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Manganese oxide affects nitrification and ammonia oxidizers in subtropical and temperate acid forest soils
Catena 137 (2016) 24–30
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Manganese oxide affects nitrification and ammonia oxidizers in subtropical and temperate acid forest soils Xiaoping Xin a, Xianjun Jiang a,⁎, Jing Su a, Xiaojuan Yan a, Jiupai Ni a, Sarwee J. Faeflen a, Xueru Huang a, Alan L. Wright b a b
College of Resources and Environment, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400715, China Everglades Research & Education Center, University of Florida, Belle Glade, FL 33430, United States
a r t i c l e
i n f o
Article history: Received 24 May 2015 Received in revised form 1 September 2015 Accepted 6 September 2015 Available online xxxx Keywords: Nitrification Ammonia oxidizing bacteria Ammonia oxidizing archaea Manganese
a b s t r a c t Variations in nitrification dynamics were observed both in temperate and subtropical acidic forest soils. The effect of Mn on nitrification was studied to explain variation in nitrification between different soil types. Weakly and highly acidic soils in subtropical and temperate forests were treated with 0% or 3% birnessite. The nitrification process was simulated by kinetic model. Dynamic changes of amoA gene abundance of ammonia monooxygenase (AMO) for ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were estimated by qPCR. Nitrification rates were significantly decreased by Mn addition in 3 days both for the weakly acidic subtropical and temperate soils. However, the total nitrification rate did not change for either soil by Mn addition after 10 days' incubation. Nitrification was best fitted by a firstorder kinetic model for both weakly acidic soils. However, it was best fitted with a zero-order model after MnO2 addition. Numbers of AMO amoA gene copy decreased after Mn addition. No significant nitrification was observed for highly acidic soils and Mn had a minimal effect. Soil nitrification was retarded by 3% MnO2 for both subtropical soil and temperate soils. Nitrification dynamics were altered by MnO2 in weakly acidic soils, probably due to Mn toxicity. © 2015 Published by Elsevier B.V.
1. Introduction Great variation of nitrification potential in acid soils has been reported (Compton et al., 2004; De Boer and Kowalchuk, 2001; Nugroho et al., 2009) due to numerous biotic and abiotic factors. It is now accepted that nitrification can occur in a wide range of acid soils (De Boer and Kowalchuk, 2001), but there are also many acid soils for which nitrification appears to be minimal or absent (Robertson, 1982). Biotic and abiotic factors such as pH, N availability and microbial community structure are responsible in part but not completely for the variation in nitrification rates in acid soils (Bäckman and Klemedtsson, 2003; Compton et al., 2004; Nugroho et al., 2007; Tolli and King, 2005). For example, it was reported that net nitrification rates could vary 3-fold in two acid soils within a given pH (Nugroho et al., 2007). NH+ 4 -N content also did not constrain net nitrification, as some temperate acidic forest soils having high NH+ 4 -N concentrations did not experience net nitrification (Nugroho et al., 2007; Zhao et al., 2007). Nugroho et al. (2009) also concluded that low net nitrification rates in acid Scots pine forest soils cannot be solely explained by unfavorable abiotic soil conditions
⁎ Corresponding author. E-mail address: [email protected] (X. Jiang).
http://dx.doi.org/10.1016/j.catena.2015.09.004 0341-8162/© 2015 Published by Elsevier B.V.
after observing the bacterial community structure and interactions between members of the bacterial community. Therefore, there are still uncaptured biotic factors or abiotic factors that contribute to suppression of nitrification in acid soils. Manganese is one of the elements released during the weathering of primary minerals, which explains its common accumulation in most soils in the range of 0.07–4.0% (Aller, 1990; Fujimoto and Sherman, 1948). Manganese activity is closely related to soil pH, as its concentrations in soil solution increase 100-fold for each pH unit decrease (Hue et al., 2001). Hence, Mn toxicity may be one of the important abiotic stresses on nitrification in acidic soils (Marschner, 1991). Stimulation, retardation, or inhibitation of nitrification by Mn has been observed in different ecosystems and a variety of soil types depending on Mn concentration (Leeper, 1970; Premi and Cornfield, 1969; Wilson, 1977). Manganese toxicity to microorganisms may be responsible for the nitrification inhibition, but no data are as yet available to substantiate this hypothesis. Furthermore, the mechanistic patterns of nitrification altered by Mn are poorly understood. We hypothesize that the effects of Mn on nitrification occur in different temperate and subtropical soils. Both highly acidic and weakly acidic forest soil samples from temperate area (brown soils) and subtropical area (purple soils) were selected to test this hypothesis by investigating the effect of Mn on the nitrification processes and nitrifying microorganisms.
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2. Material and methods 2.1. Site description and soil sampling The subtropical soil samples (purple soil, Inceptisols) were collected from a subtropical forestland in Yongchuan, Chongqing, southwest of China (29°16′N, 105°84′E). This region has the annual mean temperature 19.7 °C and annual mean rainfall 1400 mm, with middle subtropical evergreen broad-leaved forests. Two sites were chosen to obtain weakly acidic soil and highly acidic soil. Temperate soil samples (brown soil, Luvisols) were collected from Dahei Mountain in Dalian, Liaoning Province, northeast of China (38°43′N, 120°58′E) where the climate is between monsoon and continental with annual mean temperature 10.2 °C (ranging from − 19.1 °C to 37.8 °C) and annual mean rainfall 810 mm. The two sampling sites were weakly acidic and highly acidic soils. Five soil cores (0–20 cm) were collected from a 4 m × 4 m plot using a soil corer (Ø = 13 cm). Portion of the soil samples collected was pooled and homogenized to reduce heterogeneity. The samples were air-dried and separated into two parts. One was ground to pass a 2-mm sieve and stored at 4 °C prior to make subsamples for incubation; another was ground to pass a 1-mm sieve and used for chemical analyses. 2.2. Preparation of birnessite (&-MnO2) and samples Birnessite (&-MnO2) is a naturally occurring, poorly crystalline oxide of tetravalent manganese. It is one of the most common forms of mineralized Mn in soils (Taylor et al., 1964). Birnessite was prepared by dropwise addition of HCl to KMnO4 (McKenzie, 1971). The precipitate was washed, dried, and aged at 60 °C for 12 h, and again washed with water. Obtained birnessite was ground to pass through a 1-mm sieve and stored at 4 °C prior before use. The obtained birnessite was checked by X-ray powder diffractometer (XRD). Subsamples were prepared by amendment with 0% (unmended) or 3% birnessite by weight. Eight subsamples were obtained as follows: weakly acidic subtropical soil, weakly acidic subtropical soil + 3% birnessite, highly acidic subtropical soil, highly acidic subtropical soil +3% birnessite; weakly acidic temperate soil, weakly acidic temperate soil +3% birnessite, highly acidic temperate soil, highly acidic temperate soil + 3% birnessite. Each subsample was mixed thoroughly and ground to pass a 1-mm sieve and stored at 4 °C for 2 months before use. 2.3. Physical and chemical analysis Soil properties were determined following Soil Agro-Chemical Analyses procedures (Lu, 2000). Soil pH was measured in a soil to water ratio of 1:2.5 (v/v) by a DMP-2 mV/pH detector (Quark Ltd., Chongqing, China). Soil organic matter (SOM), total N and kjeldahl N contents were determined by a Macro Elemental Analyzer (Elementary Analysensysteme GmbH, Hanau, Germany). Total soil Fe and Mn were determined by digesting the soil with HNO3–HF–HClO4, and HClextractable Fe and Mn in soil were extracted by shaking 150 g of soil in 300 ml of 0.002 M HCl for 1 h. The concentration of Fe and Mn in the digesters and extracts was determined by atomic absorption spectrophotometry with a graphite furnace (GFAAS) using a model Z-8200 spectrophotometer. 2.4. Incubation For each subsample, 20 g soil was placed into a 250-ml plastic bottle. Distilled water was added to adjust the moisture content to 20% by weight (50% of water holding capacity). All bottles were covered with polyethylene film punctured with needle holes to maintain aerobic conditions, and pre-incubated at 28 °C in the dark for 7 days. After preincubation, 120 mg N kg−1 dry soils were added as (NH4)2SO4 to assess
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nitrification rate. The loss of water through evaporation was compensated by addition of distilled water daily. At the interval of 0, 1, 3, 7 and 10 days after incubation days, subsamples in four replicates were taken and extracted with 100 ml of 2 M KCl for 1 h (Keeney and Nelson, 1982). The concentrations of inorganic N forms in the extracts were determined using SKLAR continuous-flow analyzer (SKLAR San ++, Netherland, 2003). The net nitrification rate was calculated using the following equation (Robertson et al., 1999): Net nitrification rate ¼
− NO− 3 −N f − NO3 −N i =T d
where the subscripts i and f indicate concentrations measured before and after aerobic incubation, respectively and Td indicates incubation time in days. Net ammonification and net nitrification were expressed as ammonia-oxidizing archaea mg N kg−1 day−1 dry soil per day (Francis et al., 2005). 2.5. DNA extraction and quantitative PCR assay Right after pre-incubation, 4 replicate bottles of each treatment were randomly selected to extract DNA and analysis of amoA genes was performed by quantitative PCR (qPCR). The DNA was extracted from 0.50 g of soil with the Fast DNA Spin Kit for soil (MP Biomedicals, United States), according to the protocol of the manufacturer. The quality and quantity of the extracted DNA were analyzed with a spectrophotometer (Nanodrop, PeqLab, Germany), and pooled and stored at −20 °C until use. Quantitative PCR of amoA genes was performed to estimate the abundance of the ammonia-oxidizing bacterial and archaeal communities, respectively. The primers amoA-1 F (5′-GGGGTTTCTACTGGTGGT3′) and amoA-2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) were used for ammonia-oxidizing bacteria generating a 491 bp fragment; ArchamoA F (5′-STAATGGTCTGGCTTAGACG-3′) and Arch amoA R (5′-GCGG CCATCCATCTGTATGT-3′) were used for generating a 635 bp fragment (Francis et al., 2005). Quantification was based on the fluorescence intensity of the SYBR Green dye and reactions for each sample were carried out in a Bio-Rad CFX-96 thermal cycler. The quantification of amoA genes was performed in a total volume of 25 μl reaction mixtures with 12.5 μl of SYBR Premix Ex Taq™ as described by the suppliers (Takara Bio, Otsu, Shiga, Japan), 0.25 μl of each primer (50 μm), 1 μl of soil DNA template, with a final content of 1–10 ng in each reaction mixture, and 11 μl ddH2O. The fragments for the AOB and AOA were both amplified using an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C, 30 s at 72 °C for AOB, and 45 s at 72 °C for AOA for the collection of fluorescence data. All reactions were finished with a melting curve starting at 65 °C with an increase of 0.5 °C up to 95 °C to verify amplicon specificity. The PCR reaction runs had an efficiency of 90% and 94% for the AOB and AOA, respectively. Standard curves for the AOB and AOA were obtained using serial dilutions of linearized plasmids (pGEM-T, Promega) containing cloned amoA genes amplified from environmental clones (r2 N 0.995 for both standard curves). 2.6. Statistical analysis The changes in NO− 3 N content with incubation time were modeled with a first-order reaction kinetic model, expressed as NNO3 = N0 + NP (1 − exp.(− k1t), or by a zero-order reaction kinetic model, expressed as NNO3 = N0 = k0t, where NNO3 was NO− 3 N content at incubation time t; N0 was NO− 3 N content after pre-incubation (t = 0); Np was nitrification potential; and k1 and k0 were rate constants of firstand zero-order reactions, respectively. The potential nitrification rate (Vp) was calculated from first-order kenetics as Vp = k1 * Np (Oorts et al., 2007). The mean actual net nitrification rate (Va) was calculated as Va = (N10 − N0)/10, here N10 was NO− 3 N content after 10 days' incubation (t = 10).
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Table 1 Selected soil properties. Soil species
Subtropical soil Temperate soil
pH
4.60 6.30 4.50 6.10
SOM
Total N
Kjeldahl nitrogen
HCl-extractable Mn
Total Mn
HCl-extractable Fe
Total Fe
(g kg−1)
(g kg−1)
(mg kg−1)
(mg kg−1)
(g kg−1)
(mg kg−1)
(g kg−1)
11.9 12.9 47.8 36.6
0.75 0.97 0.85 1.01
90.6 76.0 115 96
13.5 11.7 20.6 14.0
0.33 0.45 0.66 0.45
233 11.8 256 14.2
21.1 34.9 46.9 50.7
SOM represents soil organic matter.
Data (measured or calculated) were subjected to one-way ANOVA and mean values were separated using Duncan's New Multiple Range Test at P b 0.05. All statistical analyses were performed by the SPSS statistical package.
soil, which was about half of the content of temperate soil. 3% birnessite significantly increased the concentration of HCl-extractable Mn in highly acidic soils to 294 mg kg−1 for subtropical soil and 266 mg kg−1 for temperate soil, respectively (Fig. 1).
3. Results
3.2. Effects of Mn oxides on soil net nitrification rate and kinetic patterns
3.1. Selected soil properties
−1 soil after preThe NO− 3 N concentration was 9.67 mg N kg incubation for weakly acidic subtropical soil. The largest net nitrification rate (4.76 mg N kg−1 day−1) was observed in the first 3 days' incuba−1 soil in tion (Fig. 2). The NH+ 4 -N concentration was 104.3 mg N kg −1 dry soil, and sharply one hour after addition of 120 mg N kg decreased to 15.7 mg N kg− 1 after 3 days' incubation. At the end of −1 (Fig. 3). the incubation, NH+ 4 -N concentration was 12.7 mg N kg − Though the NO3 N concentration increased linearly by 3% birnessite addition during the whole incubation period, the largest net nitrification rate decreased significantly to 1.94 mg N kg− 1 day−1 in the first 3 days' incubation, and average net nitrification rate by 10 days (1.67 mg N kg−1 day−1) did not differ significantly. NH+ 4 -N concentration did not differ significantly at the end of the incubation with or without Mn oxide addition in weakly acidic subtropical soil. Significant nitrification also occurred in weakly acidic temperate soil. A higher net nitrification rate (2.76 mg N kg− 1 day− 1) was observed in the first 3 days' incubation, compared to the average net nitrification rate (1.13 mg N kg− 1 day− 1) for the whole 10 days' incubation (Fig. 2). −1 soil one hour after addiThe NH+ 4 -N concentration was 113 mg N kg tion of 120 mg N kg−1 dry soil, and decreased to 19.5 mg N kg−1 at the end of incubation (Fig. 3). 3% birnessite addition significantly decreased net nitrification rate to 1.04 mg N kg−1 day−1 in the first 3 days' incubation, but did not change the average net nitrification rate for the whole 10 days' incubation period. The NH+ 4 -N concentration did not differ during the 10 day's incubation regardless of Mn oxide addition in weakly acidic temperate soil. The nitrification process in soils could be described by a zero- or first-order kinetic model (Cheng et al., 2004; Jiang et al., 2013; Mary
Selected soil properties for weakly acidic and highly acidic soil in subtropical and temperate areas are listed in Table 1. For weakly acidic subtropical and temperate soil, pH averaged 6.3 and 6.1, respectively. Soil organic matter (SOM) content was 12.9 g kg−1 for subtropical soil and 36.6 g kg−1 for temperate soil. Total N was 1.0 g kg−1 for both subtropical and temperate soils, while kjeldahl N for subtropical soil was 76.0 mg kg−1 which was lower than temperate soil (96.3 mg kg−1). HCl-extractable Mn was 11.7 mg kg− 1 for subtropical soil and 14.0 mg kg−1 for temperate soil, while total Mn content was the same for subtropical and temperate soil (0.45 g kg−1). HCl-extractable Fe was 11.8 mg kg−1 for subtropical soil and 14.2 mg kg−1 for temperate soil. Total Fe was 34.9 and 50.7 g kg−1 for subtropical soil and temperate soil, respectively. After the addition of 3% birnessite, the concentration of HCl-extractable Mn increased to 149 mg kg− 1 for subtropical soil and 96.3 mg kg−1 for temperate soil, which was 12 and 6 times higher compared with unamended samples, respectively (Fig. 1). For highly acidic soils, the pH was 4.6 and 4.5 for subtropical and temperate soil, respectively. Similar to weakly acidic soils, SOM was about 4-fold higher for temperate than subtropical soil. However, total N did not differ significantly between the two soil types (Table 1). Kjeldahl N for subtropical soil was 90.6 mg kg−1 and 115 mg kg−1 for temperate soil. HCl-extractable Mn was 13.5 mg kg−1 for subtropical soil and 20.6 mg kg−1 for temperate soil. Total Mn was 2 times higher in temperate than subtropical soil (0.33 g kg−1). The concentration of HCl-extractable Fe was 233 mg kg−1 for subtropical and 256 mg kg−1 for temperate soil. Total Fe content was 21.1 g kg− 1 for subtropical
Fig. 1. Concentration of HCl-extractable Mn after the addition of 3% birnessite for subtropical and temperate soils. Lowercase letters indicate statistically significant differences (P b 0.05). Control represents natural soils; + 3% Mn represents the addition of 3% birnessite; Error bars represent standard deviation, n = 3.
X. Xin et al. / Catena 137 (2016) 24–30
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Fig. 2. Impacts of 3% birnessite addition on NO− 3 N dynamics for subtropical and temperate soils during the 10 days' incubation at 28 °C with soil moisture of 20% (m/m) (soils were preincubated in dark at 28 °C with soil moisture of 20% (m/m) for 7 days, then added 120 mg N kg−1 dry soil as (NH4)2SO4). pH 4.6, highly acidic subtropical soil; pH 4.6 + Mn, highly acidic subtropical soil +3% birnessite; pH 6.3, weakly acidic subtropical soil; pH 6.3 + Mn, weakly acidic subtropical soil +3% birnessite; pH 4.5, highly acidic temperate soil; pH 4.5 + Mn, highly acidic temperate soil +3% birnessite; pH 6.1, weakly acidic temperate soil; pH 6.1 + Mn, weakly acidic temperate soil +3% birnessite. Error bars represent standard deviation, n = 3.
et al., 1998; Oorts et al., 2007; Pansu and Thuriès, 2003). In the present study, the time-dependent kinetics of net nitrification was best fitted by a first-order kinetic model for weakly acidic subtropical soil and weakly acidic temperate soil. However, net nitrification kinetics was changed to be best fitted by a zero-order model after Mn oxide addition for both subtropical and temperate soils (r2 N 0.991). Simulated parameters of nitrification are listed in Table 1. Both potential nitrification (Np) and nitrification rates (Vp) in subtropical soil (17.9 mg N kg−1 and 10.2 mg N kg− 1 day− 1, respectively) were significantly higher than for temperate soil (11.7 mg N kg−1 and 4.46 mg N kg−1 day−1, respectively). However, the average nitrification rate (Va) was not significantly affected in both subtropical and temperate soils by Mn oxide at the end of 10 days' incubation (Table 2). No significant nitrification was observed for highly acidic subtropical or temperate soils, and NO− 3 N concentrations averaged 2.68 and 2.87 mg N kg− 1 soil, respectively after pre-incubation. At the end of −1 soil for the incubation, NO− 3 N concentrations were 2.52 mg N kg subtropical soil and 2.87 mg N kg− 1 soil for temperate soil (Fig. 2). Therefore, net nitrification rates were −0.16 and 0 mg N kg−1 day−1 for highly acidic subtropical soil and temperate soil, respectively. After −1 for subpre-incubation, NH+ 4 -N concentrations were 113 mg N kg −1 tropical soil and 110 mg N kg for temperate soil in one hour after addition of 120 mg N kg− 1 dry soil (Fig. 3). In the first day, NH+ 4 -N concentrations slightly decreased for subtropical soil (111 mg N kg−1)
and temperate soil (107 mg N kg− 1). However, after one day's incubation, NH+ 4 -N concentration sharply decreased to 51.0 and 34.9 mg N kg−1 for subtropical soil and temperate soil, whose levels were maintained at the end of the incubation. After 3% birnessite addition, net nitrification rates were −0.14 and 0.09 mg N kg−1 day−1 for subtropical soil and temperate soil, respectively. Conversely, NH+ 4 -N concentration decreased significantly from 110 mg N kg− 1 to 50.7 mg N kg− 1 for subtropical soil and from 107 mg N kg−1 to 42.5 mg N kg−1 for temperate soil during the 10 days' incubation. 3.3. pH changes in nitrification dynamics After pre-incubation, soil pH was 6.3 for weakly acidic subtropical soil and 6.1 for weakly acidic temperate soil. One day after incubation, pH decreased to 5.7 and 5.1 for subtropical soil and temperate soil, respectively, but soil pH did not change for the rest of incubation time (Fig. 4). 3% birnessite addition increased soil pH about 0.5 units for both subtropical soil and temperate soils. In the first day's incubation, pH decreases occurred for subtropical soil (0.9 units) and temperate soil (1.3 units). Soil pH was unchanged for the rest of the incubation (P b 0.05). For highly acidic soils, after pre-incubation, soil pH was 4.3 for subtropical soil and 4.5 for temperate soil, while 3% birnessite addition significantly increased soil pH (0.7 and 0.5 units for subtropical soil and
Fig. 3. Impacts of 3% birnessite addition on NH+ 4 -N concentrations for subtropical and temperate soils during the 10 days incubation at 28 °C with soil moisture of 20% (m/m) (soils were pre-incubated in dark at 28 °C with soil moisture of 20% (m/m) for 7 days, then added 120 mg N kg−1 dry soil as (NH4)2SO4). pH 4.6, highly acidic subtropical soil; pH 4.6 + Mn, highly acidic subtropical soil +3% birnessite; pH 6.3, weakly acidic subtropical soil; pH 6.3 + Mn, weakly acidic subtropical soil +3% birnessite; pH 4.5, highly acidic temperate soil; pH 4.5 + Mn, highly acidic temperate soil +3% birnessite; pH 6.1, weakly acidic temperate soil; pH 6.1 + Mn, weakly acidic temperate soil +3% birnessite. Error bars represent standard deviation, n = 3.
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Table 2 Parameters of zero or first-order kinetics model fitting NO-3-N accumulation for weakly acidic soils during 10 days of incubation. Soil species
Model
Np (mg N kg−1)
k0 (mg N kg−1 day−1) or k1 (day−1)
R2
Vp (mg N kg−1 day−1)
Subtropical soil Subtropical soil + 3% birnessite Temperate soil Temperate soil + 3% birnessite
First-order Zero-order First-order Zero-order
17.9 ± 1.21
0.57 ± 0.10 1.62 ± 0.20 0.38 ± 0.07 1.09 ± 0.20
0.99 0.99 1.00 0.99
10.2 ± 1.27
11.7 ± 0.70
4.46 ± 0.51
Mean values (n = 4) of nitrate-N of four replicates were used in fitting zero or first-order kinetics model. Np was potential nitrification; and k0 or k1 was the rate constant of zero or first-order kinetics model; Vp was potential nitrification rate calculated from first-order kinetics as Vp = k1* Np.
temperate soil, respectively). Nevertheless, no significant changes of pH were observed during the 10 day's incubation regardless of Mn oxide addition for these highly acidic soils.
3.4. Ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies Abundance of AOB and AOA was estimated by quantifying their respective amoA gene copy numbers immediately after preincubation (Fig. 5). AOB and AOA amoA gene copy numbers were significantly higher for weakly acidic soils than for highly acidic soils (P b 0.05). For subtropical soils, the AOB amoA gene copy numbers were 0.92 and 4.73 * 106 copies g−1 dry soil for highly acidic and weakly acidic soil, respectively. The addition of Mn oxide significantly decreased AOB abundance to 0.48 and 2.72 * 106 copies g−1 dry soil for highly acidic soils and weakly acidic soils, respectively (P b 0.05). For temperate soils, the AOB amoA gene copy numbers were 2.05 and 12.8 * 106 copies g−1 dry soil for highly acidic soil and weakly acidic soil, respectively, while the addition of Mn oxide significantly decreased AOB amoA gene copy numbers by 18.6% for highly acidic soil and 35.7% for weakly acidic soil (P b 0.05). Similar to AOB, AOA abundances in both subtropical soil and temperate soil were all decreased by Mn oxide addition. The AOA amoA gene copy numbers in subtropical soils averaged 8.09 and 19.5 * 106 copies g−1 dry soil for highly acidic soil and weakly acidic soils, respectively. The addition of Mn oxide decreased AOA amoA gene copy numbers about 13.3% for highly acidic soil (P N 0.05) and 48.2% for weakly acidic soil (P b 0.05). For temperate soils, the AOA amoA gene copy numbers were 12.4 and 32.9 * 106 copies g−1 dry soil for highly acidic soil and weakly acidic soil, respectively. The addition of Mn oxide significantly decreased AOA abundance to 10.8 and 20.5 * 106 copies g−1 dry soil for highly acidic soils and weakly acidic soils, respectively (P b 0.05).
4. Discussion Soil nitrification was retarded by Mn oxide addition for both subtropical soil and temperate soil despite their contrasting properties. Simulated results from nitrification dynamics further indicated that Mn addition changed the pattern of nitrification from first-order to zero-order model in weakly acidic purple soil and brown soils but had no effect on highly-acid soils. The adverse effects of Mn on nitrification have been observed in several agricultural soils. The addition of 100 mg kg−1 Mn significantly retarded nitrification, and 1000 mg kg−1 of Mn completely inhibited nitrification in an Arenic Plinthaquic Paleudults soil (Wilson, 1977), while 10,000 mg kg−1 Mn was required to completely inhibit nitrification, and nitrification was unaffected at 100 and 1000 mg kg− 1 Mn levels in an alluvial sandy soil (Premi and Cornfield, 1969). The nitrification process was retarded by the addition of 3% birnessite (&-MnO2) in the weakly acidic subtropical soil and temperate soil in the present study, although subtropical and temperate soil showed great differences in their properties. While adverse effects of Mn on nitrification were observed, the mechanisms responsible for retarded nitrification by Mn were not well understood. Mn toxicity to microorganisms may be responsible for the nitrification retardation or inhibition (Pittman, 2005), but no data were available to confirm this hypothesis. The nitrification process in soils could be simulated by zero- or firstorder kinetics (Cheng et al., 2004; Jiang et al., 2013; Mary et al., 1998; Oorts et al., 2007; Pansu and Thuriès, 2003). Factors affecting NH3 and nitrifying microorganisms influence nitrification process. They include soil organic matter (especially readily mineralized organic N), pH (controls the reaction of NH+ 4 to NH3), O2, and perhaps soil surface properties as well (Jiang et al., 2011). In the present study, time-dependent kinetics of net nitrification was best fitted by a first-order kinetic model for both weakly acidic subtropical soil and temperate soil. This indicated that the substrate (NH3) is insufficient compared to the oxidizing capacity of the ammonia oxidizers, and the rate of nitrification was proportional to the first power of substrate concentration.
Fig. 4. Impacts of 3% birnessite addition on pH for subtropical and temperate soils during the 10 days incubation at 28 °C with soil moisture of 20% (m/m) (soils were pre-incubated in dark at 28 °C with soil moisture of 20% (m/m) for 7 days, then added 120 mg N kg−1 dry soil as (NH4)2SO4). pH 4.6, highly acidic subtropical soil; pH 4.6 + Mn, highly acidic subtropical soil +3% birnessite; pH 6.3, weakly acidic subtropical soil; pH 6.3 + Mn, weakly acidic subtropical soil +3% birnessite; pH 4.5, highly acidic temperate soil; pH 4.5 + Mn, highly acidic temperate soil +3% birnessite; pH 6.1, weakly acidic temperate soil; pH 6.1 + Mn, weakly acidic temperate soil +3% birnessite. Error bars represent standard deviation, n = 3.
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Fig. 5. Effects of 3% birnessite addition on abundance of ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies for subtropical and temperate soils. Lowercase letters indicate statistically significant differences (P b 0.05). Control represents natural soils; +3% Mn represents the addition of 3% birnessite; error bars represent standard deviation, n = 4.
Nitrification patterns were changed from first-order to zero-order kinetic models by the addition of Mn oxide for both weakly acidic subtropical and temperate soils. This indicated that the substrate for nitrification (NH3) was sufficient relative to the oxidizing capacity of the ammonia oxidizers, and nitrification rates were limited by ammonia oxidizers rather than the substrate (NH3) supply. Present results showed that both amoA genes for the ammonia monooxygense (AMO) of AOB and AOA were decreased by Mn oxide addition. For example, amoA gene copy numbers of AMO decreased by 39.1% and 48.2% for AOB and AOA, respectively, after Mn oxide addition in the weakly acidic subtropical soil. While nitrification patterns were changed by Mn oxide in weakly acidic soils, the average net nitrification rate (Va) did not change significantly for both subtropical soil and temperate soil during the 10 days' incubation. This indicated that soil nitrification was retarded rather than inhibited by addition of 3% of birnessite. However, nitrification was not changed by Mn oxide addition in highly acidic purple soil or brown soils in the present study, probably because nitrification was slow in low pH environment. Differences in Mn effects on nitrification were also observed in different soil ecosystems (Leeper, 1970; Premi and Cornfield, 1969; Wilson, 1977), suggesting that Mn plays different roles in different environments. The chemistry of Mn in soils is extremely complicated (Leeper, 1970) and greatly affected by soil type, pH and moisture content. Manganese oxides (birnessite), which occur in soils and sediments, are very reactive (McKenzie, 1971). They can act as final electron acceptors in oxidizing organic compounds and NH4 (Shindo and Huang, 1982). Luther et al. (1997) reported the oxidation of NH3 and organic-N to N2 by Mn4+, and the reduction of NO− 3 N to N2 by Mn2+. Denitrification was also observed in Mn-rich sediments in aerobic places (Jenkins and Kemp, 1984; Seitzinger, 1988). Therefore, Mn may play an important role in N cycling (Aller, 1990; Murray et al., 1995; Schulz et al., 1994; Shindo and Huang, 1984). 5. Conclusions Soil nitrification was retarded by the addition of 3% Mn oxide for weakly acidic subtropical and temperate soils with contrasting
properties. Nitrification dynamics was altered by MnO2 in weakly acidic soils, probably due to toxicity to nitrifying microorganisms, but not affected in highly acidic soils, suggesting that Mn plays a different role in different environment, which has important implication in N cycling. Acknowledgments We thank Dr. Zhenli He, University of Florida, for reviewing this manuscript. We wish to acknowledge the useful suggestions by the reviewers and the editor. This research was financially supported by the Natural Science Foundation of China (No. 41271267). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.catena.2015.09.004. References Aller, R., 1990. Bioturbation and manganese cycling in hemipelagic sediments. Philos. Trans. R. Soc. Lond. Ser. A Math. Phys. Sci. 331, 51–68. Bäckman, J.S., Klemedtsson, Å.K., 2003. Increased nitrification in acid coniferous forest soil due to high nitrogen deposition and liming. Scand. J. For. Res. 18, 514–524. Cheng, W., Tsuruta, H., Chen, G., Yagi, K., 2004. N2O and NO production in various Chinese agricultural soils by nitrification. Soil Biol. Biochem. 36, 953–963. Compton, J.E., Watrud, L.S., Arlene Porteous, L., DeGrood, S., 2004. Response of soil microbial biomass and community composition to chronic nitrogen additions at Harvard forest. For. Ecol. Manag. 196, 143–158. De Boer, W., Kowalchuk, G., 2001. Nitrification in acid soils: micro-organisms and mechanisms. Soil Biol. Biochem. 33, 853–866. Francis, C.A., Roberts, K.J., Beman, J.M., Santoro, A.E., Oakley, B.B., 2005. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proc. Natl. Acad. Sci. U. S. A. 102, 14683–14688. Fujimoto, C.K., Sherman, G.D., 1948. Behavior of manganese in the soil and the manganese cycle. Soil Sci. 66, 131–145. Hue, N.V., Vega, S., Silva, J.A., 2001. Manganese toxicity in a Hawaiian oxisol affected by soil pH and organic amendments. Soil Sci. Soc. Am. J. 65, 153–160. Jenkins, M.C., Kemp, W.M., 1984. The coupling of nitrification and denitrification in two estuarine sediments l v2. Limnol. Oceanogr. 29, 609–619. Jiang, X., Shi, X., Liu, W., Wright, A.L., 2011. Kinetics of net nitrification associated with soil aggregates under conventional and no-tillage in a subtropical rice soil. Plant Soil 347, 305–312.
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Jiang, X., et al., 2013. Soil N mineralization, nitrification and dynamic changes in abundance of ammonia-oxidizing bacteria and archaea along a 2000 year chronosequence of rice cultivation. Plant Soil 365, 59–68. Keeney, D.R., Nelson, D., 1982. Nitrogen—inorganic forms. Methods of soil analysis. Part 2. Chemical and microbiological properties. pp. 643–698. Leeper, G.W., 1970. Six Trace Elements in Soils: Their Chemistry As Micro-Nutrients. Melbourne University Press. Lu, R., 2000. Soil agro-chemical analyses. Agric. Tech. Press of China, Beijing (Chinese). Luther, G.W., Sundby, B., Lewis, B.L., Brendel, P.J., Silverberg, N., 1997. Interactions of manganese with the nitrogen cycle: alternative pathways to dinitrogen. Geochim. Cosmochim. Acta 61, 4043–4052. Marschner, H., 1991. Mechanisms of adaptation of plants to acid soils, plant–soil interactions at low pH. Springer, pp. 683–702. Mary, B., Recous, S., Robin, D., 1998. A model for calculating nitrogen fluxes in soil using 15 N tracing. Soil Biol. Biochem. 30, 1963–1979. McKenzie, R., 1971. The synthesis of birnessite, cryptomelane, and some other oxides and hydroxides of manganese. Mineral. Mag. 38, 493–502. Murray, J.W., Codispoti, L.A., Friederich, G.E., 1995. Oxidation-reduction environments: the suboxic zone in the black sea. ACS Adv. Chem. Ser. 244, 157–176. Nugroho, R., Röling, W., Laverman, A., Verhoef, H., 2007. Low nitrification rates in acid scots pine forest soils are due to pH-related factors. Microb. Ecol. 53, 89–97. Nugroho, R.A., Röling, W.F., van Straalen, N.M., Verhoef, H.A., 2009. Changes in nitrification and bacterial community structure upon cross-inoculation of scots pine forest soils with different initial nitrification rates. Soil Biol. Biochem. 41, 243–250. Oorts, K., Merckx, R., Gréhan, E., Labreuche, J., Nicolardot, B., 2007. Determinants of annual fluxes of CO2 and N2O in long-term no-tillage and conventional tillage systems in northern France. Soil Tillage Res. 95, 133–148. Pansu, M., Thuriès, L., 2003. Kinetics of C and N mineralization, N immobilization and N volatilization of organic inputs in soil. Soil Biol. Biochem. 35, 37–48.
Pittman, J.K., 2005. Managing the manganese: molecular mechanisms of manganese transport and homeostasis. New Phytol. 167, 733–742. Premi, P., Cornfield, A., 1969. Effects of addition of copper, manganese, zinc and chromium compounds on ammonification and nitrification during incubation of soil. Plant Soil 31, 345–352. Robertson, G.P., 1982. Nitrification in forested ecosystems. Philos. Trans. R. Soc. Lond. Ser. B 296, 445–457. Robertson, G.P., Coleman, D.C., Bledsoe, C.S., Sollins, P., 1999. Standard Soil Methods for Long-Term Ecological Research. Oxford University Press. Schulz, H.D., Dahmke, A., Schinzel, U., Wallmann, K., Zabel, M., 1994. Early diagenetic processes, fluxes, and reaction rates in sediments of the south Atlantic. Geochim. Cosmochim. Acta 58, 2041–2060. Seitzinger, S.P., 1988. Denitrification in freshwater and coastal marine ecosystems: ecological and geochemical significance. Limnol. Oceanogr. 33, 702–724. Shindo, H., Huang, P., 1982. Role of Mn (IV) oxide in abiotic formation of humic substances in the environment. Nature 298, 363–365. Shindo, H., Huang, P., 1984. Catalytic effects of manganese (IV), iron (III), aluminum, and silicon oxides on the formation of phenolic polymers. Soil Sci. Soc. Am. J. 48, 927–934. Taylor, R., McKenzie, R., Norrish, K., 1964. The mineralogy and chemistry of manganese in some Australian soils. Aust. J. Soil Res. 2, 235–248. Tolli, J., King, G.M., 2005. Diversity and structure of bacterial chemolithotrophic communities in pine forest and agroecosystem soils. Appl. Environ. Microbiol. 71, 8411–8418. Wilson, D., 1977. Nitrification in soil treated with domestic and industrial sewage sludge. Environ. Pollut. 12, 73–82. Zhao, W., Cai, Z., Xu, Z., 2007. Does ammonium-based N addition influence nitrification and acidification in humid subtropical soils of China? Plant Soil 297, 213–221.
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Manganese oxide affects nitrification and ammonia oxidizers in subtropical and temperate acid forest soils Xiaoping Xin a, Xianjun Jiang a,⁎, Jing Su a, Xiaojuan Yan a, Jiupai Ni a, Sarwee J. Faeflen a, Xueru Huang a, Alan L. Wright b a b
College of Resources and Environment, Southwest University, 2 Tiansheng Road, Beibei, Chongqing 400715, China Everglades Research & Education Center, University of Florida, Belle Glade, FL 33430, United States
a r t i c l e
i n f o
Article history: Received 24 May 2015 Received in revised form 1 September 2015 Accepted 6 September 2015 Available online xxxx Keywords: Nitrification Ammonia oxidizing bacteria Ammonia oxidizing archaea Manganese
a b s t r a c t Variations in nitrification dynamics were observed both in temperate and subtropical acidic forest soils. The effect of Mn on nitrification was studied to explain variation in nitrification between different soil types. Weakly and highly acidic soils in subtropical and temperate forests were treated with 0% or 3% birnessite. The nitrification process was simulated by kinetic model. Dynamic changes of amoA gene abundance of ammonia monooxygenase (AMO) for ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) were estimated by qPCR. Nitrification rates were significantly decreased by Mn addition in 3 days both for the weakly acidic subtropical and temperate soils. However, the total nitrification rate did not change for either soil by Mn addition after 10 days' incubation. Nitrification was best fitted by a firstorder kinetic model for both weakly acidic soils. However, it was best fitted with a zero-order model after MnO2 addition. Numbers of AMO amoA gene copy decreased after Mn addition. No significant nitrification was observed for highly acidic soils and Mn had a minimal effect. Soil nitrification was retarded by 3% MnO2 for both subtropical soil and temperate soils. Nitrification dynamics were altered by MnO2 in weakly acidic soils, probably due to Mn toxicity. © 2015 Published by Elsevier B.V.
1. Introduction Great variation of nitrification potential in acid soils has been reported (Compton et al., 2004; De Boer and Kowalchuk, 2001; Nugroho et al., 2009) due to numerous biotic and abiotic factors. It is now accepted that nitrification can occur in a wide range of acid soils (De Boer and Kowalchuk, 2001), but there are also many acid soils for which nitrification appears to be minimal or absent (Robertson, 1982). Biotic and abiotic factors such as pH, N availability and microbial community structure are responsible in part but not completely for the variation in nitrification rates in acid soils (Bäckman and Klemedtsson, 2003; Compton et al., 2004; Nugroho et al., 2007; Tolli and King, 2005). For example, it was reported that net nitrification rates could vary 3-fold in two acid soils within a given pH (Nugroho et al., 2007). NH+ 4 -N content also did not constrain net nitrification, as some temperate acidic forest soils having high NH+ 4 -N concentrations did not experience net nitrification (Nugroho et al., 2007; Zhao et al., 2007). Nugroho et al. (2009) also concluded that low net nitrification rates in acid Scots pine forest soils cannot be solely explained by unfavorable abiotic soil conditions
⁎ Corresponding author. E-mail address: [email protected] (X. Jiang).
http://dx.doi.org/10.1016/j.catena.2015.09.004 0341-8162/© 2015 Published by Elsevier B.V.
after observing the bacterial community structure and interactions between members of the bacterial community. Therefore, there are still uncaptured biotic factors or abiotic factors that contribute to suppression of nitrification in acid soils. Manganese is one of the elements released during the weathering of primary minerals, which explains its common accumulation in most soils in the range of 0.07–4.0% (Aller, 1990; Fujimoto and Sherman, 1948). Manganese activity is closely related to soil pH, as its concentrations in soil solution increase 100-fold for each pH unit decrease (Hue et al., 2001). Hence, Mn toxicity may be one of the important abiotic stresses on nitrification in acidic soils (Marschner, 1991). Stimulation, retardation, or inhibitation of nitrification by Mn has been observed in different ecosystems and a variety of soil types depending on Mn concentration (Leeper, 1970; Premi and Cornfield, 1969; Wilson, 1977). Manganese toxicity to microorganisms may be responsible for the nitrification inhibition, but no data are as yet available to substantiate this hypothesis. Furthermore, the mechanistic patterns of nitrification altered by Mn are poorly understood. We hypothesize that the effects of Mn on nitrification occur in different temperate and subtropical soils. Both highly acidic and weakly acidic forest soil samples from temperate area (brown soils) and subtropical area (purple soils) were selected to test this hypothesis by investigating the effect of Mn on the nitrification processes and nitrifying microorganisms.
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2. Material and methods 2.1. Site description and soil sampling The subtropical soil samples (purple soil, Inceptisols) were collected from a subtropical forestland in Yongchuan, Chongqing, southwest of China (29°16′N, 105°84′E). This region has the annual mean temperature 19.7 °C and annual mean rainfall 1400 mm, with middle subtropical evergreen broad-leaved forests. Two sites were chosen to obtain weakly acidic soil and highly acidic soil. Temperate soil samples (brown soil, Luvisols) were collected from Dahei Mountain in Dalian, Liaoning Province, northeast of China (38°43′N, 120°58′E) where the climate is between monsoon and continental with annual mean temperature 10.2 °C (ranging from − 19.1 °C to 37.8 °C) and annual mean rainfall 810 mm. The two sampling sites were weakly acidic and highly acidic soils. Five soil cores (0–20 cm) were collected from a 4 m × 4 m plot using a soil corer (Ø = 13 cm). Portion of the soil samples collected was pooled and homogenized to reduce heterogeneity. The samples were air-dried and separated into two parts. One was ground to pass a 2-mm sieve and stored at 4 °C prior to make subsamples for incubation; another was ground to pass a 1-mm sieve and used for chemical analyses. 2.2. Preparation of birnessite (&-MnO2) and samples Birnessite (&-MnO2) is a naturally occurring, poorly crystalline oxide of tetravalent manganese. It is one of the most common forms of mineralized Mn in soils (Taylor et al., 1964). Birnessite was prepared by dropwise addition of HCl to KMnO4 (McKenzie, 1971). The precipitate was washed, dried, and aged at 60 °C for 12 h, and again washed with water. Obtained birnessite was ground to pass through a 1-mm sieve and stored at 4 °C prior before use. The obtained birnessite was checked by X-ray powder diffractometer (XRD). Subsamples were prepared by amendment with 0% (unmended) or 3% birnessite by weight. Eight subsamples were obtained as follows: weakly acidic subtropical soil, weakly acidic subtropical soil + 3% birnessite, highly acidic subtropical soil, highly acidic subtropical soil +3% birnessite; weakly acidic temperate soil, weakly acidic temperate soil +3% birnessite, highly acidic temperate soil, highly acidic temperate soil + 3% birnessite. Each subsample was mixed thoroughly and ground to pass a 1-mm sieve and stored at 4 °C for 2 months before use. 2.3. Physical and chemical analysis Soil properties were determined following Soil Agro-Chemical Analyses procedures (Lu, 2000). Soil pH was measured in a soil to water ratio of 1:2.5 (v/v) by a DMP-2 mV/pH detector (Quark Ltd., Chongqing, China). Soil organic matter (SOM), total N and kjeldahl N contents were determined by a Macro Elemental Analyzer (Elementary Analysensysteme GmbH, Hanau, Germany). Total soil Fe and Mn were determined by digesting the soil with HNO3–HF–HClO4, and HClextractable Fe and Mn in soil were extracted by shaking 150 g of soil in 300 ml of 0.002 M HCl for 1 h. The concentration of Fe and Mn in the digesters and extracts was determined by atomic absorption spectrophotometry with a graphite furnace (GFAAS) using a model Z-8200 spectrophotometer. 2.4. Incubation For each subsample, 20 g soil was placed into a 250-ml plastic bottle. Distilled water was added to adjust the moisture content to 20% by weight (50% of water holding capacity). All bottles were covered with polyethylene film punctured with needle holes to maintain aerobic conditions, and pre-incubated at 28 °C in the dark for 7 days. After preincubation, 120 mg N kg−1 dry soils were added as (NH4)2SO4 to assess
25
nitrification rate. The loss of water through evaporation was compensated by addition of distilled water daily. At the interval of 0, 1, 3, 7 and 10 days after incubation days, subsamples in four replicates were taken and extracted with 100 ml of 2 M KCl for 1 h (Keeney and Nelson, 1982). The concentrations of inorganic N forms in the extracts were determined using SKLAR continuous-flow analyzer (SKLAR San ++, Netherland, 2003). The net nitrification rate was calculated using the following equation (Robertson et al., 1999): Net nitrification rate ¼
− NO− 3 −N f − NO3 −N i =T d
where the subscripts i and f indicate concentrations measured before and after aerobic incubation, respectively and Td indicates incubation time in days. Net ammonification and net nitrification were expressed as ammonia-oxidizing archaea mg N kg−1 day−1 dry soil per day (Francis et al., 2005). 2.5. DNA extraction and quantitative PCR assay Right after pre-incubation, 4 replicate bottles of each treatment were randomly selected to extract DNA and analysis of amoA genes was performed by quantitative PCR (qPCR). The DNA was extracted from 0.50 g of soil with the Fast DNA Spin Kit for soil (MP Biomedicals, United States), according to the protocol of the manufacturer. The quality and quantity of the extracted DNA were analyzed with a spectrophotometer (Nanodrop, PeqLab, Germany), and pooled and stored at −20 °C until use. Quantitative PCR of amoA genes was performed to estimate the abundance of the ammonia-oxidizing bacterial and archaeal communities, respectively. The primers amoA-1 F (5′-GGGGTTTCTACTGGTGGT3′) and amoA-2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) were used for ammonia-oxidizing bacteria generating a 491 bp fragment; ArchamoA F (5′-STAATGGTCTGGCTTAGACG-3′) and Arch amoA R (5′-GCGG CCATCCATCTGTATGT-3′) were used for generating a 635 bp fragment (Francis et al., 2005). Quantification was based on the fluorescence intensity of the SYBR Green dye and reactions for each sample were carried out in a Bio-Rad CFX-96 thermal cycler. The quantification of amoA genes was performed in a total volume of 25 μl reaction mixtures with 12.5 μl of SYBR Premix Ex Taq™ as described by the suppliers (Takara Bio, Otsu, Shiga, Japan), 0.25 μl of each primer (50 μm), 1 μl of soil DNA template, with a final content of 1–10 ng in each reaction mixture, and 11 μl ddH2O. The fragments for the AOB and AOA were both amplified using an initial denaturation step at 95 °C for 3 min, followed by 35 cycles of 30 s at 95 °C, 30 s at 55 °C, 30 s at 72 °C for AOB, and 45 s at 72 °C for AOA for the collection of fluorescence data. All reactions were finished with a melting curve starting at 65 °C with an increase of 0.5 °C up to 95 °C to verify amplicon specificity. The PCR reaction runs had an efficiency of 90% and 94% for the AOB and AOA, respectively. Standard curves for the AOB and AOA were obtained using serial dilutions of linearized plasmids (pGEM-T, Promega) containing cloned amoA genes amplified from environmental clones (r2 N 0.995 for both standard curves). 2.6. Statistical analysis The changes in NO− 3 N content with incubation time were modeled with a first-order reaction kinetic model, expressed as NNO3 = N0 + NP (1 − exp.(− k1t), or by a zero-order reaction kinetic model, expressed as NNO3 = N0 = k0t, where NNO3 was NO− 3 N content at incubation time t; N0 was NO− 3 N content after pre-incubation (t = 0); Np was nitrification potential; and k1 and k0 were rate constants of firstand zero-order reactions, respectively. The potential nitrification rate (Vp) was calculated from first-order kenetics as Vp = k1 * Np (Oorts et al., 2007). The mean actual net nitrification rate (Va) was calculated as Va = (N10 − N0)/10, here N10 was NO− 3 N content after 10 days' incubation (t = 10).
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Table 1 Selected soil properties. Soil species
Subtropical soil Temperate soil
pH
4.60 6.30 4.50 6.10
SOM
Total N
Kjeldahl nitrogen
HCl-extractable Mn
Total Mn
HCl-extractable Fe
Total Fe
(g kg−1)
(g kg−1)
(mg kg−1)
(mg kg−1)
(g kg−1)
(mg kg−1)
(g kg−1)
11.9 12.9 47.8 36.6
0.75 0.97 0.85 1.01
90.6 76.0 115 96
13.5 11.7 20.6 14.0
0.33 0.45 0.66 0.45
233 11.8 256 14.2
21.1 34.9 46.9 50.7
SOM represents soil organic matter.
Data (measured or calculated) were subjected to one-way ANOVA and mean values were separated using Duncan's New Multiple Range Test at P b 0.05. All statistical analyses were performed by the SPSS statistical package.
soil, which was about half of the content of temperate soil. 3% birnessite significantly increased the concentration of HCl-extractable Mn in highly acidic soils to 294 mg kg−1 for subtropical soil and 266 mg kg−1 for temperate soil, respectively (Fig. 1).
3. Results
3.2. Effects of Mn oxides on soil net nitrification rate and kinetic patterns
3.1. Selected soil properties
−1 soil after preThe NO− 3 N concentration was 9.67 mg N kg incubation for weakly acidic subtropical soil. The largest net nitrification rate (4.76 mg N kg−1 day−1) was observed in the first 3 days' incuba−1 soil in tion (Fig. 2). The NH+ 4 -N concentration was 104.3 mg N kg −1 dry soil, and sharply one hour after addition of 120 mg N kg decreased to 15.7 mg N kg− 1 after 3 days' incubation. At the end of −1 (Fig. 3). the incubation, NH+ 4 -N concentration was 12.7 mg N kg − Though the NO3 N concentration increased linearly by 3% birnessite addition during the whole incubation period, the largest net nitrification rate decreased significantly to 1.94 mg N kg− 1 day−1 in the first 3 days' incubation, and average net nitrification rate by 10 days (1.67 mg N kg−1 day−1) did not differ significantly. NH+ 4 -N concentration did not differ significantly at the end of the incubation with or without Mn oxide addition in weakly acidic subtropical soil. Significant nitrification also occurred in weakly acidic temperate soil. A higher net nitrification rate (2.76 mg N kg− 1 day− 1) was observed in the first 3 days' incubation, compared to the average net nitrification rate (1.13 mg N kg− 1 day− 1) for the whole 10 days' incubation (Fig. 2). −1 soil one hour after addiThe NH+ 4 -N concentration was 113 mg N kg tion of 120 mg N kg−1 dry soil, and decreased to 19.5 mg N kg−1 at the end of incubation (Fig. 3). 3% birnessite addition significantly decreased net nitrification rate to 1.04 mg N kg−1 day−1 in the first 3 days' incubation, but did not change the average net nitrification rate for the whole 10 days' incubation period. The NH+ 4 -N concentration did not differ during the 10 day's incubation regardless of Mn oxide addition in weakly acidic temperate soil. The nitrification process in soils could be described by a zero- or first-order kinetic model (Cheng et al., 2004; Jiang et al., 2013; Mary
Selected soil properties for weakly acidic and highly acidic soil in subtropical and temperate areas are listed in Table 1. For weakly acidic subtropical and temperate soil, pH averaged 6.3 and 6.1, respectively. Soil organic matter (SOM) content was 12.9 g kg−1 for subtropical soil and 36.6 g kg−1 for temperate soil. Total N was 1.0 g kg−1 for both subtropical and temperate soils, while kjeldahl N for subtropical soil was 76.0 mg kg−1 which was lower than temperate soil (96.3 mg kg−1). HCl-extractable Mn was 11.7 mg kg− 1 for subtropical soil and 14.0 mg kg−1 for temperate soil, while total Mn content was the same for subtropical and temperate soil (0.45 g kg−1). HCl-extractable Fe was 11.8 mg kg−1 for subtropical soil and 14.2 mg kg−1 for temperate soil. Total Fe was 34.9 and 50.7 g kg−1 for subtropical soil and temperate soil, respectively. After the addition of 3% birnessite, the concentration of HCl-extractable Mn increased to 149 mg kg− 1 for subtropical soil and 96.3 mg kg−1 for temperate soil, which was 12 and 6 times higher compared with unamended samples, respectively (Fig. 1). For highly acidic soils, the pH was 4.6 and 4.5 for subtropical and temperate soil, respectively. Similar to weakly acidic soils, SOM was about 4-fold higher for temperate than subtropical soil. However, total N did not differ significantly between the two soil types (Table 1). Kjeldahl N for subtropical soil was 90.6 mg kg−1 and 115 mg kg−1 for temperate soil. HCl-extractable Mn was 13.5 mg kg−1 for subtropical soil and 20.6 mg kg−1 for temperate soil. Total Mn was 2 times higher in temperate than subtropical soil (0.33 g kg−1). The concentration of HCl-extractable Fe was 233 mg kg−1 for subtropical and 256 mg kg−1 for temperate soil. Total Fe content was 21.1 g kg− 1 for subtropical
Fig. 1. Concentration of HCl-extractable Mn after the addition of 3% birnessite for subtropical and temperate soils. Lowercase letters indicate statistically significant differences (P b 0.05). Control represents natural soils; + 3% Mn represents the addition of 3% birnessite; Error bars represent standard deviation, n = 3.
X. Xin et al. / Catena 137 (2016) 24–30
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Fig. 2. Impacts of 3% birnessite addition on NO− 3 N dynamics for subtropical and temperate soils during the 10 days' incubation at 28 °C with soil moisture of 20% (m/m) (soils were preincubated in dark at 28 °C with soil moisture of 20% (m/m) for 7 days, then added 120 mg N kg−1 dry soil as (NH4)2SO4). pH 4.6, highly acidic subtropical soil; pH 4.6 + Mn, highly acidic subtropical soil +3% birnessite; pH 6.3, weakly acidic subtropical soil; pH 6.3 + Mn, weakly acidic subtropical soil +3% birnessite; pH 4.5, highly acidic temperate soil; pH 4.5 + Mn, highly acidic temperate soil +3% birnessite; pH 6.1, weakly acidic temperate soil; pH 6.1 + Mn, weakly acidic temperate soil +3% birnessite. Error bars represent standard deviation, n = 3.
et al., 1998; Oorts et al., 2007; Pansu and Thuriès, 2003). In the present study, the time-dependent kinetics of net nitrification was best fitted by a first-order kinetic model for weakly acidic subtropical soil and weakly acidic temperate soil. However, net nitrification kinetics was changed to be best fitted by a zero-order model after Mn oxide addition for both subtropical and temperate soils (r2 N 0.991). Simulated parameters of nitrification are listed in Table 1. Both potential nitrification (Np) and nitrification rates (Vp) in subtropical soil (17.9 mg N kg−1 and 10.2 mg N kg− 1 day− 1, respectively) were significantly higher than for temperate soil (11.7 mg N kg−1 and 4.46 mg N kg−1 day−1, respectively). However, the average nitrification rate (Va) was not significantly affected in both subtropical and temperate soils by Mn oxide at the end of 10 days' incubation (Table 2). No significant nitrification was observed for highly acidic subtropical or temperate soils, and NO− 3 N concentrations averaged 2.68 and 2.87 mg N kg− 1 soil, respectively after pre-incubation. At the end of −1 soil for the incubation, NO− 3 N concentrations were 2.52 mg N kg subtropical soil and 2.87 mg N kg− 1 soil for temperate soil (Fig. 2). Therefore, net nitrification rates were −0.16 and 0 mg N kg−1 day−1 for highly acidic subtropical soil and temperate soil, respectively. After −1 for subpre-incubation, NH+ 4 -N concentrations were 113 mg N kg −1 tropical soil and 110 mg N kg for temperate soil in one hour after addition of 120 mg N kg− 1 dry soil (Fig. 3). In the first day, NH+ 4 -N concentrations slightly decreased for subtropical soil (111 mg N kg−1)
and temperate soil (107 mg N kg− 1). However, after one day's incubation, NH+ 4 -N concentration sharply decreased to 51.0 and 34.9 mg N kg−1 for subtropical soil and temperate soil, whose levels were maintained at the end of the incubation. After 3% birnessite addition, net nitrification rates were −0.14 and 0.09 mg N kg−1 day−1 for subtropical soil and temperate soil, respectively. Conversely, NH+ 4 -N concentration decreased significantly from 110 mg N kg− 1 to 50.7 mg N kg− 1 for subtropical soil and from 107 mg N kg−1 to 42.5 mg N kg−1 for temperate soil during the 10 days' incubation. 3.3. pH changes in nitrification dynamics After pre-incubation, soil pH was 6.3 for weakly acidic subtropical soil and 6.1 for weakly acidic temperate soil. One day after incubation, pH decreased to 5.7 and 5.1 for subtropical soil and temperate soil, respectively, but soil pH did not change for the rest of incubation time (Fig. 4). 3% birnessite addition increased soil pH about 0.5 units for both subtropical soil and temperate soils. In the first day's incubation, pH decreases occurred for subtropical soil (0.9 units) and temperate soil (1.3 units). Soil pH was unchanged for the rest of the incubation (P b 0.05). For highly acidic soils, after pre-incubation, soil pH was 4.3 for subtropical soil and 4.5 for temperate soil, while 3% birnessite addition significantly increased soil pH (0.7 and 0.5 units for subtropical soil and
Fig. 3. Impacts of 3% birnessite addition on NH+ 4 -N concentrations for subtropical and temperate soils during the 10 days incubation at 28 °C with soil moisture of 20% (m/m) (soils were pre-incubated in dark at 28 °C with soil moisture of 20% (m/m) for 7 days, then added 120 mg N kg−1 dry soil as (NH4)2SO4). pH 4.6, highly acidic subtropical soil; pH 4.6 + Mn, highly acidic subtropical soil +3% birnessite; pH 6.3, weakly acidic subtropical soil; pH 6.3 + Mn, weakly acidic subtropical soil +3% birnessite; pH 4.5, highly acidic temperate soil; pH 4.5 + Mn, highly acidic temperate soil +3% birnessite; pH 6.1, weakly acidic temperate soil; pH 6.1 + Mn, weakly acidic temperate soil +3% birnessite. Error bars represent standard deviation, n = 3.
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Table 2 Parameters of zero or first-order kinetics model fitting NO-3-N accumulation for weakly acidic soils during 10 days of incubation. Soil species
Model
Np (mg N kg−1)
k0 (mg N kg−1 day−1) or k1 (day−1)
R2
Vp (mg N kg−1 day−1)
Subtropical soil Subtropical soil + 3% birnessite Temperate soil Temperate soil + 3% birnessite
First-order Zero-order First-order Zero-order
17.9 ± 1.21
0.57 ± 0.10 1.62 ± 0.20 0.38 ± 0.07 1.09 ± 0.20
0.99 0.99 1.00 0.99
10.2 ± 1.27
11.7 ± 0.70
4.46 ± 0.51
Mean values (n = 4) of nitrate-N of four replicates were used in fitting zero or first-order kinetics model. Np was potential nitrification; and k0 or k1 was the rate constant of zero or first-order kinetics model; Vp was potential nitrification rate calculated from first-order kinetics as Vp = k1* Np.
temperate soil, respectively). Nevertheless, no significant changes of pH were observed during the 10 day's incubation regardless of Mn oxide addition for these highly acidic soils.
3.4. Ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies Abundance of AOB and AOA was estimated by quantifying their respective amoA gene copy numbers immediately after preincubation (Fig. 5). AOB and AOA amoA gene copy numbers were significantly higher for weakly acidic soils than for highly acidic soils (P b 0.05). For subtropical soils, the AOB amoA gene copy numbers were 0.92 and 4.73 * 106 copies g−1 dry soil for highly acidic and weakly acidic soil, respectively. The addition of Mn oxide significantly decreased AOB abundance to 0.48 and 2.72 * 106 copies g−1 dry soil for highly acidic soils and weakly acidic soils, respectively (P b 0.05). For temperate soils, the AOB amoA gene copy numbers were 2.05 and 12.8 * 106 copies g−1 dry soil for highly acidic soil and weakly acidic soil, respectively, while the addition of Mn oxide significantly decreased AOB amoA gene copy numbers by 18.6% for highly acidic soil and 35.7% for weakly acidic soil (P b 0.05). Similar to AOB, AOA abundances in both subtropical soil and temperate soil were all decreased by Mn oxide addition. The AOA amoA gene copy numbers in subtropical soils averaged 8.09 and 19.5 * 106 copies g−1 dry soil for highly acidic soil and weakly acidic soils, respectively. The addition of Mn oxide decreased AOA amoA gene copy numbers about 13.3% for highly acidic soil (P N 0.05) and 48.2% for weakly acidic soil (P b 0.05). For temperate soils, the AOA amoA gene copy numbers were 12.4 and 32.9 * 106 copies g−1 dry soil for highly acidic soil and weakly acidic soil, respectively. The addition of Mn oxide significantly decreased AOA abundance to 10.8 and 20.5 * 106 copies g−1 dry soil for highly acidic soils and weakly acidic soils, respectively (P b 0.05).
4. Discussion Soil nitrification was retarded by Mn oxide addition for both subtropical soil and temperate soil despite their contrasting properties. Simulated results from nitrification dynamics further indicated that Mn addition changed the pattern of nitrification from first-order to zero-order model in weakly acidic purple soil and brown soils but had no effect on highly-acid soils. The adverse effects of Mn on nitrification have been observed in several agricultural soils. The addition of 100 mg kg−1 Mn significantly retarded nitrification, and 1000 mg kg−1 of Mn completely inhibited nitrification in an Arenic Plinthaquic Paleudults soil (Wilson, 1977), while 10,000 mg kg−1 Mn was required to completely inhibit nitrification, and nitrification was unaffected at 100 and 1000 mg kg− 1 Mn levels in an alluvial sandy soil (Premi and Cornfield, 1969). The nitrification process was retarded by the addition of 3% birnessite (&-MnO2) in the weakly acidic subtropical soil and temperate soil in the present study, although subtropical and temperate soil showed great differences in their properties. While adverse effects of Mn on nitrification were observed, the mechanisms responsible for retarded nitrification by Mn were not well understood. Mn toxicity to microorganisms may be responsible for the nitrification retardation or inhibition (Pittman, 2005), but no data were available to confirm this hypothesis. The nitrification process in soils could be simulated by zero- or firstorder kinetics (Cheng et al., 2004; Jiang et al., 2013; Mary et al., 1998; Oorts et al., 2007; Pansu and Thuriès, 2003). Factors affecting NH3 and nitrifying microorganisms influence nitrification process. They include soil organic matter (especially readily mineralized organic N), pH (controls the reaction of NH+ 4 to NH3), O2, and perhaps soil surface properties as well (Jiang et al., 2011). In the present study, time-dependent kinetics of net nitrification was best fitted by a first-order kinetic model for both weakly acidic subtropical soil and temperate soil. This indicated that the substrate (NH3) is insufficient compared to the oxidizing capacity of the ammonia oxidizers, and the rate of nitrification was proportional to the first power of substrate concentration.
Fig. 4. Impacts of 3% birnessite addition on pH for subtropical and temperate soils during the 10 days incubation at 28 °C with soil moisture of 20% (m/m) (soils were pre-incubated in dark at 28 °C with soil moisture of 20% (m/m) for 7 days, then added 120 mg N kg−1 dry soil as (NH4)2SO4). pH 4.6, highly acidic subtropical soil; pH 4.6 + Mn, highly acidic subtropical soil +3% birnessite; pH 6.3, weakly acidic subtropical soil; pH 6.3 + Mn, weakly acidic subtropical soil +3% birnessite; pH 4.5, highly acidic temperate soil; pH 4.5 + Mn, highly acidic temperate soil +3% birnessite; pH 6.1, weakly acidic temperate soil; pH 6.1 + Mn, weakly acidic temperate soil +3% birnessite. Error bars represent standard deviation, n = 3.
X. Xin et al. / Catena 137 (2016) 24–30
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Fig. 5. Effects of 3% birnessite addition on abundance of ammonia-oxidizing bacterial (AOB) and ammonia-oxidizing archaeal (AOA) amoA gene copies for subtropical and temperate soils. Lowercase letters indicate statistically significant differences (P b 0.05). Control represents natural soils; +3% Mn represents the addition of 3% birnessite; error bars represent standard deviation, n = 4.
Nitrification patterns were changed from first-order to zero-order kinetic models by the addition of Mn oxide for both weakly acidic subtropical and temperate soils. This indicated that the substrate for nitrification (NH3) was sufficient relative to the oxidizing capacity of the ammonia oxidizers, and nitrification rates were limited by ammonia oxidizers rather than the substrate (NH3) supply. Present results showed that both amoA genes for the ammonia monooxygense (AMO) of AOB and AOA were decreased by Mn oxide addition. For example, amoA gene copy numbers of AMO decreased by 39.1% and 48.2% for AOB and AOA, respectively, after Mn oxide addition in the weakly acidic subtropical soil. While nitrification patterns were changed by Mn oxide in weakly acidic soils, the average net nitrification rate (Va) did not change significantly for both subtropical soil and temperate soil during the 10 days' incubation. This indicated that soil nitrification was retarded rather than inhibited by addition of 3% of birnessite. However, nitrification was not changed by Mn oxide addition in highly acidic purple soil or brown soils in the present study, probably because nitrification was slow in low pH environment. Differences in Mn effects on nitrification were also observed in different soil ecosystems (Leeper, 1970; Premi and Cornfield, 1969; Wilson, 1977), suggesting that Mn plays different roles in different environments. The chemistry of Mn in soils is extremely complicated (Leeper, 1970) and greatly affected by soil type, pH and moisture content. Manganese oxides (birnessite), which occur in soils and sediments, are very reactive (McKenzie, 1971). They can act as final electron acceptors in oxidizing organic compounds and NH4 (Shindo and Huang, 1982). Luther et al. (1997) reported the oxidation of NH3 and organic-N to N2 by Mn4+, and the reduction of NO− 3 N to N2 by Mn2+. Denitrification was also observed in Mn-rich sediments in aerobic places (Jenkins and Kemp, 1984; Seitzinger, 1988). Therefore, Mn may play an important role in N cycling (Aller, 1990; Murray et al., 1995; Schulz et al., 1994; Shindo and Huang, 1984). 5. Conclusions Soil nitrification was retarded by the addition of 3% Mn oxide for weakly acidic subtropical and temperate soils with contrasting
properties. Nitrification dynamics was altered by MnO2 in weakly acidic soils, probably due to toxicity to nitrifying microorganisms, but not affected in highly acidic soils, suggesting that Mn plays a different role in different environment, which has important implication in N cycling. Acknowledgments We thank Dr. Zhenli He, University of Florida, for reviewing this manuscript. We wish to acknowledge the useful suggestions by the reviewers and the editor. This research was financially supported by the Natural Science Foundation of China (No. 41271267). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.catena.2015.09.004. References Aller, R., 1990. Bioturbation and manganese cycling in hemipelagic sediments. Philos. Trans. R. Soc. Lond. Ser. A Math. Phys. Sci. 331, 51–68. Bäckman, J.S., Klemedtsson, Å.K., 2003. Increased nitrification in acid coniferous forest soil due to high nitrogen deposition and liming. Scand. J. For. Res. 18, 514–524. Cheng, W., Tsuruta, H., Chen, G., Yagi, K., 2004. N2O and NO production in various Chinese agricultural soils by nitrification. Soil Biol. Biochem. 36, 953–963. Compton, J.E., Watrud, L.S., Arlene Porteous, L., DeGrood, S., 2004. Response of soil microbial biomass and community composition to chronic nitrogen additions at Harvard forest. For. Ecol. Manag. 196, 143–158. De Boer, W., Kowalchuk, G., 2001. Nitrification in acid soils: micro-organisms and mechanisms. Soil Biol. Biochem. 33, 853–866. Francis, C.A., Roberts, K.J., Beman, J.M., Santoro, A.E., Oakley, B.B., 2005. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proc. Natl. Acad. Sci. U. S. A. 102, 14683–14688. Fujimoto, C.K., Sherman, G.D., 1948. Behavior of manganese in the soil and the manganese cycle. Soil Sci. 66, 131–145. Hue, N.V., Vega, S., Silva, J.A., 2001. Manganese toxicity in a Hawaiian oxisol affected by soil pH and organic amendments. Soil Sci. Soc. Am. J. 65, 153–160. Jenkins, M.C., Kemp, W.M., 1984. The coupling of nitrification and denitrification in two estuarine sediments l v2. Limnol. Oceanogr. 29, 609–619. Jiang, X., Shi, X., Liu, W., Wright, A.L., 2011. Kinetics of net nitrification associated with soil aggregates under conventional and no-tillage in a subtropical rice soil. Plant Soil 347, 305–312.
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