Soil Biology & Biochemistry 113 (2017) 161e172
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Ammonium availability and temperature control contributions of ammonia oxidizing bacteria and archaea to nitrification in an agricultural soil Yang Ouyang a, 1, Jeanette M. Norton a, *, John M. Stark b a b
Department of Plants, Soils and Climate, Utah State University, Logan, UT, USA Department of Biology, Utah State University, Logan, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 March 2017 Received in revised form 24 May 2017 Accepted 4 June 2017
Soil ammonia-oxidizing bacteria and archaea (AOB and AOA) convert ammonium/ammonia to nitrite in the process of nitrification. However, the potentially differential responses of these AO to substrate and temperature and the effects of conventional and organic nitrogen management on these responses remains poorly understood. We determined the response of nitrification to ammonium substrate concentration and temperature using an AOB specific inhibitor to distinguish the contribution of AOB and AOA to nitrification. Soils were sampled from cornfield plots that had been treated for four years with contrasting nitrogen sources: control (no additional N), ammonium sulfate at two rates and compost. Nitrification potential and net rates were stimulated for one month after fertilization with ammonium sulfate compared to relatively lower and stable rates in control and compost treated soils. For soils that had been fertilized with ammonium sulfate, the proportion of nitrification mediated by AOB in slurry assays was over 90% at 1.0 mM but less than 50% at 0.01 mM. Kinetic analysis showed maximum nitrification activity (Vmax) for AOB ranged from 0.32 to 4.8 mmol N kg1d1 with a half saturation constant (Km) of 14e160 mM ammonium; parameters were higher for soils from ammonium sulfate treated plots. Vmax and Km for AOA averaged 0.24 mmol N kg1d1 and 4.28 mM ammonium with no effect of field treatment. The proportion of nitrification due to AOA was lowest at 5 C, increased with temperature, and was near to 100% at 50 C; optimum temperature was 41 C for AOA versus 31 C for AOB. Understanding the kinetic and temperature response of microbes responsible for nitrification may allow ecosystem models to include these populations as dynamic components driving nitrogen flux. © 2017 Published by Elsevier Ltd.
Keywords: Ammonia oxidizing archaea Nitrosospira Octyne Niche differentiation Nitrification kinetics Nitrogen fertilizer use efficiency
1. Introduction Nitrification transforms ammonium (NHþ 4 ) into nitrate (NO3 ) and increases the likelihood of N loss from agroecosystems. Estimates of NO 3 leaching from agricultural systems worldwide are projected to reach 61.5 Tg N year1 by 2050 (Schlesinger, 2009) and agriculture contributes around 70% of nitrous oxide emissions (Hu et al., 2015; Robertson and Vitousek, 2009). Improved management of nitrification may decrease impacts from these N losses and increase N fertilizer use efficiency (Subbarao et al., 2013). The first step of autotrophic nitrification is primarily mediated
* Corresponding author. 4820 Old Main Hill, Logan, UT 84322-4820, USA. E-mail address:
[email protected] (J.M. Norton). 1 Current address: Department of Crop, Soil and Microbial Sciences, Michigan State University, East Lansing, MI, USA. http://dx.doi.org/10.1016/j.soilbio.2017.06.010 0038-0717/© 2017 Published by Elsevier Ltd.
by ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA). AOB and AOA have developed distinct ammonia € nneke et al., 2014; Kozlowski et al., 2016; oxidation pathways (Ko Sayavedra-Soto and Arp, 2011), and physiological responses to substrate availability (Martens-Habbena et al., 2009; Prosser and Nicol, 2012; Qin et al., 2014). AOB and AOA co-exist in most soils and their distinct physiologies may affect their contribution to nitrification. Previous studies showed that AOB dominate nitrification in agricultural soils with high ammonium, while AOA were dominant in soils with low ammonium (Carey et al., 2016; Jia and Conrad, 2009; Offre et al., 2009; Ouyang, 2016; Taylor et al., 2010, 2013; Xia et al., 2011; Zhang et al., 2010). Included in AOB may be members of the Nitrospira that perform the recently discovered comammox process of complete ammonia oxidation to nitrate (Daims et al., 2016) although their importance in agricultural soils remains to be investigated. Both abundance and relative activity of
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AOA and AOB vary with cropping treatment, the time since N fertilization and environmental conditions (Giguere et al., 2015; Ouyang et al., 2016; Taylor et al., 2012). Ammonium concentrations and temperature range widely in cropped soils in the field. Soil NHþ 4 concentration peaks shortly after the application of mineral ammonium fertilizers but this þ available NHþ 4 is consumed quickly and subsequently soil NH4 is supplied slowly through mineralization of organic nitrogen (Habteselassie et al., 2006, 2013; Shi et al., 2004). AOA and AOB may respond differently to these fluctuating conditions; however, previous studies have usually evaluated the relative contribution of AOA and AOB to nitrification under high NHþ 4 availability and within a limited temperature range. Given the differences in physiologies of AOA and AOB, predictive models of nitrification will likely be improved by considering nitrifier community characteristics in the choice of kinetic parameters. However, this choice requires more information on the relative contribution of AOA and AOB to nitrification and on their response to NHþ 4 concentrations and temperatures typical of soils. Current models often use Michaelis-Menten kinetic rate equations but the approach for parameter estimation remains crucial (Inselsbacher et al., 2013; Vogeler et al., 2013). Greater efforts are needed to determine AOA and AOB specific kinetic parameters appropriate for agricultural soils. In a previous study, we assessed relative contributions of AOA and AOB to nitrification under organic and conventional N management. AOB dominantly contributed to nitrification in soil slurries at 1 mM NHþ 4 , while AOA dominated gross nitrification activity in soils at ambient NHþ 4 (Ouyang et al., 2016). The objective of this study is to determine the contribution of AOA and AOB to nitrification shortly after fertilization, and to determine if their kinetics and temperature responses differ in soils with contrasting N management history. We hypothesized that AOA and AOB would have distinct kinetic parameters for substrate and temperature response and that these would be affected by N management. Understanding the link between microbial communities and biogeochemical functions such as nitrification may allow models to incorporate microbial communities as dynamic components driving N flux. 2. Materials and methods 2.1. Soil characterization The agricultural site (North Logan, Utah, USA), experimental design, and treatments, have been previously described (Ouyang et al., 2016). Briefly, the experiment was initiated in April 2011 with four blocks and four nitrogen treatments: control (no N fertilization), ammonium sulfate (AS 100 and 200 kg N ha1), and steer-waste compost (200 kg N ha1) in a randomized complete block design. Treatments were applied in May of each year, incorporated immediately and corn planted within one week. The soil is an irrigated, very strongly calcareous Millville silt loam. Nearby precipitation and soil temperature (0e10 cm) data were retrieved from the Utah Climate Center (https://climate.usurf.usu.edu/ agweather.php?station_num¼424) and are shown in Fig. S1. Soils were sampled from the field at 16 days (d) before, and at 28, 57, 83, 120, and 154 d after fertilization in 2014. In 2015, samples were collected at 20 d before, and 3, 7, 14, 21, 35, and 80 d after fertilization. Six soil cores (0e15 cm depth, three cores between rows and three cores within row between plants) were taken per plot, composited, thoroughly mixed, brought to the laboratory and a sub-sample was stored at 80 C immediately. Samples from deeper in the soil profile were taken after corn harvest at 154 d after fertilization in 2014. These soil core samples were collected from
the center of each plot with a Giddings soil corer (5 cm diam.) (Giddings Machine Co., Windsor, CO) to 90-cm depth, and the core sample was split into 4 depth intervals: 0e15, 15e30, 30e60, 60e90 cm. Subsamples from each depth interval were extracted in þ 2 M KCl and analyzed for NHþ 4 and NO3 . Soil NH4 /NH3 and NO2 þ NO3 were measured with a flow injection analyzer (QuikChem 8500, methods 12-107-06-1-A, 12-107-04-1-J Lachat Instrument, Loveland CO). Since in the soil pH range over 95% of the þ NHþ 4 /NH3 is expected to be in the form of NH4 we refer to ammonium (NHþ ) concentrations throughout. The soil gravimetric 4 moisture was determined by drying soils at 105 ºC for 24 h. Soil samples were sieved (2.0 mm) and stored briefly at 4 C before nitrification potential assays. 2.2. Net nitrification For all soil sampled in 2015, net nitrification was determined by a 2-d incubation at the ambient soil moisture (Fig. S1 for moisture) using the modified method of Taylor et al. (2013). On the day of sampling, soil sub-samples (4.5 g) were placed into 150-ml glass serum bottles, capped with grey butyl stoppers and sealed. These bottles were treated with: (i) no amendment for control, (ii) acetylene (6 mM aqueous concentration (Caq)), or (iii) 1-octyne (4 mM Caq). Acetylene was used to block ammonia oxidation and to evaluate acetylene-insensitive heterotrophic nitrification. The 1-octyne acts on AOB as a differential inhibitor and was used to distinguish the contributions of AOA and AOB (Taylor et al., 2013, 2015). Samples were incubated in the dark at 25 C for 2 d. At the beginning and end of the incubation, NHþ 4 and NO2 þ NO3 concentrations were determined. No significant NO þ NO accumulation was 2 3 observed under acetylene treatment. Nitrification activity in the presence of octyne was considered to be carried out by AOA (which are octyne-resistant), and the difference between nitrification activity with and without octyne was considered to be due to AOB (octyne-sensitive). 2.3. Nitrification potential and nitrification kinetics Nitrification potentials (NP) were determined by the shaken soil slurry method (1.0 mM NHþ 4 -N) (Norton and Stark, 2011; Ouyang et al., 2016). Briefly, 4.5 g sieved soil samples were placed in 150ml bottles with caps fitted with butyl stoppers, and then 30 ml of phosphate buffer (1.0 mM phosphate at pH 7.2) with ammonium sulfate (1.0 mM final NHþ 4 -N) was added. Bottles were shaken for 2 h to equilibrate the soil and solution at 30 C. Then the differential AOB inhibitor 1-octyne was added to distinguish the contributions of AOA and AOB (Taylor et al., 2013, 2015). Five ml aliquots were sampled at 2 and 24 h after the beginning of shaking. The nitrification potentials were determined by linear regression of NO 2 þ NO3 accumulation over time. Nitrification activity by AOA and AOB was calculated as described above. Nitrification kinetics were determined for soils sampled at 28 d and 83 d after fertilization in 2014. Nitrification kinetics were assessed within two weeks of sample collection. Excess inorganic N was removed prior to assay to achieve lower starting concentrations and reduced variability of substrates and products (Koper et al., 2010). We added 4.5 g soil into 40-ml centrifuge tubes with 30 ml of buffer (1.0 mM phosphate at pH 7.2), vortexed briefly, centrifuged at 16,000g for 10 min, and then decanted the supernatant. Soil pellets were resuspended in 30 ml buffer, which þ differed in NHþ 4 concentrations, ranging from 0 to 20.0 mM NH4 , (target concentrations 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10 and 20 mM NHþ 4) generated by adding (NH4)2SO4 in solution. These slurries were transferred to 150-ml bottles and capped with butyl stoppers. Bottles were shaken continuously (200 rpm) in an orbital incubator
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shaker at 30 C for 2 h to equilibrate the soil and solution. Slurry pH was not adjusted but was buffered by the soil carbonates throughout the experiment. After 2 h shaking, sufficient 1-octyne (4 mM Caq) was added to one set of samples, and the other set served as a control. Since preliminary experiments showed that nitrification activity could be completely blocked by acetylene indicating that acetylene-insensitive heterotrophic nitrification was absent, we did not include an acetylene control in our assay. Five-ml aliquots were sampled at 2 and 24 h after the beginning of shaking. Aliquots were centrifuged at 8000 g for 8 min at 4 C, and supernatants frozen until analyzed. The concentration of NHþ 4 and NO 2 þ NO3 were measured with flow injection colorimetric methods (Ouyang et al., 2016). Nitrification rate and NHþ 4 substrate concentration data from each soil sample were fit to two kinetic models: Michaelis-Menten (Eq. (1)) and Haldane (Eq. (2)) (Koper et al., 2010; Stark and Firestone, 1996).
V ¼ Vmax S=ðKm þ SÞ
(1)
. . V ¼ Vmax S Km þ S þ S2 Ki
(2)
Where V is the nitrification rate, S is the substrate concentrations, Vmax is the apparent maximum nitrification rate, Km is apparent half-saturation constant, and Ki is apparent substrate inhibition constant. 2.4. Temperature response of nitrification potential Soil samples collected in 2014 at 83d after fertilization were used to evaluate the temperature response of NP. The NP assay was as described above, but performed at seven temperatures (5, 9, 20, 30, 35, 40 and 50 C). Q10 was calculated from the change in NP between 9 and 20 C. The generalized Poisson density function equation (Eq. (3)) was used fit to the rate and temperature data (Stark, 1996). Temperature optima of total NP, octyne-resistant NP, and octyne-sensitive NP were estimated.
V¼
( " #) c bT c bT d exp 1 ba d ba
(3)
Where V is the nitrification potential, T is the temperature, a is the temperature optimum, b is the temperature maximum, and c and d are shape parameters for portions of the curve to the right and left of the temperature optimum. 2.5. Soil DNA extraction and real-time quantitative PCR Soil DNA was extracted from field soils samples in 2014 and 2015 using the MoBio Power Soil DNA kit (MoBio Laboratories Inc, Carlsbad, USA) using 0.25 g moist soil. Quantitative PCR of AOA and AOB amoA genes was performed using the SsoAdvanced SYBR Green Supermix and a CFX CONNECT Real-Time PCR Detection System (Bio-Rad laboratories, Hercules, CA, USA). Primers, PCR conditions and calibration standards were described in Ouyang et al. (2016). Reaction efficiencies ranged from 94% to 105%, and R2 values ranged from 0.990 to 0.999. The log number of amoA copies g1 dry soil was calculated. 2.6. Statistical analysis Statistical analysis for the multiple dates for NP, amoA gene copy numbers (log transformed), and nitrification kinetic parameters were carried out using repeated measures analysis of variance (ANOVA) with the Proc Mixed model of SAS 9.2 (SAS Institute, Inc.,
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Cary, NC, USA). Treatment and sampling time were used as fixed effects and block as a random effect. Data were log transformed as necessary to meet normality assumptions. One-way ANOVA followed by Tukey's HSD was used to compare Q10 and temperature optima for treatment effects. The temperature optima of octynesensitive and octyne-resistant NP were compared by t-test. Differences were considered statistically significant at p 0.05. Kinetic models parameters were determined with SigmaPlot 11.0 (SPSS Inc., Chicago, IL). 3. Results 3.1. Inorganic N dynamics In 2014, N treatment had no significant effect on extractable NHþ 4 for dates evaluated over the growing season (Fig. 1A and Table S1). In 2015, NHþ 4 in AS treatments was highest at 3-d after fertilization, and then decreased gradually over the following five 1 weeks (Fig. 1B). Extractable NHþ 4 ranged from 0.2 to 59.2 mg N kg soil in 2014 and 2015. Extractable NO was highest across all N 3 treatments at 28 d in 2014 (Fig. 1C) and was always highest in the AS200 treatment, regardless of sampling time. Extractable NO 3 accumulated in fertilized treatments for three weeks after fertilization in 2015 (Fig. 1D). 3.2. Seasonal dynamics of AOA and AOB abundance AOA amoA copy numbers ranged from 2.1 107 to 1.1 108 copies g1 soil throughout 2014 and 2015, were not affected by treatment (Fig. 2 A&B, Table S1), and were higher than AOB copy numbers at all times (Fig. 2). AOA amoA copy numbers fluctuated by sampling time. In 2014, AOA amoA copy numbers were higher at 28d and 154d following fertilization (Fig. 2A) while in 2015, they were lowest at 35d, but highest at 80d (Fig. 2B). AOB amoA copies, ranged from 3.0 105 to 3.1 107 copies g1 soil, were significantly affected by both treatment and sampling time (Fig. 2, Table S1). AOB amoA copy numbers for AS treatments were often higher than those in control and compost treated soils. In 2014, AOB amoA copy numbers in compost treated soils increased throughout the season but were relatively stable in the other treatments. AOB amoA copy numbers in control and compost were higher in 2015 than those in 2014. AOB amoA copy numbers in AS treatments gradually increased 3e4 fold in the first two weeks after fertilization in 2015. The ratios of AOA to AOB amoA gene copy numbers, varied from 6 to 242, were strongly affected by treatment and time, and were higher under control and compost treatments (Table S2). AOA amoA copy numbers were not significantly correlated with total NP (r ¼ 0.001, p ¼ 0.99) or octyne-resistant NP (r ¼ 0.07, p ¼ 0.14). However, AOB amoA copy numbers were positively correlated with total NP (r ¼ 0.66, p < 0.001) and octyne-sensitive NP (r ¼ 0.64, p < 0.001). 3.3. Nitrification kinetics The Vmax and Km compared in the text are those predicted by the Michaelis-Menten model (Table 1; Fig. 3 shows results at 28d and Fig. S2 shows results at 83d). The Vmax for octyne-sensitive nitrification activity ranged from 0.32 to 4.8 mmol N kg1 d1 and was affected by both treatment and sampling time. The Vmax of octynesensitive nitrification activity in AS treatments were significantly higher than those from control and compost treatments (Table 1 and Table S4). Furthermore, octyne-sensitive nitrification Vmax in AS treatments were higher at 28d than at 83d, while the Vmax in control and compost treated soils did not change between 28d and 83d. Vmax of octyne-resistant nitrification activity was not affected by treatments (Table S4), and the mean Vmax was 0.24 mM N kg1
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Fig. 1. Soil KCl-extractable ammonium in 2014 (A) and 2015 (B), and KCl-extractable nitrate in 2014 (C) and 2015 (D) across four N treatments (control (no N fertilization), ammonium sulfate (AS, 100 & 200 kg N ha1), and compost (200 kg N ha1)). Arrows denote fertilizer application. Error bars represent standard errors (n ¼ 4). Different lowercases above the bars indicate a significant difference among treatments in a specific sampling time (p 0.05), based on repeated measures ANOVA.
d1. Vmax of total nitrification activity ranged from 0.54 to 5.00 mmol N kg1 d1, with the same pattern as the Vmax of octynesensitive nitrification activity. The Vmax for octyne-sensitive nitrification was significantly correlated with AOB amoA copy numbers (r ¼ 0.48, p ¼ 0.006, n ¼ 32), while Vmax for octyne-resistant nitrification was not correlated with AOA amoA copy numbers (r ¼ 0.22, p ¼ 0.23, n ¼ 32). The Km of octyne-sensitive nitrification activity showed significant treatment and time effects (Table S4). Octyne-sensitive nitrification Km was highest in AS200 and lowest in control treatment, ranging from 14 to 161 mM NHþ 4 . Octyne-sensitive nitrification Km were higher at 28d than at 83d among all N treatments (Table 1). The Km of octyne-resistant nitrification activity was not significantly different among the treatments and time, and the mean Km was 4.28 mM NHþ 4 across all treatments and times (Table 1). The Km of total nitrification activity ranged from 6.8 to 136 mM NHþ 4 , with the same pattern as the changes in Km of octynesensitive nitrification activity. Haldane model was also used to predict nitrification kinetic parameters. Haldane model provided a slightly better fit (higher R2) for the octyne-resistant nitrification rates than the MichaelisMenten model (Table S3). Octyne-resistant nitrification activity showed a slight inhibition by high NHþ 4 concentration both at 28d and 83d, but Ki predicted by the Haldane model was highly variable and Ki was not significantly different among the treatments and
time (Table S3). The Ki values indicated that octyne-sensitive nitrification activity by AOB was more tolerant to high NHþ 4 concentrations than the octyne-resistant nitrification activity by AOA. Octyne-resistant fraction of NP was close to 100% when no or very low ( 35 C. 3.5. Seasonal dynamics of nitrification potential and net nitrification Treatment and sampling time had significant effects on octynesensitive NP in 2014 and 2015 (Table S1). Octyne-sensitive NPs ranged from 0.3 to 4.6 mM N kg1 d1 throughout 2014 and 2015 (Fig. 5). Soil octyne-sensitive NP rates in fertilized soils (AS and compost) were higher than those in the control soil. In 2014, octyne-sensitive NPs in AS treatments were highest at 28 d and lowest at 57 d, when the soil temperature was highest during the growing season. In 2015, octyne-sensitive NPs in AS treatments were highest at one week after fertilization, and then declined gradually during the five weeks after fertilization. Octyne-sensitive NPs in control and compost treatments were relatively stable throughout 2014 and 2015. Total NP activity showed the same pattern as the changes in the octyne-sensitive NP (Fig. 5). Octyne-resistant NPs, ranged from 0.15 to 0.47 mM N kg1 d1, were always lower than octyne-sensitive NPs throughout the growing season in 2014 and 2015 (Fig. 5). In 2014, octyne-resistant NP showed no significant treatment effect, but significant sampling time effect (Table S1). Octyne-resistant NPs slightly increased through the season in both years. Octyne-resistant fraction of NP was often lower than 50% (Table S5), indicating that AOB contribute dominantly to NP under all N treatments. However, octyne-
resistant fraction of NP was significantly affected by treatment and sampling time (Table S1). In AS treatments, the octyneresistant fraction of NP ranged from 7.2 to 20.8%, while it ranged from 18.6 to 47.7% in control and compost treatments, reflecting higher AOA contribution. AOA amoA copy numbers were not significantly correlated with total NP (r ¼ 0.001, p ¼ 0.99) or octyne-resistant NP (r ¼ 0.07, p ¼ 0.14). However, AOB amoA copy numbers were positively correlated with total NP (r ¼ 0.66, p < 0.001) and octyne-sensitive NP (r ¼ 0.64, p < 0.001). Net nitrification rates were stimulated 10e18-fold in AS treatments the first week after fertilization, and they were often higher in fertilized treatments than that in control treatment (Fig. 6). Octyne-sensitive net nitrification rates in AS treatments significantly increased the first week after fertilization (Table S1), and they contributed more than 80% of total net nitrification rates. Octyne-resistant net nitrification rates in AS treatments also increased at two weeks after fertilization. Octyne-resistant net nitrification dominated net nitrification for most sampling times except at three and seven days after fertilization. 3.6. Nitrification potential through the soil profile The NP of both AOA and AOB decreased with soil depth, theAOB potential activity usually played a dominant role in surface soils, while AOA potential activity was more dominant lower in the profile (Table S6 Fig. 7). Octyne-sensitive NP showed statistical
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Fig. 4. Temperature response of octyne-sensitive (A), octyne-resistant (B), and total (C) nitrification potential, and octyne-resistant fraction of the nitrification potential (D) in soils sampled at 83 days after fertilization in 2014. Points represent means of four replicates. Lines (A, B & C) were predicted by generalized Poisson density equation. Error bars represent standard errors (n ¼ 4).
Fig. 5. Effect of N treatment and sampling time on octyne-sensitive (AOB) nitrification potential, ocytne-resistant (AOA) nitrification potential, and total nitrification potential during the growing season in 2014 (A) and 2015 (B). Arrows denote fertilizer application. Octyne-resistant nitrification rates are the shaded bottom portion of the bar, octynesensitive nitrification rates are the lighter top portion of each bar. Error bars represent standard errors (n ¼ 4). Different lowercases above the bars indicate a significant difference for total nitrification potential among treatments in a specific sampling time (p 0.05), based on repeated measures ANOVA.
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Fig. 6. Effect of N treatment and sampling time on octyne-sensitive (AOB) net nitrification, octyne-resistant (AOA) net nitrification, and total net nitrification during the growing season in 2015. Octyne-resistant nitrification rates are the shaded bottom portion of the bar, octyne-sensitive nitrification rates are the lighter top portion of each bar. Error bars represent standard errors (n ¼ 4) of each portion. Different lowercases above the bars indicate a significant difference for total net nitrification among treatments in a specific sampling time (p 0.05), based on repeated measures ANOVA.
differences among treatments and depths (Table S6). Octynesensitive NP in AS200 was always higher than other treatments at all soil depths (Fig. 7). Octyne-sensitive NPs decreased significantly from 0 to 15 to 15e30 cm in AS treatments, while they did
not change in control and compost treatments. Octyne-resistant NP decreased significantly with soil depth, but were not different by treatment (Fig. 7, Table S6), total NP showed the same pattern as the changes in the octyne-sensitive NP activity. Octyne-resistant
Fig. 7. Effect of N treatment and depth on octyne-sensitive (AOB) nitrification potential (A), ocytne-resistant (AOA) nitrification potential (B), total nitrification potential (C), and octyne-resistant fraction of the nitrification potential (D) in soils sampled at 154 days after fertilization in 2014. Error bars represent standard errors (n ¼ 4). Different lowercases above the bars indicate a significant difference among treatments in a specific sampling time (p 0.05), based on repeated measures ANOVA. A complete summary of the repeated measurement ANOVA result is shown in Table S6.
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fraction of NP did not increase deeper in the soil profile for the AS200 treatment, the higher amount of AS application stimulated octyne-sensitive NP in subsurface soils (Table S6). Octyne-resistant fractions were lower (20e48%) in surface soils (0e30 cm), but higher (47e77%) in deeper soils (30e90 cm) in the control, AS100 and compost treatments, signifying a relative increase in AOA contribution to NP deeper in the profile. 4. Discussion 4.1. Ammonia oxidization kinetics and temperature response Ammonium concentration is a key factor determining the relative contribution of AOA and AOB to nitrification in this agricultural soil. AOB dominantly contribute to nitrification under nonlimiting ammonium while AOA dominate nitrification at low ammonium concentration. Although previous studies specifically examined the soil AOA and AOB in response to NHþ 4 addition (Giguere et al., 2015; Ouyang et al., 2016; Taylor et al., 2013), the current study is the first direct measurements of the kinetic parameters for soil AOA and AOB communities exposed to NHþ 4 concentrations spanning the range from below Km to high enough to cause substrate inhibition. In the agricultural soil environment immediately following ammonium fertilization, AOB activity increases and growth occurs. We observe that at higher than 0.2 mM NHþ 4 concentrations AOB are responsible for about 60e90% of the NP (Fig. 3) because at these concentrations AOB respond to additional NHþ 4 while AOA's capacity is saturated. At lower concentrations of NHþ 4 (i.e. 80d post fertilization), AOA are functioning and responsible for a higher relative amount of nitrification activity. AOA do have a high affinity for NHþ 4 as indicated by Km, but their activity reaches its maximum below 0.03 mM NHþ 4 while nitrification by AOB continues to increase (Fig. 8). Therefore, AOA's relative contributions to nitrification activity decreases rapidly with increasing NHþ 4 availability such as occurs in recently fertilized
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soils. The Michaelis-Menten model was an appropriate model for nitrification in this agricultural soil. The modeled Vmax for total nitrification activity across four N treatments ranged from 0.54 to 5.00 mmol N kg1 d1. Similarly high levels of nitrification activity have been seen in other agricultural soils (Koper et al., 2010; Norton and Stark, 2011; Taylor et al., 2010). Although the modeled Vmax for soil AOB activity was much higher in AS treatments than that in control and compost treatments, there was no difference when the rate was expressed on a per AOB cell basis. Assuming each AOB cell has 2.5 amoA gene copy, and using the modeled Vmax of soil AOB activity, we calculated ammonia oxidation rate of AOB was about 95 fmol NH3 oxidized cell1 h1. This rate was higher than ammonia oxidation rates (4e23 fmol NH3 oxidized cell1 h1) of AOB cultures (Belser and Schmidt, 1980; Jiang and Bakken, 1999). One contributing reason was that our measurements were carried out at 30 C, which is higher than the temperature (22 C) used for the pure culture measurements. Our temperature response assays indicated that AOB potential rates strongly increased with temperature in the range of 22e30 C. The Vmax for AOA activity was not affected by N treatments, regardless of rates expressed in per g soil or per cell. We calculated ammonia oxidation rate of AOA was about 0.19 fmol NH3 oxidized cell1 h1, with the assumption that each AOA cell has one amoA gene copy. This ammonia oxidation rate of AOA is within the range of 0.02e0.35 fmol NH3 oxidized €nneke et al., cell1 h1 that has been reported for AOA cultures (Ko 2005; Lehtovirta-Morley et al., 2011; Tourna et al., 2011). Using the calculation above, AOB per cell rate was about 500 times higher than AOA. Interestingly, half saturation constant Km for AOB activity was affected by N treatment. Since Km differed among AOB populations and among AOB communities from different soil types, we would expect that AOB communities were different among treatments. In the previous study on communities of ammonia oxidizers we demonstrated that AOB community structure was affected by N
Fig. 8. Effect of NHþ 4 concentration on octyne-sensitive nitrification (AOB) and octyne-resistant nitrification (AOA) described by the Michaelis-Menten model across four N treatments: control (A), AS100 (B), AS200 (C), and compost (D) in soils sampled at 28 days after fertilization in 2014. Vmax and Km parameters of AOB nitrification are used from Table 2 for each treatment. Vmax and Km of AOA nitrification averaged 0.24 mmol N kg1 d1 and 4.28 mM NHþ 4.
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treatments, and that soils in the AS treatments harbored different AOB communities compared to control and compost treatments (Ouyang et al., 2016). In the current study, Km for AOB activity was higher in AS treatments than in control and compost treatments; however, the Km for AOA activity was not influenced by N treatments. Estimated half-saturation constant (Km) were about 3e40 times higher for AOB activity than for AOA activity. The lower Km for AOA is consistent with Martens-Habbena et al. (2009), who showed that AOA have a significantly higher substrate affinity. This lower Km of AOA may be explained by surface area to enzyme ratios or possible transporters for NH3/NHþ 4 (Prosser and Nicol, 2012; Urakawa et al., 2011). It is reported that Km of cultured AOB ranged from 50 to 1780 mM NHþ 4 (Koper et al., 2010), and specifically, Km of Nitrosospira strains ranged from 167 to 380 mM NHþ 4 (Bollmann et al., 2005; Jiang and Bakken, 1999), which is slightly higher than Km observed in our soil slurry assay. In our soils, most AOB populations were closely related to Nitrosospira multiformis, Nitrosospira sp. NP39-19, and Nitrosospira sp.Wyke8 (Ouyang et al., 2016), but no Km has been determined for those specific AOB cultures. The Km of enriched or pure AOA cultures ranged from 0.13 to 0.69 mM ammonium (Jung et al., 2011; Martens-Habbena et al., 2009; Park et al., 2010). Our Km of soil octyne-resistant nitrification activity (2.9e6.0 mM) was much higher than those AOA cultures but may be similar to soil Nitrososphaera (Tourna et al., 2011). The AOA community in our soils is likely distinct from the enrichment or pure culture AOA from aquatic or marine systems. Some AOB cultures and all the currently available AOA cultures are sensitive to high concentration of NHþ 4 (He et al., 2012; Prosser and Nicol, 2012; Tourna et al., 2010). In the current study, we did not observe significant NHþ 4 sensitivity for AOB activity, and there was only slight reduction of AOB activity at 83d when 20 mM NHþ 4 was added to the soils. In contrast to AOB, AOA activity was sensitive to high NHþ 4 concentration, and its activity was slightly inhibited when >5 mM NHþ 4 was added to the soil slurry. Specific growth rates of two soil isolated AOA cultures, Nitrosotalea devanaterra and Nitrososphaera viennensis, also decreased with increasing NHþ 4 concentration from 1 to 10 or 15 mM, and their growth was completely inhibited at 50 mM and 20 mM NHþ 4, respectively (Lehtovirta-Morley et al., 2011, 2016; Tourna et al., 2011). Our nitrification kinetics analysis also showed that fertilization significantly influenced the response of AOB but not AOA to NHþ 4 concentration, and this larger AOB community in the repeatedly AS treated soils would be competitive for NHþ 4 with AOA even in the low NHþ 4 range (Fig. 8B&C). This is in contrast to the AOB communities under control and compost treatments (Fig. 8A&D) which would not be as competitive for low concentrations of NHþ 4 . For our þ soils using soil water content, KCl-extracted NHþ 4 , and NH4 sorption capacity based on a Langmuir model (Venterea et al., 2015) (Fig. S4), we estimated concentrations of NHþ 4 in the soil solution ranged from 6 to 50 mM (with mean value of 28 mM) at most sampling times, but from 117 to 1728 mM for the three weeks immediately after fertilization with AS. Our kinetic assays indicated that AOB could be effective competitors in low NHþ 4 ranges, especially under AS treatments (Fig. 8). However, net and gross nitrification rates in whole soils confirmed that AOA were dominant in carrying out nitrification at these low soil solution ranges. This contradictory result may suggest a strong limitation of NHþ 4 diffusion for AOB activity in whole soils, especially at the low soil NHþ 4 concentration. The larger AOB cell with a faster per cell rate of oxidation would develop larger depletion zones and experience lower than the bulk soil concentration of substrate. In this current study, we observed a preference for higher temperatures for AOA than AOB as indicated by 1) higher optimum temperature for AOA activity (40.8 C) than for AOB activity
(31.2 C); 2) increased relative contribution of AOA to NP from 5 to 50 C; and c) higher Q10 for AOA activity. AOA are thought to originate from a thermophilic ancestors; the broad distribution of AOA in various mesophilic environments is thought to be the result of secondary adaptations to mesophily (Brochier-Armanet et al., 2012). For example, two soil isolates, Nitrososphaera viennensis and Candidatus Nitrosocosmicus franklandus, have optimum temperature for growth at 37 C and 40 C, respectively (LehtovirtaMorley et al., 2016; Tourna et al., 2011). A recent study in Oregon soils also found that the optimum temperatures of soil AOA were more than 12 C higher than soil AOB (Taylor et al., 2017). In contrast to AOA, AOB may prefer lower temperature environments with the optimum temperatures range 20e31 C (Table 2). Currently there are no literature reports of AOB in environments where temperature remains higher than 40 C (Hatzenpichler, 2012). AOB communities were mainly affiliated with the Nitrosospira lineage in our soils (Ouyang et al., 2016), strains of Nitrosospira isolated from soil environments have temperature optimum between 25 C and 31 C (Table 2). Avrahami et al. (2003) found that the maximum ammonia oxidation activity was between 10 C and 25 C in a Nitrosospira dominated soil. In this study, Q10 values for soil AOB (determined from 9 to 20 C) fall within the range of 1.7e2.7 found in AOB (Stark and Firestone, 1996). However, the average Q10 values for soil AOA (determined from 9 to 20 C) of 5.3, were much higher than those for AOB, suggesting global warming may have a much stronger effect on the activity of soil AOA than AOB. Temperature optimum of total NP activity was significant higher in control and compost treatments (35.5 C) than that in AS treatments (32.2 C). Stark and Firestone (1996) found significant difference of temperature optima of NP between soils from open grassy interspaces (35.9 C) and soils from beneath oak canopies (31.8 C), and they attributed the difference in temperature optima to seasonal differences. Our results suggest that the observed differences in temperature optimum for soil NP were due to different contributions from AOB and AOA to nitrification. To our knowledge, this is the first study to estimate differential nitrification kinetic parameters for soil AOA and AOB in soil. Evidence indicated AOA and AOB showed distinct responses to ammonium concentrations and to temperature. Our results may be used to parameterize mechanistic models of nitrification. Although current nitrification models try to incorporate mechanistic considerations of nitrifier characteristics, the approaches vary widely (Cannavo et al., 2008; Vogeler et al., 2013). Michaelis-Menten kinetics may be appropriate for nitrification since changes in substrate concentration cause shifts from first order to pseudo zero order rate relationships (Inselsbacher et al., 2013). Our observations suggest that these shifts may occur at different substrate concentrations for AOB versus AOA. Nitrification is a process that would benefit from incorporating microbial community characteristics (i.e., the abundance or relative abundance of AOB versus AOA) into kinetic parameters for models. 4.2. Variability in abundance and activity of AOA and AOB AOA abundance showed no long-term or short-term fertilization effect during this two-year study, while AOB abundance were significantly higher after four or five years of repeated AS treatments. Interestingly, we also observed a significant further increase in AOB abundance in short periods (less than one month) after AS application. This is consistent with other field studies, which reported 3e10-fold increases in AOB population size two or four weeks after AS application (Dai et al., 2013; Okano et al., 2004; Taylor et al., 2012). AOB potential rates were more responsive than AOA, peaking
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Table 2 Temperature parameters for AOA and AOB soil isolates or enrichments. AO
Organisms
Soil habitat
AOA AOA AOA AOA AOA AOA AOA AOA AOB AOB AOB AOB AOB AOB
Nitrosoarchaeum koreensis MY1 Nitrosoteuis. chungbukensis MY2 Nitrososphaera sp. JGI Nitrosotalea devanaterra Nd1 Nitrosotalea sp. Nd2 Nitrosocosmicus franklandus C13 Nitrososphaera viennensis EN76 Nitrososphaera viennensis EN123 Nitrosomonas europaea Nm35 Nitrosomonas europaea Nm35 Nitrososphira 40K1 Nitrososphira AF Nitrosomonas europaea ATCC 19178 Nitrosospira multiformis ATCC 25196 Nitrosospira briensis C-128
Agricultural Agricultural Agricultural Acidic soils Agricultural Agricultural Garden soil Garden soil Soil isolate Soil isolate Agricultural Agricultural Agricultural Soil isolate
AOB
soils soils soils soils soils
soils soils soils
Soil isolate
Enriched temperature
Topt
Growth range
References
25 25 37 28 37 37 37 37 30 20 22 22 25 25e30
25 30 35e40 25 35 40 35 37 30 20 26 31 25 28
20e25 25e40 25e40 20e30 20e42 30e45 28e47 32e37 0e40 0e40 3e36 3e36 20e25 15e30
Jung et al., 2011 Jung et al., 2014 Kim et al., 2012 Lehtovirta-Morley et al., 2011 Lehtovirta-Morley et al., 2014 Lehtovirta-Morley et al., 2016 Tourna et al., 2011 Tourna et al., 2011 Groeneweg et al., 1994 Groeneweg et al., 1994 Jiang and Bakken, 1999 Jiang and Bakken, 1999 Jung et al., 2011 Norton et al., 2008
25e30
25e28
15e30
Rice et al., 2016
*Topt indicates the optimum temperature. The unit for temperature is C.
quickly after AS fertilization, suggesting a quick response of AMO synthesis or activation in AOB cells. As NHþ 4 was consumed, AOB potential rates were reduced to the pre-fertilization level. Interestingly, AOB potential rates were lowest in July, suggesting high soil temperatures may be an important factor constraining activity of AOB in the field. Although AOA potential rates were resistant to change by N fertilization and soil temperature, they were often highest at the end of the season. Our results suggest that AOB quickly consume most of the ammonium shortly after fertilization, and strategies to immediately inhibit AOB activity after mineral N fertilization would be effective to delay nitrification in the field. Our observations indicated that nitrification activity by both the AOB and AOA decrease with soil depth. The fraction of nitrification activity due to AOA often increased in the deeper soils except for the AS200 treatment, implying the higher amount of AS application stimulated octyne-sensitive NP for these subsurface soils. Several studies have demonstrated that both AOA and AOB abundance decrease with soil depth, the ratio of AOA to AOB increase and NP decreases (Jia and Conrad, 2009; Wang et al., 2014). In summary, mineral fertilizers quickly stimulated AOB abundances and activity, compared to control and compost. AOA abundances and their response to ammonium were not affected by N treatments. AOB dominantly contribute to nitrification under nonlimiting ammonium while AOA dominate nitrification at low ammonium concentration. The per cell rate of ammonium oxidation and the half-saturation constant for AOA activity were much lower than those for AOB. The relative contribution of AOA to NP increased with increasing temperature, and AOA had a higher temperature optimum than AOB. Understanding the differential response of AOB and AOA to substrate and temperature may allow the AO microbial community to be included into simulation models as dynamic components driving N flux. Conflict of interest The authors declare no conflict of interest. Acknowledgments We would like to thank Danielle Barandiaran, Brittany Johnson, Cory Ortiz, Marlen Craig Rice for laboratory and field assistance. This research was supported by grants from the USDA NIFA Awards 2011-67019-30178, 2016-35100-25091 and by the Utah Agricultural Experiment Station, Utah State University and approved as journal paper #8912.
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