nitrite oxidizers. For instance, N additions could have a direct effect on soil NH4+ availability, the substrate for nitrification, and thus, on the soil nitrifiers. But elevated CO2 and N addition could also have indirect effects on soil nitrifiers by increasing shoot and root biomass and associated root exudates, with impacts on soil moisture and soil carbon (C) and oxygen contents, variables to which soil nitrifiers are sensitive [1]. Elevated CO2 and N addition may induce different responses between the soil ammonia and nitrite oxidizers, given the different ecological requirements of these two functional groups, as ammonia oxidizers are predominately autotrophs [33], while different nitrite oxidizers can also be mixotrophs [34]. Furthermore, among soil ammonia oxidizers, AOA and AOB are thought to occupy different ecological niches, e.g., in terms of soil pH, oxygen
concentration, and nutrient conditions [35–37]. Similarly, among soil nitrite oxidizers, Nitrobacter- and Nitrospira-like NOB have different ecological requirements, Nitrobacter being adapted to high-nitrite and high-oxygen environments, while Nitrospira are adapted to low-nitrite and low-oxygen environments [38]. Hence, the responses of the soilnitrifying microbial communities to the combined effects of elevated CO2 and N addition are difficult to predict given the multi-factor parameters in play and could differ depending on the microbial group considered. Here, we investigated the short-term and combined effects of elevated CO2 and inorganic N supply on nitrifier abundances in grassland mesocosms planted with Dactylis glomerata. We measured the abundances of the four main soil microbial groups involved in nitrification (AOA, AOB, Nitrobacter, and Nitrospira) using quantitative PCR and analyzed their responses to elevated CO2 and/or N additions. We then explored the relationships between these abundances and key plant/soil variables to identify the main drivers of the observed responses to the treatments. We also examined the relationships between gross and potential nitrification rates and soil ammonia- and nitrite-oxidizer abundances to determine whether changes in nitrifier abundances could explain changes in nitrification rates. Finally, we investigated the links between and among ammonia- and nitrite-oxidizer abundances to decipher the coupling of the different microbial groups involved in nitrification under the combined effects of elevated CO2 and inorganic N supply.
Materials and Methods Experimental Design, Treatments, and Soil Sampling Experimental pots (15×20×50 cm) were filled with a sandyloam soil and assigned to one of 12 growth chambers, set up inside a large greenhouse at the Université Paris Sud (Orsay, France;see [39] for full description). The soil had a total N content of 0.23 g kg−1, a total C content of 2.46 g kg−1, an organic matter content of 4.26 g kg−1, a cation exchange capacity of 1.81 cmol kg−1, and a pH of 8.5 (INRA, Arras, France). The experimental pots were sown with D. glomerata, a fast-growing perennial grass common in a wide variety of habitats worldwide [40], at a density of 2,000 seeds per square meter. Maximum daily photosynthetically active radiation (PAR) values recorded during the study ranged between 150 and 1,680 μmoL s−1 m−2. One month later, when the seedlings had fully emerged, two CO2 treatments (ambient vs. elevated) crossed with two N treatments (no N addition vs. N addition) were applied. Six chambers were ventilated with ambient air taken from outside the greenhouse (CO2 =381±6 μmol mol−1) and the six others with ambient air enriched with CO2 (CO2 = 645±9 μmol mol−1). The elevated CO2 concentration chosen
[18]
[19] [20]
[21]
[22]
[23] [24]
[25]
[26]
[27] [16]
N
N N
N
N
N N
N
N
N CO2 × N
Upland grassland (China) Mediterranean grassland (USA)
Upland grassland (China)
Temperate grassland (New Zealand) Mediterranean grassland (USA) Upland grassland (Switzerland) Temperate grassland (New Zealand) Semiarid temperate grassland (China)
Icelandic grassland (Iceland) Temperate grassland (New Zealand)
Upland grassland (China)
Temperate grassland (Germany)
6 years
AOA and AOB 5 years AOB 2 years
AOA and AOB 6 years
AOA and AOB 6 years
AOA and AOB 2 years AOA and AOB 56 years
AOB
AOA and AOB 97 years
AOA and AOB 4 years AOA and AOB 83 years
AOA and AOB 6 years
Effects reported
Increases in the AOA and AOB abundances No effect on the AOA abundance; increase in the AOB abundance
30 g N m−2 (as NH4NO3) 600 kg N ha−1 year−1 (as bovine urine) 0, 1, 2, 4, 8, 16, 32, and 64 g N m−2 year−1 (as urea)
No effect on the AOA abundance, except a decrease with the highest N loading; increase in the AOB abundance with increasing N concentrations 1.5 and 7.5 g N m−2 year−1 Increases in the AOA and AOB abundances in the high (as NH4, NO3, or NH4NO3) N treatments whatever the N form 10 g N m−2 year−1 (as NH4NO3) No effect on the AOA abundance; increase in the AOB abundance 680 ppm 7 g N m−2 year−1 (as Ca(NO3)2) Decrease in the AOB abundance under elevated CO2; no effect of N addition on the AOB abundance; no interactive effect of the CO2 and N treatments
Increase in the AOB abundance
g N m−2 year−1 (as Ca(NO3)2)
440 ppm (in dry, medium, and wet plots) Increase in the AOA abundance in the dry plots and slight decreases in the medium and wet plots; no effect on the AOB abundance 10 g N m−2 year−1 (as urea and NH4NO3) Decrease in the AOA abundance at one of the two sampling dates; increase in the AOB abundance −2 −1 10 g N m year (as urea) Increase in the AOA abundance; AOB were undetectable. 50 and 100 kg N ha−1 year−1 No effect on the AOA abundance; increase in the AOB abundance only with the highest concentration of animal urine (as urea) or 300 and 600 kg N ha−1 year−1 (as animal urine) 1,000 kg N ha−1 year−1 (as animal urine) Decrease in the AOA abundance and increase in the AOB abundance
Duration of Treatment level treatment
AOA and AOB 10 years
Nitrifier community
No published data were found on the soil nitrite oxidizers
[17]
CO2
Treatment References Grassland type
Table 1 A review of the effects of elevated CO2, organic or inorganic N supply, and of the combined effects of elevated CO2 and N supply on the abundance of the soil ammonia oxidizers (AOA and AOB) in grassland ecosystems
Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms
M. Simonin et al.
was close to the middle of the range of the IPCC scenarios for the second half of the twenty-first century [7]. Within each chamber, two N treatments were applied—one pot received 10 g N-NH4NO3 m−2 through six applications of an NH4NO3 solution at 2-week intervals, while the other pot received the equivalent amount of distilled water alone. The total amount of N added was equivalent to 100 kg N ha−1, which is in the range of common fertilization regimes in grasslands [41] and comparable to very high N deposition rates that already occur in a few industrialized regions of the world [6, 42]. Each of the four treatments (Ctrl, +CO2, +N, and +CO2+N) was replicated six times, resulting in a total of 24 experimental mesocosms (2 CO2 treatments × 2 N treatments × 6 replicates). After 10 weeks of plant growth under treatments, when roots had fully colonized, soil and shoots were well developed (about 50 cm high), plants were harvested and separated into belowand above-ground material (see [40] for further details). Soil from the top 10 cm was collected in each pot, homogenized, and sieved (2 mm). Part of the freshly sieved soil was immediately used for measurements of microbial activities and soil variables, while the rest of the soil was stored at −20 °C before DNA extraction and measurements of microbial abundances.
Soil DNA Extraction and Quantification of the Abundance of Soil Ammonia Oxidizers DNA was extracted from 0.7 g of frozen soil using the Power SoilTM DNA Isolation Kit (MO BIO laboratories, Carlsbad, CA, USA), following the manufacturer’s instructions. The abundances of AOA and AOB were measured by quantitative PCR targeting the amoA functional gene encoding for ammonia monooxygenase which is specific of these groups. Amplification was performed using gene primers CrenamoA23f and CrenamoA616r for the AOA [43] and amoA_1F and amoA_2R for the AOB [44]. The final reaction volume was 20 μL and contained (final concentrations) 0.75 μM of CrenamoA616r and 1 μM of CrenamoA23f for the archaeal amoA or 0.5 μM of each primer for the bacterial amoA, 2 % bovine serum albumin (BSA), 1× of QuantiTect SybrGreen PCR Master Mix (Qiagen, Courtaboeuf, France), and 10 ng of soil DNA extract or 107–102 gene copies number per microliters of an in-house plasmid containing cloned archaeal (54d9 fosmide fragment [45]) and bacterial (Nitrosomonas europaea, GenBank accession number:L08050) amoA genes. The samples were run in duplicate (n=6 in all treatments) on a Lightcycler 480 (Roche Diagnostics, Meylan, France) as follows: for archaeal amoA, 15 min at 95 °C, 45 amplification cycles (45 s at 94 °C, 45 s at 55 °C, and 45 s at 72 °C) and 10 s at 40 °C; for bacterial amoA, 15 min at 95 °C, 45 amplification cycles (30 s at 95 °C, 45 s at 54 °C, 45 s at 72 °C, and 15 s at 80 °C), and 30 s at 40 °C.
Melting curve analysis confirmed the specificity of amplification for both AOA and AOB. Amplification efficiencies of 91 and 94 % were obtained for AOA and AOB quantification, respectively. Dilution series were performed to control for possible PCR inhibition by co-extracted compounds, and no inhibition was observed. The ratio of the abundance of AOA to AOB was calculated. This ratio enables the comparison of the relative numbers of AOA and AOB amoA gene copies between treatments, but does not reflect the exact cell proportion of AOA to AOB in soils as the mean amoA gene copy numbers per cell may vary between these two groups.
Quantification of the Abundance of Soil Nitrite Oxidizers The abundance of Nitrobacter-like NOB was measured by quantitative PCR targeting the functional gene nxrA [38], which encodes for the nitrite oxidoreductase. The amplification was performed using the gene primers F1norA [46] and R2norA [47]. The final reaction volume was 20 μL and contained (final concentrations) 0.5 μM of each primer, 1× of QuantiTect SybrGreen PCR Master Mix (Qiagen, Courtaboeuf, France), and 40 ng of soil DNA extract or 107–102 copies using a linearized plasmid containing cloned nxrA gene of Nitrobacter hamburgensis X14 (DSMZ 10229). The samples (n=6 in all treatments, except in the high N treatment where n=5) were run twice on a Lightcycler 480 (Roche Diagnostics, Meylan, France) as follows: 15 min at 95 °C and 45 amplification cycles (30 s at 95 °C, 45 s at 55 °C, and 45 s at 72 °C). The abundance of Nitrospira was measured by quantitative PCR targeting 16S rRNA gene sequences specific for this group [38]. The amplification was performed using the gene primers Ns675f and Ns746r [48]. The final reaction volume was 25 μL and contained (final concentrations) 0.4 μM of each primer, 1× of QuantiTect SybrGreen PCR Master Mix (Qiagen, Courtaboeuf, France), and 10 ng of soil DNA extract or 107–102 Nitrospira copies of linearized plasmid DNA (GenBank accession number: FJ529918). The samples (n=6 in all treatments) were run twice on a Lightcycler 480 (Roche Diagnostics, Meylan, France) as follows: 15 min at 95 °C and 45 amplification cycles (30 s at 95 °C, 30 s at 66 °C, and 1 min at 72 °C). For both genes, melting curves confirmed the specificity of the amplification and amplification efficiencies were of 95– 96 %, with no PCR inhibition observed. The ratio of the abundance of Nitrospira to Nitrobacter-like NOB was calculated. This ratio allows the comparison of the numbers of Nitrospira 16S gene copies and Nitrobacter-like nxrA gene copies between treatments, but does not reflect the exact cell proportion of Nitrospira to Nitrobacter-like NOB in soils, as the mean nxrA or 16S gene copy numbers per cell may vary between these two groups.
Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms
Plant/Soil Variables and Nitrification Rates
Results
Previously measured plant/soil variables [39] were used to determine whether changes in the abundances of the soil ammonia and nitrite oxidizers in response to the treatments were related to changes in plant biomass (shoot and root biomass) or soil variables (soil respiration, gross N mineralization and soil water content). Soil respiration was used as an indicator of soil heterotrophic activity and oxygen status, gross N mineralization rates as a proxy of soil N availability, and soil water content as an indicator of soil oxygen status [39]. In addition to the soil variables that were previously measured, we conducted measurements of Olsen phosphorus (P) concentration (at the INRA, Arras, France) and used this variable as a proxy of soil P availability. Potential nitrification rates (determined using nitrifying enzyme activity assays) and gross nitrification rates (determined using 15N pool dilution technique; for further details, see [39]) were used to determine whether changes in the abundances of each of the four microbial groups involved in nitrification were related to changes in potential and gross nitrification rates.
Ammonia-Oxidizer Abundance
Statistical Analysis We assessed the interactive effects of elevated CO2 and inorganic N supply on the abundances of soil ammonia oxidizers (AOA and AOB), soil nitrite oxidizers (Nitrobacter-like NOB and Nitrospira), and on the AOA/AOB and Nitrospira/ Nitrobacter-like NOB abundance ratios. Statistical analyses were carried out using PROC MIXED in SAS 9.3 (SAS Institute, Cary, NC). The experiment was analyzed as a full factorial split-plot design, with two factors (CO2 and N) at two levels (ambient and elevated) and their interaction. The CO2 treatment was included as a whole-plot factor, and the N treatment as a split-plot factor. Where necessary, data were logtransformed prior to the analysis to ensure conformity with the assumptions of normality and homogeneity of variances. Effects with P
SOIL MICROBIOLOGY
Coupling Between and Among Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms Submitted to Elevated CO2 and Nitrogen Supply Marie Simonin 1,2 & Xavier Le Roux 2 & Franck Poly 2 & Catherine Lerondelle 2 & Bruce A. Hungate 3 & Naoise Nunan 1 & Audrey Niboyet 1
Received: 29 July 2014 / Accepted: 23 March 2015 # Springer Science+Business Media New York 2015
Abstract Many studies have assessed the responses of soil microbial functional groups to increases in atmospheric CO2 or N deposition alone and more rarely in combination. However, the effects of elevated CO2 and N on the (de)coupling between different microbial functional groups (e.g., different groups of nitrifiers) have been barely studied, despite potential consequences for ecosystem functioning. Here, we investigated the short-term combined effects of elevated CO2 and N supply on the abundances of the four main microbial groups involved in soil nitrification: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitriteoxidizing bacteria (belonging to the genera Nitrobacter and Nitrospira) in grassland mesocosms. AOB and AOA abundances responded differently to the treatments: N addition increased AOB abundance, but did not alter AOA abundance. Nitrobacter and Nitrospira abundances also showed contrasted responses to the treatments: N addition increased Nitrobacter abundance, but decreased Nitrospira abundance. Our results support the idea of a niche differentiation between Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0604-9) contains supplementary material, which is available to authorized users. * Audrey Niboyet [email protected] 1
Institute of Ecology and Environmental Sciences - Paris, UMR 7618 Université Pierre et Marie Curie/CNRS/AgroParisTech, 78850 Thiverval Grignon, France
2
Microbial Ecology Center, Université de Lyon/Université Lyon 1/CNRS/INRA, UMR CNRS 5557, USC INRA 1364, 69622 Villeurbanne, France
3
Center for Ecosystem Science and Society and Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA
AOB and AOA, and between Nitrobacter and Nitrospira. AOB and Nitrobacter were both promoted at high N and C conditions (and low soil water content for Nitrobacter), while AOA and Nitrospira were favored at low N and C conditions (and high soil water content for Nitrospira). In addition, Nitrobacter abundance was positively correlated to AOB abundance and Nitrospira abundance to AOA abundance. Our results suggest that the couplings between ammonia and nitrite oxidizers are influenced by soil N availability. Multiple environmental changes may thus elicit rapid and contrasted responses between and among the soil ammonia and nitrite oxidizers due to their different ecological requirements. Keywords Global change . Grasslands . Nitrification . Ammonia oxidizers . Nitrite oxidizers . Niche differentiation
Introduction Environmental changes, such as increases in atmospheric carbon dioxide (CO2) and in nitrogen (N) deposition, are expected to alter the functioning of terrestrial ecosystems, partly through modifications of soil microbial community abundance, structure, and activity [1–5]. Previous studies have focused on the effects of increases in atmospheric CO2 or N deposition in isolation, even though atmospheric CO2 and N deposition are increasing simultaneously [6, 7]. As a result, little information is available about the combined effects of elevated CO2 and N addition on soil microbial communities and the ecosystem functions they perform. Some microbial processes, such as organic matter decomposition, are carried out by organisms that span a broad range of phylogenies [8], likely with high functional redundancy and a degree of resistance to environmental changes [9, 10]. Other processes, such as nitrification and some trace gas
M. Simonin et al.
transformations, are performed by organisms with restricted phylogenies [8] and might, therefore, be more sensitive to environmental changes. For instance, several steps of the N cycle are mediated by only a few microbial groups. Among these, nitrification influences the availability of ammonium (NH4+) and nitrate (NO3−) in soils, the two major N sources for plants and microbes [11, 12]. Because the end product of nitrification, NO3−, is mobile in soils and is the substrate for denitrification, nitrification also contributes to N losses from ecosystems [13]. Nitrification is a two-step process: the first step (the oxidation of NH4+ to nitrite, NO2−) is mediated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) [14], while the second step (the oxidation of NO2− to NO3−) is mediated by nitrite-oxidizing bacteria (NOB), with two main genera of NOB present in soils: Nitrobacter and Nitrospira [15]. The effects of inorganic or organic N additions on soil ammonia oxidizers in grassland ecosystems are well documented; in contrast, very few studies have investigated the effects of elevated CO2 (alone or combined with N additions) [16, 17] (see Table 1). Most of these studies have been conducted in long-term experiments, i.e., for 2 to 10 years under field conditions (Table 1). It is, thus, difficult to know whether the reported effects on soil ammonia oxidizers are progressively induced after several years of treatment or are generated quickly after treatment application. Furthermore, no studies have reported the effects of elevated CO2 and N addition on soil nitrite oxidizers in grassland ecosystems. Most studies have focused solely on the first step of nitrification, as it is considered to be the rate-limiting step [28]. However, NO2− can accumulate in disturbed soil systems [29–31], which indicates that nitrite oxidation may sometimes be limiting for nitrification. Moreover, it has been demonstrated that the two steps of nitrification can respond differently to environmental changes [32]: in a grassland ecosystem exposed to simulated global changes, N additions, and elevated precipitation affected ammonia and nitrite oxidation differently. Elevated CO2 and N additions are expected to have both direct and indirect effects on soil ammonia and nitrite oxidizers. For instance, N additions could have a direct effect on soil NH4+ availability, the substrate for nitrification, and thus, on the soil nitrifiers. But elevated CO2 and N addition could also have indirect effects on soil nitrifiers by increasing shoot and root biomass and associated root exudates, with impacts on soil moisture and soil carbon (C) and oxygen contents, variables to which soil nitrifiers are sensitive [1]. Elevated CO2 and N addition may induce different responses between the soil ammonia and nitrite oxidizers, given the different ecological requirements of these two functional groups, as ammonia oxidizers are predominately autotrophs [33], while different nitrite oxidizers can also be mixotrophs [34]. Furthermore, among soil ammonia oxidizers, AOA and AOB are thought to occupy different ecological niches, e.g., in terms of soil pH, oxygen
concentration, and nutrient conditions [35–37]. Similarly, among soil nitrite oxidizers, Nitrobacter- and Nitrospira-like NOB have different ecological requirements, Nitrobacter being adapted to high-nitrite and high-oxygen environments, while Nitrospira are adapted to low-nitrite and low-oxygen environments [38]. Hence, the responses of the soilnitrifying microbial communities to the combined effects of elevated CO2 and N addition are difficult to predict given the multi-factor parameters in play and could differ depending on the microbial group considered. Here, we investigated the short-term and combined effects of elevated CO2 and inorganic N supply on nitrifier abundances in grassland mesocosms planted with Dactylis glomerata. We measured the abundances of the four main soil microbial groups involved in nitrification (AOA, AOB, Nitrobacter, and Nitrospira) using quantitative PCR and analyzed their responses to elevated CO2 and/or N additions. We then explored the relationships between these abundances and key plant/soil variables to identify the main drivers of the observed responses to the treatments. We also examined the relationships between gross and potential nitrification rates and soil ammonia- and nitrite-oxidizer abundances to determine whether changes in nitrifier abundances could explain changes in nitrification rates. Finally, we investigated the links between and among ammonia- and nitrite-oxidizer abundances to decipher the coupling of the different microbial groups involved in nitrification under the combined effects of elevated CO2 and inorganic N supply.
Materials and Methods Experimental Design, Treatments, and Soil Sampling Experimental pots (15×20×50 cm) were filled with a sandyloam soil and assigned to one of 12 growth chambers, set up inside a large greenhouse at the Université Paris Sud (Orsay, France;see [39] for full description). The soil had a total N content of 0.23 g kg−1, a total C content of 2.46 g kg−1, an organic matter content of 4.26 g kg−1, a cation exchange capacity of 1.81 cmol kg−1, and a pH of 8.5 (INRA, Arras, France). The experimental pots were sown with D. glomerata, a fast-growing perennial grass common in a wide variety of habitats worldwide [40], at a density of 2,000 seeds per square meter. Maximum daily photosynthetically active radiation (PAR) values recorded during the study ranged between 150 and 1,680 μmoL s−1 m−2. One month later, when the seedlings had fully emerged, two CO2 treatments (ambient vs. elevated) crossed with two N treatments (no N addition vs. N addition) were applied. Six chambers were ventilated with ambient air taken from outside the greenhouse (CO2 =381±6 μmol mol−1) and the six others with ambient air enriched with CO2 (CO2 = 645±9 μmol mol−1). The elevated CO2 concentration chosen
[18]
[19] [20]
[21]
[22]
[23] [24]
[25]
[26]
[27] [16]
N
N N
N
N
N N
N
N
N CO2 × N
Upland grassland (China) Mediterranean grassland (USA)
Upland grassland (China)
Temperate grassland (New Zealand) Mediterranean grassland (USA) Upland grassland (Switzerland) Temperate grassland (New Zealand) Semiarid temperate grassland (China)
Icelandic grassland (Iceland) Temperate grassland (New Zealand)
Upland grassland (China)
Temperate grassland (Germany)
6 years
AOA and AOB 5 years AOB 2 years
AOA and AOB 6 years
AOA and AOB 6 years
AOA and AOB 2 years AOA and AOB 56 years
AOB
AOA and AOB 97 years
AOA and AOB 4 years AOA and AOB 83 years
AOA and AOB 6 years
Effects reported
Increases in the AOA and AOB abundances No effect on the AOA abundance; increase in the AOB abundance
30 g N m−2 (as NH4NO3) 600 kg N ha−1 year−1 (as bovine urine) 0, 1, 2, 4, 8, 16, 32, and 64 g N m−2 year−1 (as urea)
No effect on the AOA abundance, except a decrease with the highest N loading; increase in the AOB abundance with increasing N concentrations 1.5 and 7.5 g N m−2 year−1 Increases in the AOA and AOB abundances in the high (as NH4, NO3, or NH4NO3) N treatments whatever the N form 10 g N m−2 year−1 (as NH4NO3) No effect on the AOA abundance; increase in the AOB abundance 680 ppm 7 g N m−2 year−1 (as Ca(NO3)2) Decrease in the AOB abundance under elevated CO2; no effect of N addition on the AOB abundance; no interactive effect of the CO2 and N treatments
Increase in the AOB abundance
g N m−2 year−1 (as Ca(NO3)2)
440 ppm (in dry, medium, and wet plots) Increase in the AOA abundance in the dry plots and slight decreases in the medium and wet plots; no effect on the AOB abundance 10 g N m−2 year−1 (as urea and NH4NO3) Decrease in the AOA abundance at one of the two sampling dates; increase in the AOB abundance −2 −1 10 g N m year (as urea) Increase in the AOA abundance; AOB were undetectable. 50 and 100 kg N ha−1 year−1 No effect on the AOA abundance; increase in the AOB abundance only with the highest concentration of animal urine (as urea) or 300 and 600 kg N ha−1 year−1 (as animal urine) 1,000 kg N ha−1 year−1 (as animal urine) Decrease in the AOA abundance and increase in the AOB abundance
Duration of Treatment level treatment
AOA and AOB 10 years
Nitrifier community
No published data were found on the soil nitrite oxidizers
[17]
CO2
Treatment References Grassland type
Table 1 A review of the effects of elevated CO2, organic or inorganic N supply, and of the combined effects of elevated CO2 and N supply on the abundance of the soil ammonia oxidizers (AOA and AOB) in grassland ecosystems
Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms
M. Simonin et al.
was close to the middle of the range of the IPCC scenarios for the second half of the twenty-first century [7]. Within each chamber, two N treatments were applied—one pot received 10 g N-NH4NO3 m−2 through six applications of an NH4NO3 solution at 2-week intervals, while the other pot received the equivalent amount of distilled water alone. The total amount of N added was equivalent to 100 kg N ha−1, which is in the range of common fertilization regimes in grasslands [41] and comparable to very high N deposition rates that already occur in a few industrialized regions of the world [6, 42]. Each of the four treatments (Ctrl, +CO2, +N, and +CO2+N) was replicated six times, resulting in a total of 24 experimental mesocosms (2 CO2 treatments × 2 N treatments × 6 replicates). After 10 weeks of plant growth under treatments, when roots had fully colonized, soil and shoots were well developed (about 50 cm high), plants were harvested and separated into belowand above-ground material (see [40] for further details). Soil from the top 10 cm was collected in each pot, homogenized, and sieved (2 mm). Part of the freshly sieved soil was immediately used for measurements of microbial activities and soil variables, while the rest of the soil was stored at −20 °C before DNA extraction and measurements of microbial abundances.
Soil DNA Extraction and Quantification of the Abundance of Soil Ammonia Oxidizers DNA was extracted from 0.7 g of frozen soil using the Power SoilTM DNA Isolation Kit (MO BIO laboratories, Carlsbad, CA, USA), following the manufacturer’s instructions. The abundances of AOA and AOB were measured by quantitative PCR targeting the amoA functional gene encoding for ammonia monooxygenase which is specific of these groups. Amplification was performed using gene primers CrenamoA23f and CrenamoA616r for the AOA [43] and amoA_1F and amoA_2R for the AOB [44]. The final reaction volume was 20 μL and contained (final concentrations) 0.75 μM of CrenamoA616r and 1 μM of CrenamoA23f for the archaeal amoA or 0.5 μM of each primer for the bacterial amoA, 2 % bovine serum albumin (BSA), 1× of QuantiTect SybrGreen PCR Master Mix (Qiagen, Courtaboeuf, France), and 10 ng of soil DNA extract or 107–102 gene copies number per microliters of an in-house plasmid containing cloned archaeal (54d9 fosmide fragment [45]) and bacterial (Nitrosomonas europaea, GenBank accession number:L08050) amoA genes. The samples were run in duplicate (n=6 in all treatments) on a Lightcycler 480 (Roche Diagnostics, Meylan, France) as follows: for archaeal amoA, 15 min at 95 °C, 45 amplification cycles (45 s at 94 °C, 45 s at 55 °C, and 45 s at 72 °C) and 10 s at 40 °C; for bacterial amoA, 15 min at 95 °C, 45 amplification cycles (30 s at 95 °C, 45 s at 54 °C, 45 s at 72 °C, and 15 s at 80 °C), and 30 s at 40 °C.
Melting curve analysis confirmed the specificity of amplification for both AOA and AOB. Amplification efficiencies of 91 and 94 % were obtained for AOA and AOB quantification, respectively. Dilution series were performed to control for possible PCR inhibition by co-extracted compounds, and no inhibition was observed. The ratio of the abundance of AOA to AOB was calculated. This ratio enables the comparison of the relative numbers of AOA and AOB amoA gene copies between treatments, but does not reflect the exact cell proportion of AOA to AOB in soils as the mean amoA gene copy numbers per cell may vary between these two groups.
Quantification of the Abundance of Soil Nitrite Oxidizers The abundance of Nitrobacter-like NOB was measured by quantitative PCR targeting the functional gene nxrA [38], which encodes for the nitrite oxidoreductase. The amplification was performed using the gene primers F1norA [46] and R2norA [47]. The final reaction volume was 20 μL and contained (final concentrations) 0.5 μM of each primer, 1× of QuantiTect SybrGreen PCR Master Mix (Qiagen, Courtaboeuf, France), and 40 ng of soil DNA extract or 107–102 copies using a linearized plasmid containing cloned nxrA gene of Nitrobacter hamburgensis X14 (DSMZ 10229). The samples (n=6 in all treatments, except in the high N treatment where n=5) were run twice on a Lightcycler 480 (Roche Diagnostics, Meylan, France) as follows: 15 min at 95 °C and 45 amplification cycles (30 s at 95 °C, 45 s at 55 °C, and 45 s at 72 °C). The abundance of Nitrospira was measured by quantitative PCR targeting 16S rRNA gene sequences specific for this group [38]. The amplification was performed using the gene primers Ns675f and Ns746r [48]. The final reaction volume was 25 μL and contained (final concentrations) 0.4 μM of each primer, 1× of QuantiTect SybrGreen PCR Master Mix (Qiagen, Courtaboeuf, France), and 10 ng of soil DNA extract or 107–102 Nitrospira copies of linearized plasmid DNA (GenBank accession number: FJ529918). The samples (n=6 in all treatments) were run twice on a Lightcycler 480 (Roche Diagnostics, Meylan, France) as follows: 15 min at 95 °C and 45 amplification cycles (30 s at 95 °C, 30 s at 66 °C, and 1 min at 72 °C). For both genes, melting curves confirmed the specificity of the amplification and amplification efficiencies were of 95– 96 %, with no PCR inhibition observed. The ratio of the abundance of Nitrospira to Nitrobacter-like NOB was calculated. This ratio allows the comparison of the numbers of Nitrospira 16S gene copies and Nitrobacter-like nxrA gene copies between treatments, but does not reflect the exact cell proportion of Nitrospira to Nitrobacter-like NOB in soils, as the mean nxrA or 16S gene copy numbers per cell may vary between these two groups.
Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms
Plant/Soil Variables and Nitrification Rates
Results
Previously measured plant/soil variables [39] were used to determine whether changes in the abundances of the soil ammonia and nitrite oxidizers in response to the treatments were related to changes in plant biomass (shoot and root biomass) or soil variables (soil respiration, gross N mineralization and soil water content). Soil respiration was used as an indicator of soil heterotrophic activity and oxygen status, gross N mineralization rates as a proxy of soil N availability, and soil water content as an indicator of soil oxygen status [39]. In addition to the soil variables that were previously measured, we conducted measurements of Olsen phosphorus (P) concentration (at the INRA, Arras, France) and used this variable as a proxy of soil P availability. Potential nitrification rates (determined using nitrifying enzyme activity assays) and gross nitrification rates (determined using 15N pool dilution technique; for further details, see [39]) were used to determine whether changes in the abundances of each of the four microbial groups involved in nitrification were related to changes in potential and gross nitrification rates.
Ammonia-Oxidizer Abundance
Statistical Analysis We assessed the interactive effects of elevated CO2 and inorganic N supply on the abundances of soil ammonia oxidizers (AOA and AOB), soil nitrite oxidizers (Nitrobacter-like NOB and Nitrospira), and on the AOA/AOB and Nitrospira/ Nitrobacter-like NOB abundance ratios. Statistical analyses were carried out using PROC MIXED in SAS 9.3 (SAS Institute, Cary, NC). The experiment was analyzed as a full factorial split-plot design, with two factors (CO2 and N) at two levels (ambient and elevated) and their interaction. The CO2 treatment was included as a whole-plot factor, and the N treatment as a split-plot factor. Where necessary, data were logtransformed prior to the analysis to ensure conformity with the assumptions of normality and homogeneity of variances. Effects with P
