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The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils
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Soil Biology & Biochemistry xxx (2014) 1e6
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The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils Q5
Jinbo Zhang a, b, c, *, Weijun Sun a, Wenhui Zhong a, c, Zucong Cai a, c a
School of Geography Sciences, Nanjing Normal University, Nanjing 210023, China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, China c Jiangsu Key Laboratory of Environmental Change and Ecological Construction, Nanjing 210023, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 January 2014 Received in revised form 28 April 2014 Accepted 1 May 2014 Available online xxx
In this study, a 15N tracer experiment was carried out to investigate the relative availability of different nitrogen (N) substrates for heterotrophic nitrifiers and to determine the significance of heterotrophic nitrification in two acidic forest soils in eastern China. Five 15N labeled substrates were applied, i.e. Glycine (4.90 atom% 15N excess), L-glutamic acid (4.89 atom% 15N excess), maize straw (3.63 atom% 15N excess), (NH4)2SO4 (4.97 atom% 15N excess), and control and were incubated for 4 days without or with C2H2 at 1 KPa (1%) in laboratory. The results showed that accumulation of 15N-NO 3 significantly increased with the incubation time in all 15N labeled treatments in the presence of acetylene, indicating that NO 3 produced from heterotrophic nitrification convincingly occurred and heterotrophic nitrifiers could use both ammonium and organic N compounds substrates for nitrification in the studied soils. The 15 N-NO 3 production in the Glycine and L-glutamic acid treatments in the presence of acetylene was obviously higher than that in the (NH4)2SO4 and maize straw treatments (p < 0.05), indicating availability of N substrates for heterotrophic nitrifiers was different. The heterotrophic nitrification of amino organic N compounds could occur via a combined organic and inorganic pathway. However, for complicated organic N substrate, e.g. maize straw in this study, almost all 15N-NO 3 was produced by organic N pathway. The contribution of heterotrophic nitrification to total nitrification varied from 23% to 93% in the different N substrate treatments as follows: (NH4)2SO4 < amino acid < maize straw. The substrate was an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils. Ó 2014 Published by Elsevier Ltd.
Keywords: Acidic forest soil Heterotrophic nitrification 15 N tracer experiment Substrates
1. Introduction It is widely accepted that nitrate (NO 3 ) could be produced by two pathways in soils (Pedersen et al., 1999; De Boer and Kowalchuk, 2001; Huygens et al., 2008; Zhang et al., 2013a,b). One is the oxidation of ammonia to NO 3 driving by chemoautotrophic nitrifiers, i.e. autotrophic nitrification. The other is the heterotrophic nitrification, which is driven by heterotrophic nitrifying bacteria or fungal. Because autotrophic nitrifiers are sensitive to low pH (Weber and Gainey, 1962), it is thought that NO 3 is produced predominantly via heterotrophic nitrification process in acidic soils (Kreitinger et al., 1985; Killham, 1990; Wood, 1990; Huygens et al., 2008; Zhang et al., 2011, 2013a,b). However,
* Corresponding author. School of Geography Sciences, Nanjing Normal University, Nanjing 210023, China. Tel.: þ86 25 8589 1203; fax: þ86 25 8589 1745. E-mail address: [email protected] (J. Zhang).
the significance of heterotrophic nitrification process in soil N transformation is largely unknown (Killham, 1986; De Boer and Kowalchuk, 2001), although the presence of this process has been demonstrated in some pure culture experiments for several decades (Schmidt, 1960; Killham, 1986; Honda et al., 1998). Some studies, using inhibition methods (mostly acetylene), have provided quantitative data on the relative importance of heterotrophic and autotrophic nitrification in acidic pasture or forest soils (Barraclough and Puri, 1995; Pedersen et al., 1999; Zhang et al., 2013b). Previous investigations showed a very large variation of the relative contribution of heterotrophic to nitrification in acidic soils. Barraclough and Puri (1995) reported that about only 8% of nitrification could come from heterotrophic nitrification in an acidic woodland soil (pH 3.8). However, Pedersen et al. (1999) observed that the importance of heterotrophic nitrification varied in the different land use soils, which was responsible for about 18%, 67%, 78%, 92% of total nitrification in Clear cut area soil (pH 5.7), mature forest organic horizon (pH 5.2), young forest (pH 5.9) and
http://dx.doi.org/10.1016/j.soilbio.2014.05.001 0038-0717/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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mature forest mineral soil (pH 5.8), respectively. Similarly, Zhang et al. (2013b) reported autotrophic nitrification was the predominant nitrification process (more than 99%) in acidic arable soils (pH 4.6), while, the heterotrophic nitrification accounted for more than 95% of the total nitrification in the acidic coniferous forest soil (pH 4.5). Above mentioned results suggested that low soil pH could not be the only factor controlling the importance of heterotrophic nitrification. For modeling nitrification in acidic soils accurately, more studies on the relative importance of heterotrophic and autotrophic nitrification and its controlling factors are needed. Previous investigation has suggested that the character and availability of substrates may be more important than pH in affecting heterotrophic nitrification process in soils (Killham, 1986). Many reports have suggested that heterotrophic nitrifiers can use both inorganic and organic substrates for nitrification (Honda et al., 1998; De Boer and Kowalchuk, 2001). Pure culture experiments in cuvette have showed that various N compounds can act as substrates for heterotrophic nitrification (Focht and Verstraete, 1977; Rho, 1986; Stroo et al., 1986). However, some investigations observed that unidentified organic N compounds, not NHþ 4 , were the substrate for heterotrophic nitrification in acidic soils (Barraclough and Puri, 1995; Pedersen et al., 1999; Islam et al., 2007). To date, very few studies were carried out to investigate the relative availability of N substrates for heterotrophic nitrification in the acidic soils. The main objective of this study was to investigate the relative availability of different N substrates for heterotrophic nitrifiers and to determine the significance of heterotrophic nitrification in two acidic forest soils in eastern China.
water). One mL of inorganic or organic N (amino acid) solution was added to each of the conical flask at a rate of 240 mg N kg1 soil. The powder of maize straw was added to the flask and well mixed with soil. Because the turnover of amino acids is very rapid in soil (Levi cnik-Höfferle et al., 2012), the soils were incubated at 25 C and 60% of the water-hold capacity for 4 days without or with C2H2 at 1 KPa (1%) under dark conditions. Previous investigations, using the method of DNA-based stable isotope probing (SIP), have shown that low concentration of acetylene 10 Pa (0.01%) can inhibit autotrophic nitrification completely in the same soil samples (Wang and Zhong, unpublished data) and in acidic soils in the same region (Lu and Jia, 2013). To make sure that autotrophic nitrification can be inhibited completely in the studied soils, soil samples for the acetylene treatment was previously exposed to 1 KPa (1%) acetylene for one day (De Boer and Kowalchuk, 2001). The acetylene (1%) was kept continuously in the headspace during the experiment. The samples were collected at 2 and 4 days after substrates application. Three flasks were randomly selected from each substrate treatment, and the soil was extracted using 2 M KCl to determine the NHþ 4 and NO3 concentrations by a continuous-flow analyzer (Skalar, Breda, Netherlands) and their 15N enrichment by Isotope Ratio Mass Spectrometry (IRMS 20e22, SerCon, Crewe, UK). The details were given by Zhang et al. (2011). Three flasks were immediately, randomly selected from each substrate treatment after addition of substrates. Soil samples were immediately mixed with deionized water at a soil:water ratio of 1:2.5 (v/v) and shook on a mechanical shaker for 5 min at 300 rpm at 25 C. Soil pH was immediately measured in soil-water suspension using a DMP-2 mV/pH detector (Quark Ltd, Nanjing, China).
2. Materials and methods 2.3.
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N pool dilution experiment
2.1. Study site and soil sample The study site was located in Wanmulin Nature Reserve in Fujian province, in eastern China (118 090 E, 27 030 ). The study region has a middle sub-tropical monsoon climate, with a mean annual air temperature of 19.4 C and 277 days annual frost-free period. The mean annual precipitation is 1731 mm, most of which falls between March to August. The soil parent material is granite and soils are classified as red soils (humic Planosols, FAO). Soil depth exceeds 1.0 m, and the depths of the O and A horizons are about 4 cm and 10 cm, respectively (Lin et al., 2011). The evergreen broadleaved forest covers approximately 189 ha in the Wanmulin Nature Reserve. Two sites different in dominant tree species were selected in the present investigation, one site was dominated by Cinnamomum chekiangense (CI), and the other was Castanopsis fargesii (CA). Soil samples were collected in July 2013. Five grids (about 4 m 4 m) were randomly staked out at each site. From each grid, the O horizon was removed firstly and three subsamples were collected from the 0e10 cm zone. All subsamples at the same site were put together, and passed through a 2 mm sieve. From the sieved sample two subsamples were collected for 1) incubation experiment and 2) analysis of soil properties. Two soils have acidic pH (4.4 and 4.7 for CI and CA, respectively). Soil organic C content was 31.5 g kg1 and 30.2 g kg1 and C to N ratio was 13.6, and 16.1, for CI and CA, respectively.
Gross nitrification rate in the studied soils was determined using the 15N pool dilution technique (Kirkham and Bartholomew, 1954). Briefly, the nitrate pool was labeled using K15NO3 (10.12 atom% excess). For each soil, six 250 ml Erlenmeyer flasks were prepared with 20 g of fresh soil (oven-dry basis). Two ml of K15NO3 solution 1 was added to each of the flasks at a rate of 2 mg NO soil. 3 -N kg The soil was adjusted to 60% water hold capacity and incubated for 24 h at 25 C. The soils (three replications) were extracted at 0.5 and 24 h after K15NO3 application to determine the concentration and isotopic composition of the NO 3 . The gross nitrification rate was calculated by the 15N isotope pool dilution method (Kirkham and Bartholomew, 1954). 2.4. Calculation and statistical analyses Based on the assumption that autotrophic nitrifiers were completely inhibited by 1 KPa acetylene, the ratio of the production of 15N-NO 3 in the presence of acetylene to the production of 15 N-NO 3 in the absence of acetylene was defined as the contribution of heterotrophic nitrification to total nitrification. One-way ANOVA was carried out to compare the results among the substrate treatments using SPSS 17.0 software (where p < 0.05, the difference was considered significant). 3. Results
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N labeled N substrates experiments 3.1. Change of soil pH after addition of substrates
For each soil, a series of 250 ml Erlenmeyer flasks, each containing 20 g of soil, was prepared. Five 15N labeled substrates were applied: Glycine (4.90 atom% 15N excess), L-glutamic acid (4.89 atom% 15N excess), maize straw (3.63 atom% 15N excess, C/N ratio 55), (NH4)2SO4 (4.97 atom% 15N excess) and control (CK, added
Soil pH was immediately measured after addition of substrates in the different treatments (Fig. 1). There were significant changes in pH after adding substrates, which ranged from 3.9 to 5.2 and were likely to affect nitrification process. Comparing with the
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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Fig. 1. Soil pH measured immediately after addition of substrates in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b). CK: control; L-g: Lglutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4. Identical letters indicate no statistical differences for the average values between the treatments.
control, addition of (NH4)2SO4 and L-glutamic acid reduced soil pH (p < 0.05), and the lowest value was observed in the L-glutamic acid treatment. While, soil pH significantly increased in the maize straw treatment (p < 0.05). Addition of Glycine did not affect soil pH. 3.2. NHþ 4 concentration Ammonium concentration rapidly increased for the Glycine and acid treatments whether in the presence or absence of acetylene (Fig. 2), which was higher in the presence of acetylene than in the absence of acetylene generally. Ammonium concentration showed no change in the control treatment, while, it significantly decreased in the maize straw treatment, especially at the end of the incubation. The 15N-NHþ 4 concentration rapidly, significantly increased with the incubation time in the treatments with the Glycine and L-glutamic acid added whether in the presence or absence of acetylene (Fig. 3). While, for the maize straw treatment, the 15N-NHþ 4 concentration was very low and did not significantly change during the L-glutamic
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incubation. About 63%e78% of added 15N-Glycine and 15N-L-glutamic acid was measured in the NHþ 4 pool after 4 days of incubation, while, the 15N-NHþ 4 in maize straw treatment was less than 2% of added 15N. The 15N-NHþ 4 concentration significantly decreased with the incubation time in the (NH4)2SO4 treatment. Although in the 15 N-NHþ 4 concentration in the (NH4)2SO4 treatment was slightly higher than in the Glycine and L-glutamic acid treatment after 2 days of incubation, there was no significant difference for the 15NNHþ 4 concentration among the (NH4)2SO4, Glycine and L-glutamic acid treatments at 2 and 4 days of incubation in the presence of acetylene due to the large variation (Fig. 3). 3.3. NO 3 production Nitrate concentration at the beginning of incubation in the presence of acetylene was lower than that in the absence of acetylene for both studied soils, due to one day pre-incubation (Fig. 4). During the incubation, NO 3 concentration only slightly increased
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Fig. 3. The dynamic of 15N-NHþ 4 concentration in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: 15 N-NHþ 4 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
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Fig. 4. Nitrate concentration in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: NO 3 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. CK: control; L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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Fig. 5. The abundance of 15N in nitrate pool in the (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: Abundance of 15 N in nitrate pool in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
for the CK, Glycine, L-glutamic acid and (NH4)2SO4 treatments in the presence of acetylene. Comparatively, NO 3 concentration significantly increased for the CK, Glycine, and L-glutamic acid treatments in the absence of acetylene, especially for the CI soil. However, for the maize straw treatment, NO 3 concentration significantly decreased during the incubation in both studied soils whether in the presence or absence of acetylene. Abundance of 15N in NO 3 pool significantly increased in all treatments whether in the presence or absence of acetylene (Fig. 5), 15 indicating that NO 3 produced from the high N abundance N pool must have entered the NO3 pool in the studied soil. The 15N-NO 3 abundance in the Glycine, L-glutamic acid and (NH4)2SO4 treatments in the presence acetylene was significantly lower than that in the absence of acetylene (p < 0.05), suggesting that the oxidation of NHþ 4 to NO3 by autotrophic nitrifiers could be inhibited. However, for the maize straw treatment, the 15N-NO 3 abundance in the presence of acetylene was not obviously different from that in the absence of acetylene.
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Fig. 6. The dynamic of 15N-NO 3 production in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: 15N-NO 3 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
Accumulation of 15N-NO 3 significantly increased with the incubation time in all 15N labeled treatments in the presence of acetylene (Fig. 6), suggesting that NO 3 produced from heterotrophic nitrification had convincingly occurred in the studied soils. The 15N-NO 3 concentration in the Glycine, L-glutamic acid and (NH4)2SO4 treatments in the presence of acetylene was significantly lower than that in the absence of acetylene (p < 0.01). In the absence of acetylene, 15N-NO 3 concentration in the Glycine, L-glutamic acid and (NH4)2SO4 treatments was significantly higher than that in the maize straw treatment (p < 0.01) (Fig. 6). However, in the presence of acetylene, 15N-NO 3 concentration in the (NH4)2SO4 treatment was similar to that in the maize straw treatment, which was obviously lower than the Glycine and L-glutamic acid treatments at 4 days of incubation (p < 0.05) (Fig. 6). The 15N-NO 3 concentration in the presence of acetylene in the maize straw treatment was not obviously different from that in the absence of acetylene. Accumulation of 15N-NO 3 linearly increased with the incubation time in the (NH4)2SO4 treatment during the incubation whether in the presence or absence of acetylene (Fig. 6), suggesting that the oxidation of NHþ 4 to NO3 rate was constant in the studied soils during the incubation. 3.4. The contribution of heterotrophic nitrification to total nitrification The average contribution of heterotrophic nitrification to total nitrification in the incubation varied from 23% to 93% in the studied acidic soils, depending on the different N substrate treatments (Fig. 7). The lowest was observed in the (NH4)2SO4 treatment, accounting for 23% and 36% in the CA and CI soils, respectively. For the amino acid treatments, the average contribution ranged from 41% to 49%. The highest contribution was measured in the maize straw treatment, with average 93% and 80% for the CA and CI soils, respectively, which were significantly higher than those of amino acid and (NH4)2SO4 treatments (p < 0.05). 4. Discussions In the present investigation, the selective inhibitor, i.e. acetylene, was used to distinguish between heterotrophic and autotrophic nitrification in the studied soils. It is well known that low concentration of acetylene (often 1 Pae1 KPa) can efficiently inhibit ammonia mono-oxygenase, but can not affect heterotrophic nitrification (Hyman and Wood, 1985; Killham, 1987; Hart et al., 1997; Pedersen et al., 1999; Stopnisek et al., 2010; Levi cnik-Höfferle et al., 2012; Zhang et al., 2013b; Lu and Jia, 2013). The previous investigations, using the method of DNA-based stable isotope probing (SIP), have shown that low concentration of acetylene 10 Pa (0.01%) or 100 Pa (0.1%) can inhibit autotrophic nitrification completely in the same soil samples (Wang and Zhong, unpublished data), for the acidic soils in same region (Lu and Jia, 2013) and in the other regions (Stopnisek et al., 2010; Leviŏnik-Höfferle et al., 2012). However, for some acidic forest soils, partial inhibition of autotrophic nitrification by acetylene was observed during short term incubations (Killham, 1987; Hart et al., 1997; Pedersen et al., 1999). The main reason could be that a fraction of autotrophic nitrifiers could not be exposed to acetylene during the short term incubation (De Boer and Kowalchuk, 2001). To make sure that autotrophic nitrification can be inhibited completely, soil samples for the acetylene treatment was exposed to 1 KPa (1%) acetylene for one day in the present study. Therefore, the demonstration of heterotrophic nitrification could be more convincing in the present investigation (De Boer and Kowalchuk, 2001). Based on this assumption, the importance of heterotrophic nitrification in the N dynamics of the studied acid soils was assessed.
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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J. Zhang et al. / Soil Biology & Biochemistry xxx (2014) 1e6
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Fig. 7. The average contribution of heterotrophic nitrification to total nitrification for the different substrates in the whole incubation: The contribution in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: Lglutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4. Identical letters indicate no statistical differences for the average values between the treatments.
The results, in the present investigation, indicated that NO 3 production via heterotrophic nitrification process convincingly occurred in the studied acidic soils. The heterotrophic nitrifiers could use both ammonium and organic N compounds substrates for nitrification, in line with some previous studies (Focht and Verstraete, 1977; Rho, 1986; Stroo et al., 1986; Honda et al., 1998; De Boer and Kowalchuk, 2001). The heterotrophic nitrifying bacteria or fungal, which possess ammonia and hydroxylamine oxidizing enzymes, could be responsible for the oxidation of ammonium by heterotrophs (Moir et al., 1996; Daum et al., 1998; Nishio et al., 1998). The results, in the present study, in which accumulation of 15N-NO 3 significantly increased with the incubation time for the (NH4)2SO4 treatment in the presence of acetylene (1 KPa), testified the existence of inorganic heterotrophic nitrification pathway. However, some investigations reported that NHþ 4 was not an important substrate for heterotrophic nitrification, while, unidentified organic N compounds were the substrate in acidic soils (Barraclough and Puri, 1995; Pedersen et al., 1999). Therefore, many previous studies used the term of heterotrophic nitrification to indicate the oxidation of organic N to NO 3 (Barraclough and Puri, 1995; Pedersen et al., 1999; Müller et al., 2007; Zhang et al., 2011). If the oxidation of ammonium by heterotrophs was not taken into account, the potentially underestimating heterotrophic nitrification, while, overestimating autotrophic nitrification could occur. The organic N substrates heterotrophic nitrification pathway, mainly driving by fungi, was the process that the organic aminonitrogen was oxidized to NO 3 (Killham, 1990; De Boer and Kowalchuk, 2001), which involved the processes that amine or amide was oxidized to a substituted hydroxylamine, which was subsequently oxidized to a nitroso-compound and then to a nitrocompound (Killham, 1990). In the present study, 15N-NO 3 production in the presence of acetylene for the Glycine and L-glutamic acid treatments was clearly observed and was obviously higher than that in the (NH4)2SO4 treatment, confirmed the occurrence of organic N heterotrophic nitrification pathway. Based on the change 15 of 15N-NO N-NHþ 3 and 4 concentration in the Glycine, L-glutamic acid and (NH4)2SO4 treatments for the studied soils, we speculated that the oxidation of amino organic N compounds (e.g. Glycine and L-glutamic acid used in this study) to NO3 by heterotrophic nitrification process could occur via a combined organic and inorganic pathway. Most of amino organic N was mineralized to ammonium firstly (about 63%e78% within 4 days of incubation), then the
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ammonium was oxidized to NO 3 via heterotrophs, i.e. inorganic heterotrophic nitrification pathway. The other part of amino organic N was oxidized directly to NO 3 via organic heterotrophic nitrification pathway. Another possible reason of the results that higher 15N-NO 3 production from amino organic N compounds than from (NH4)2SO4 might be that ammonia derived from amino organic N compounds favored nitrification in acidic soil more than ammonium (Stopnisek et al., 2010; Levi cnik-Höfferle et al., 2012). Further studies were needed to carry out to prove the organic pathway of heterotrophic nitrification and to estimate the relative contribution of organic and inorganic processes to heterotrophic nitrification in the acidic soils. In the present study, based on the assumption that the rate of the inorganic heterotrophic nitrification pathway in the Glycine and L-glutamic acid treatments was equal to the (NH4)2SO4 treatment, the relative contribution of organic and inorganic processes to heterotrophic nitrification in the amino acid treatment could be calculated. The contribution of organic N heterotrophic nitrification process ranged from 42% to 55%, and there was no significant difference between the different amino acid treatments and between the different studied soils. However, for complicated organic N substrate, e.g. maize straw in the present study, almost all 15N-NO 3 was produced by organic N heterotrophic nitrification pathway, due to the low mineralization of added 15 N-maize straw, which was supported by the results that the 15 N-NO 3 production in the presence of acetylene was not obviously different from that in the absence of acetylene in the maize straw treatment during the incubation. The fungal nitrifiers could switch to alternative nitrification pathways depending on substrate availability in the soil (Killham, 1990; Aarnio and Martikainen, 1992). It has been widely accepted that heterotrophic nitrification could make some contribution to total nitrification in acidic soils (Kreitinger et al., 1985; Killham, 1990; Wood, 1990; Huygens et al., 2008; Zhang et al., 2011, 2013a,b), because autotrophic nitrifiers are sensitive to low pH (Weber and Gainey, 1962). However, the relative contribution of heterotrophic nitrification to total nitrification varied largely among the previous investigations (Barraclough and Puri, 1995; Pedersen et al., 1999; Zhang et al., 2013b). Our results showed that the contribution of heterotrophic nitrification to total nitrification varied depending on the substrate treatments as follows: (NH4)2SO4 < amino acid < maize straw, suggested that the substrate may be an important factor in controlling the significance of heterotrophic nitrification, in line with the previous investigations (Killham, 1986; Honda et al., 1998; De Boer and Kowalchuk, 2001). The C/N ratio of N substrate could play important role in affecting the significance of heterotrophic nitrification, since the highest contribution was observed in the addition of maize straw (with the high C/N ratio, 55), while, the lowest was found in the (NH4)2SO4 treatment. Organic N compounds with high C/N ratio increasing bioavailability of C could promote the heterotrophic bacteria growth, in turn, outcompete or inhibit nitrifying bacteria growth and autotrophic nitrification activity (Fauci and Dick, 1994; Stouthamer et al., 1997; Shi and Norton, 2000). In addition, some previous studies suggested that the real significance of nitrification by the fungi could primarily lay in the competitive advantage of fungal growth strategy through this process in the soil (Killham, 1990). The by-products, produced in heterotrophic nitrification process, could well inhibit many microbial competition forms to the fungal nitrifier (Verstraete, 1975). These competition or inhibition effects could be an important reason, which resulted in the contribution of heterotrophic nitrification to total nitrification varied largely depending on substrates in the present study. Thus, the highest the contribution of heterotrophic nitrification to total nitrification was observed in the maize straw treatment with the highest C/N ratio, although soil pH significantly increased
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(pH > 5) in this treatment being more beneficial to autotrophic nitrification than the other treatments. Therefore, the substrate was a more important factor in controlling the significance of heterotrophic nitrification in acidic forest soils than soil pH. The results of 15N pool dilution experiment showed that the gross nitrification rate was 0.55 mg N kg1 d1 and 0.86 mg N kg1 d1 in the CI and CA soils, respectively. Combined the gross nitrification rate and the high contribution of heterotrophic nitrification to total nitrification, we could infer that there were the significant actual heterotrophic nitrification rates and the heterotrophic nitrification was surely an important N transformation process in the studied acidic soils. In this study, high amino organic N concentration (240 mg N kg1) was used to investigate the nitrification. However, to date, there is no information on whether there were sufficient quantities of these N compounds to support the heterotrophic nitrification process in soils (Pedersen et al., 1999). Further study should be carried out to testify heterotrophic nitrification of different N compounds in situ or in intact soil core, using low labeled N concentration. Uncited reference Waid, 1975. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (41222005, 41271255, and 41101209), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJA210002), the Natural Science Foundation of Jiangsu Province (BK2012731), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Aarnio, T., Martikainen, P.J., 1992. Nitrification in forest soil after refertilization with urea or urea and dicyandiamide. Soil Biology and Biochemistry 24, 951e954. Barraclough, D., Puri, G., 1995. The use of 15N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitrification. Soil Biology and Biochemistry 27, 17e22. Daum, M., Zimmer, W., Papen, H., Kloos, K., Nawrath, K., Bothe, H., 1998. Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida. Current Microbiology 37, 281e288. De Boer, W., Kowalchuk, G.A., 2001. Nitrification in acid soils: micro-organisms and mechanisms. Soil Biology and Biochemistry 33, 853e866. Fauci, M.F., Dick, R.P., 1994. Soil microbial dynamics: short-term and long-term effects of inorganic and organic nitrogen. Soil Science Society of America Journal 58, 801e806. Focht, D.D., Verstraete, W., 1977. Biochemical ecology of nitrification and denitrification. Advances in Microbial Ecology 1, 135e214. Hart, S.C., Binkley, D., Perry, D.A., 1997. Influence of red alder on soil nitrogen transformations in two conifer forests of contrasting productivity. Soil Biology and Biochemistry 29, 1111e1123. Honda, N., Hirai, M., Ano, T., Shoda, M., 1998. Antifungal effect of a heterotrophic nitrifier Alcaligenes faecalis. Biotechnology Letters 20, 703e705. Huygens, D., Boeckx, P., Templer, P., Paulino, L., Cleemput, O.V., Oyarzún, C., Müller, C., Godoy, R., 2008. Mechanisms for retention of bioavailable nitrogen in volcanic rainforest soils. Nature Geoscience 1, 543e548. Hyman, M.R., Wood, P.M., 1985. Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene. Biochemistry Journal 227, 719e725.
Islam, A., Chen, D., White, R.E., 2007. Heterotrophic and autotrophic nitrification in two acid pasture soils. Soil Biology and Biochemistry 39, 972e975. Killham, K., 1986. Heterotrophic nitrification. In: Presser, J.I. (Ed.), Nitrification, Special publications for Society for General Microbiology, vol. 20. IRL Press, Oxford, pp. 117e126. Killham, K., 1987. A new perfusion system for measurement and characterization of potential rates of soil nitrification. Plant and Soil 97, 267e272. Killham, K., 1990. Nitrification in coniferous forest soils. Plant and Soil 128, 31e44. Kirkham, D., Bartholomew, W.V., 1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33e34. Kreitinger, J.P., Klein, T.M., Novick, N.J., Alexander, M., 1985. Nitrification and characteristics of nitrifying microorganisms in an acid forest soil. Soil Science Society of America Journal 49, 1407e1410. Nicol, G.W., Ausec, L., Mandi Levi cnik-Höfferle, S., c-Mulec, I., Prosser, J.I., 2012. Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen. FEMS Microbiology Ecology 80, 114e123. Lin, C., Yang, Y., Guo, J., Chen, G., Xie, J., 2011. Fine root decomposition of evergreen broadleaved and coniferous tree species in mid-subtropical China: dynamics of dry mass, nutrient and organic fractions. Plant and Soil 338, 311e327. Lu, L., Jia, Z., 2013. Urease gene-containing Archaea dominate autotrophic ammonia oxidation in two acid soils. Environmental Microbiology 15, 1795e1809. Moir, J.W.B., Crossman, L.C., Spiro, S., Richardson, D.J., 1996. The purification of ammonia mono-oxygenase from Paracoccus denitrificans. FEBS Letters 387, 71e74. Müller, C., Rütting, T., Kattge, J., Laughlin, R.J., Stevens, R.J., 2007. Estimation of parameters in complex 15N tracing models via Monte Carlo sampling. Soil Biology and Biochemistry 39, 715e726. Nishio, T., Yoshikura, T., Mishima, H., Inouye, Z., Itoh, H., 1998. Conditions for nitrification and denitrification by an immobilized heterotrophic bacterium Alcaligenes faecalis OKK17. Journal of Fermentation and Bioengineering 86, 351e356. Pedersen, H., Dunkin, K.A., Firestone, M., 1999. The relative importance of autotrophic and heterotrophic nitrification in a conifer forest soil as measured by 15 N tracer and pool dilution techniques. Biogeochemistry 44, 135e150. Rho, J., 1986. Microbial interactions in heterotrophic nitrification. Canadian Journal of Microbiology 32, 243e247. Schmidt, E.L., 1960. Nitrate formation by Aspergillus flavus in pure and mixed culture environments. In: Transactions of the 7th International Congress of Soil Science, vol. 2, pp. 600e605. Shi, W., Norton, J.M., 2000. Microbial control of nitrate concentrations in an agricultural soil treated with dairy waste compost or ammonium fertilizer. Soil Biology and Biochemistry 32, 1453e1457. Nicol, G.W., Mandi Stopnisek, N., Gubry-Rangin, C., Höfferle, S., c-Mulec, I., Prosser, J.I., 2010. Thaumarchaeal ammonia oxidation in an acidic forest peat soil is not influenced by ammonium amendment. Applied and Environmental Microbiology 76, 7626e7634. Stouthamer, A.H., de Boer, A.P.N., Van der Oost, J., Van Spanning, R.J.M., 1997. Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria. Antonie van Leeuwenhoek 71, 33e41. Stroo, H.F., Klein, M.T., Alexander, M., 1986. Heterotrophic nitrification in an acid forest soil and by an acid-tolerant fungus. Applied and Environmental Microbiology 52, 107e111. Verstraete, W., 1975. Heterotrophic nitrification in soils and aqueous media e a review. Biologu Bulletin of the Academy Sciences of the U.S.S.R. 4, 515e530. Q3 Waid, J.S., 1975. Hydroxamic acids in soil systems. In: Paul, E.A., McLaren, A.D. (Eds.), Soil Biochemistry, vol. 4. Marcel Dekker, New York. Weber, D.F., Gainey, P.L., 1962. Relative sensitivity of nitrifying organisms to hydrogen ions in soils and solutions. Soil Science 94, 138e145. Wood, P.M., 1990. Autotrophic and heterotrophic mechanisms for ammonia oxidation. Soil Use and Management 6, 78e79. Zhang, J., Cai, Z., Zhu, T., Yang, W., Müller, C., 2013a. Mechanisms for the retention of inorganic N in acidic forest soils of southern China. Scientific Reports 3, 2342. http://dx.doi.org/10.1038/srep02342. Zhang, J., Zhu, T., Cai, Z., Müller, C., 2011. Heterotrophic nitrification is the predominant NO 3 production mechanism in coniferous but not broad-leaf acid forest soil in subtropical China. Biology and Fertility of Soils 47, 533e542. Zhang, Y., Zhang, J., Meng, T., Zhu, T., Müller, C., Cai, Z., 2013b. Heterotrophic nitrification is the predominant NO 3 production pathway in acid coniferous forest soil in subtropical China. Biology and Fertility of Soils 49, 955e957.
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The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils Q5
Jinbo Zhang a, b, c, *, Weijun Sun a, Wenhui Zhong a, c, Zucong Cai a, c a
School of Geography Sciences, Nanjing Normal University, Nanjing 210023, China State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, China c Jiangsu Key Laboratory of Environmental Change and Ecological Construction, Nanjing 210023, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 January 2014 Received in revised form 28 April 2014 Accepted 1 May 2014 Available online xxx
In this study, a 15N tracer experiment was carried out to investigate the relative availability of different nitrogen (N) substrates for heterotrophic nitrifiers and to determine the significance of heterotrophic nitrification in two acidic forest soils in eastern China. Five 15N labeled substrates were applied, i.e. Glycine (4.90 atom% 15N excess), L-glutamic acid (4.89 atom% 15N excess), maize straw (3.63 atom% 15N excess), (NH4)2SO4 (4.97 atom% 15N excess), and control and were incubated for 4 days without or with C2H2 at 1 KPa (1%) in laboratory. The results showed that accumulation of 15N-NO 3 significantly increased with the incubation time in all 15N labeled treatments in the presence of acetylene, indicating that NO 3 produced from heterotrophic nitrification convincingly occurred and heterotrophic nitrifiers could use both ammonium and organic N compounds substrates for nitrification in the studied soils. The 15 N-NO 3 production in the Glycine and L-glutamic acid treatments in the presence of acetylene was obviously higher than that in the (NH4)2SO4 and maize straw treatments (p < 0.05), indicating availability of N substrates for heterotrophic nitrifiers was different. The heterotrophic nitrification of amino organic N compounds could occur via a combined organic and inorganic pathway. However, for complicated organic N substrate, e.g. maize straw in this study, almost all 15N-NO 3 was produced by organic N pathway. The contribution of heterotrophic nitrification to total nitrification varied from 23% to 93% in the different N substrate treatments as follows: (NH4)2SO4 < amino acid < maize straw. The substrate was an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils. Ó 2014 Published by Elsevier Ltd.
Keywords: Acidic forest soil Heterotrophic nitrification 15 N tracer experiment Substrates
1. Introduction It is widely accepted that nitrate (NO 3 ) could be produced by two pathways in soils (Pedersen et al., 1999; De Boer and Kowalchuk, 2001; Huygens et al., 2008; Zhang et al., 2013a,b). One is the oxidation of ammonia to NO 3 driving by chemoautotrophic nitrifiers, i.e. autotrophic nitrification. The other is the heterotrophic nitrification, which is driven by heterotrophic nitrifying bacteria or fungal. Because autotrophic nitrifiers are sensitive to low pH (Weber and Gainey, 1962), it is thought that NO 3 is produced predominantly via heterotrophic nitrification process in acidic soils (Kreitinger et al., 1985; Killham, 1990; Wood, 1990; Huygens et al., 2008; Zhang et al., 2011, 2013a,b). However,
* Corresponding author. School of Geography Sciences, Nanjing Normal University, Nanjing 210023, China. Tel.: þ86 25 8589 1203; fax: þ86 25 8589 1745. E-mail address: [email protected] (J. Zhang).
the significance of heterotrophic nitrification process in soil N transformation is largely unknown (Killham, 1986; De Boer and Kowalchuk, 2001), although the presence of this process has been demonstrated in some pure culture experiments for several decades (Schmidt, 1960; Killham, 1986; Honda et al., 1998). Some studies, using inhibition methods (mostly acetylene), have provided quantitative data on the relative importance of heterotrophic and autotrophic nitrification in acidic pasture or forest soils (Barraclough and Puri, 1995; Pedersen et al., 1999; Zhang et al., 2013b). Previous investigations showed a very large variation of the relative contribution of heterotrophic to nitrification in acidic soils. Barraclough and Puri (1995) reported that about only 8% of nitrification could come from heterotrophic nitrification in an acidic woodland soil (pH 3.8). However, Pedersen et al. (1999) observed that the importance of heterotrophic nitrification varied in the different land use soils, which was responsible for about 18%, 67%, 78%, 92% of total nitrification in Clear cut area soil (pH 5.7), mature forest organic horizon (pH 5.2), young forest (pH 5.9) and
http://dx.doi.org/10.1016/j.soilbio.2014.05.001 0038-0717/Ó 2014 Published by Elsevier Ltd.
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mature forest mineral soil (pH 5.8), respectively. Similarly, Zhang et al. (2013b) reported autotrophic nitrification was the predominant nitrification process (more than 99%) in acidic arable soils (pH 4.6), while, the heterotrophic nitrification accounted for more than 95% of the total nitrification in the acidic coniferous forest soil (pH 4.5). Above mentioned results suggested that low soil pH could not be the only factor controlling the importance of heterotrophic nitrification. For modeling nitrification in acidic soils accurately, more studies on the relative importance of heterotrophic and autotrophic nitrification and its controlling factors are needed. Previous investigation has suggested that the character and availability of substrates may be more important than pH in affecting heterotrophic nitrification process in soils (Killham, 1986). Many reports have suggested that heterotrophic nitrifiers can use both inorganic and organic substrates for nitrification (Honda et al., 1998; De Boer and Kowalchuk, 2001). Pure culture experiments in cuvette have showed that various N compounds can act as substrates for heterotrophic nitrification (Focht and Verstraete, 1977; Rho, 1986; Stroo et al., 1986). However, some investigations observed that unidentified organic N compounds, not NHþ 4 , were the substrate for heterotrophic nitrification in acidic soils (Barraclough and Puri, 1995; Pedersen et al., 1999; Islam et al., 2007). To date, very few studies were carried out to investigate the relative availability of N substrates for heterotrophic nitrification in the acidic soils. The main objective of this study was to investigate the relative availability of different N substrates for heterotrophic nitrifiers and to determine the significance of heterotrophic nitrification in two acidic forest soils in eastern China.
water). One mL of inorganic or organic N (amino acid) solution was added to each of the conical flask at a rate of 240 mg N kg1 soil. The powder of maize straw was added to the flask and well mixed with soil. Because the turnover of amino acids is very rapid in soil (Levi cnik-Höfferle et al., 2012), the soils were incubated at 25 C and 60% of the water-hold capacity for 4 days without or with C2H2 at 1 KPa (1%) under dark conditions. Previous investigations, using the method of DNA-based stable isotope probing (SIP), have shown that low concentration of acetylene 10 Pa (0.01%) can inhibit autotrophic nitrification completely in the same soil samples (Wang and Zhong, unpublished data) and in acidic soils in the same region (Lu and Jia, 2013). To make sure that autotrophic nitrification can be inhibited completely in the studied soils, soil samples for the acetylene treatment was previously exposed to 1 KPa (1%) acetylene for one day (De Boer and Kowalchuk, 2001). The acetylene (1%) was kept continuously in the headspace during the experiment. The samples were collected at 2 and 4 days after substrates application. Three flasks were randomly selected from each substrate treatment, and the soil was extracted using 2 M KCl to determine the NHþ 4 and NO3 concentrations by a continuous-flow analyzer (Skalar, Breda, Netherlands) and their 15N enrichment by Isotope Ratio Mass Spectrometry (IRMS 20e22, SerCon, Crewe, UK). The details were given by Zhang et al. (2011). Three flasks were immediately, randomly selected from each substrate treatment after addition of substrates. Soil samples were immediately mixed with deionized water at a soil:water ratio of 1:2.5 (v/v) and shook on a mechanical shaker for 5 min at 300 rpm at 25 C. Soil pH was immediately measured in soil-water suspension using a DMP-2 mV/pH detector (Quark Ltd, Nanjing, China).
2. Materials and methods 2.3.
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N pool dilution experiment
2.1. Study site and soil sample The study site was located in Wanmulin Nature Reserve in Fujian province, in eastern China (118 090 E, 27 030 ). The study region has a middle sub-tropical monsoon climate, with a mean annual air temperature of 19.4 C and 277 days annual frost-free period. The mean annual precipitation is 1731 mm, most of which falls between March to August. The soil parent material is granite and soils are classified as red soils (humic Planosols, FAO). Soil depth exceeds 1.0 m, and the depths of the O and A horizons are about 4 cm and 10 cm, respectively (Lin et al., 2011). The evergreen broadleaved forest covers approximately 189 ha in the Wanmulin Nature Reserve. Two sites different in dominant tree species were selected in the present investigation, one site was dominated by Cinnamomum chekiangense (CI), and the other was Castanopsis fargesii (CA). Soil samples were collected in July 2013. Five grids (about 4 m 4 m) were randomly staked out at each site. From each grid, the O horizon was removed firstly and three subsamples were collected from the 0e10 cm zone. All subsamples at the same site were put together, and passed through a 2 mm sieve. From the sieved sample two subsamples were collected for 1) incubation experiment and 2) analysis of soil properties. Two soils have acidic pH (4.4 and 4.7 for CI and CA, respectively). Soil organic C content was 31.5 g kg1 and 30.2 g kg1 and C to N ratio was 13.6, and 16.1, for CI and CA, respectively.
Gross nitrification rate in the studied soils was determined using the 15N pool dilution technique (Kirkham and Bartholomew, 1954). Briefly, the nitrate pool was labeled using K15NO3 (10.12 atom% excess). For each soil, six 250 ml Erlenmeyer flasks were prepared with 20 g of fresh soil (oven-dry basis). Two ml of K15NO3 solution 1 was added to each of the flasks at a rate of 2 mg NO soil. 3 -N kg The soil was adjusted to 60% water hold capacity and incubated for 24 h at 25 C. The soils (three replications) were extracted at 0.5 and 24 h after K15NO3 application to determine the concentration and isotopic composition of the NO 3 . The gross nitrification rate was calculated by the 15N isotope pool dilution method (Kirkham and Bartholomew, 1954). 2.4. Calculation and statistical analyses Based on the assumption that autotrophic nitrifiers were completely inhibited by 1 KPa acetylene, the ratio of the production of 15N-NO 3 in the presence of acetylene to the production of 15 N-NO 3 in the absence of acetylene was defined as the contribution of heterotrophic nitrification to total nitrification. One-way ANOVA was carried out to compare the results among the substrate treatments using SPSS 17.0 software (where p < 0.05, the difference was considered significant). 3. Results
2.2.
15
N labeled N substrates experiments 3.1. Change of soil pH after addition of substrates
For each soil, a series of 250 ml Erlenmeyer flasks, each containing 20 g of soil, was prepared. Five 15N labeled substrates were applied: Glycine (4.90 atom% 15N excess), L-glutamic acid (4.89 atom% 15N excess), maize straw (3.63 atom% 15N excess, C/N ratio 55), (NH4)2SO4 (4.97 atom% 15N excess) and control (CK, added
Soil pH was immediately measured after addition of substrates in the different treatments (Fig. 1). There were significant changes in pH after adding substrates, which ranged from 3.9 to 5.2 and were likely to affect nitrification process. Comparing with the
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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Fig. 1. Soil pH measured immediately after addition of substrates in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b). CK: control; L-g: Lglutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4. Identical letters indicate no statistical differences for the average values between the treatments.
control, addition of (NH4)2SO4 and L-glutamic acid reduced soil pH (p < 0.05), and the lowest value was observed in the L-glutamic acid treatment. While, soil pH significantly increased in the maize straw treatment (p < 0.05). Addition of Glycine did not affect soil pH. 3.2. NHþ 4 concentration Ammonium concentration rapidly increased for the Glycine and acid treatments whether in the presence or absence of acetylene (Fig. 2), which was higher in the presence of acetylene than in the absence of acetylene generally. Ammonium concentration showed no change in the control treatment, while, it significantly decreased in the maize straw treatment, especially at the end of the incubation. The 15N-NHþ 4 concentration rapidly, significantly increased with the incubation time in the treatments with the Glycine and L-glutamic acid added whether in the presence or absence of acetylene (Fig. 3). While, for the maize straw treatment, the 15N-NHþ 4 concentration was very low and did not significantly change during the L-glutamic
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incubation. About 63%e78% of added 15N-Glycine and 15N-L-glutamic acid was measured in the NHþ 4 pool after 4 days of incubation, while, the 15N-NHþ 4 in maize straw treatment was less than 2% of added 15N. The 15N-NHþ 4 concentration significantly decreased with the incubation time in the (NH4)2SO4 treatment. Although in the 15 N-NHþ 4 concentration in the (NH4)2SO4 treatment was slightly higher than in the Glycine and L-glutamic acid treatment after 2 days of incubation, there was no significant difference for the 15NNHþ 4 concentration among the (NH4)2SO4, Glycine and L-glutamic acid treatments at 2 and 4 days of incubation in the presence of acetylene due to the large variation (Fig. 3). 3.3. NO 3 production Nitrate concentration at the beginning of incubation in the presence of acetylene was lower than that in the absence of acetylene for both studied soils, due to one day pre-incubation (Fig. 4). During the incubation, NO 3 concentration only slightly increased
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Fig. 2. Ammonium concentration in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: NHþ 4 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. CK: control; L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
M(C2H2) M
Fig. 3. The dynamic of 15N-NHþ 4 concentration in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: 15 N-NHþ 4 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
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Fig. 4. Nitrate concentration in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: NO 3 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. CK: control; L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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Fig. 5. The abundance of 15N in nitrate pool in the (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: Abundance of 15 N in nitrate pool in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
for the CK, Glycine, L-glutamic acid and (NH4)2SO4 treatments in the presence of acetylene. Comparatively, NO 3 concentration significantly increased for the CK, Glycine, and L-glutamic acid treatments in the absence of acetylene, especially for the CI soil. However, for the maize straw treatment, NO 3 concentration significantly decreased during the incubation in both studied soils whether in the presence or absence of acetylene. Abundance of 15N in NO 3 pool significantly increased in all treatments whether in the presence or absence of acetylene (Fig. 5), 15 indicating that NO 3 produced from the high N abundance N pool must have entered the NO3 pool in the studied soil. The 15N-NO 3 abundance in the Glycine, L-glutamic acid and (NH4)2SO4 treatments in the presence acetylene was significantly lower than that in the absence of acetylene (p < 0.05), suggesting that the oxidation of NHþ 4 to NO3 by autotrophic nitrifiers could be inhibited. However, for the maize straw treatment, the 15N-NO 3 abundance in the presence of acetylene was not obviously different from that in the absence of acetylene.
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Fig. 6. The dynamic of 15N-NO 3 production in the presence (solid point) and absence (hollow point) of acetylene for the different substrates during the incubation: 15N-NO 3 concentration in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: L-glutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4.
Accumulation of 15N-NO 3 significantly increased with the incubation time in all 15N labeled treatments in the presence of acetylene (Fig. 6), suggesting that NO 3 produced from heterotrophic nitrification had convincingly occurred in the studied soils. The 15N-NO 3 concentration in the Glycine, L-glutamic acid and (NH4)2SO4 treatments in the presence of acetylene was significantly lower than that in the absence of acetylene (p < 0.01). In the absence of acetylene, 15N-NO 3 concentration in the Glycine, L-glutamic acid and (NH4)2SO4 treatments was significantly higher than that in the maize straw treatment (p < 0.01) (Fig. 6). However, in the presence of acetylene, 15N-NO 3 concentration in the (NH4)2SO4 treatment was similar to that in the maize straw treatment, which was obviously lower than the Glycine and L-glutamic acid treatments at 4 days of incubation (p < 0.05) (Fig. 6). The 15N-NO 3 concentration in the presence of acetylene in the maize straw treatment was not obviously different from that in the absence of acetylene. Accumulation of 15N-NO 3 linearly increased with the incubation time in the (NH4)2SO4 treatment during the incubation whether in the presence or absence of acetylene (Fig. 6), suggesting that the oxidation of NHþ 4 to NO3 rate was constant in the studied soils during the incubation. 3.4. The contribution of heterotrophic nitrification to total nitrification The average contribution of heterotrophic nitrification to total nitrification in the incubation varied from 23% to 93% in the studied acidic soils, depending on the different N substrate treatments (Fig. 7). The lowest was observed in the (NH4)2SO4 treatment, accounting for 23% and 36% in the CA and CI soils, respectively. For the amino acid treatments, the average contribution ranged from 41% to 49%. The highest contribution was measured in the maize straw treatment, with average 93% and 80% for the CA and CI soils, respectively, which were significantly higher than those of amino acid and (NH4)2SO4 treatments (p < 0.05). 4. Discussions In the present investigation, the selective inhibitor, i.e. acetylene, was used to distinguish between heterotrophic and autotrophic nitrification in the studied soils. It is well known that low concentration of acetylene (often 1 Pae1 KPa) can efficiently inhibit ammonia mono-oxygenase, but can not affect heterotrophic nitrification (Hyman and Wood, 1985; Killham, 1987; Hart et al., 1997; Pedersen et al., 1999; Stopnisek et al., 2010; Levi cnik-Höfferle et al., 2012; Zhang et al., 2013b; Lu and Jia, 2013). The previous investigations, using the method of DNA-based stable isotope probing (SIP), have shown that low concentration of acetylene 10 Pa (0.01%) or 100 Pa (0.1%) can inhibit autotrophic nitrification completely in the same soil samples (Wang and Zhong, unpublished data), for the acidic soils in same region (Lu and Jia, 2013) and in the other regions (Stopnisek et al., 2010; Leviŏnik-Höfferle et al., 2012). However, for some acidic forest soils, partial inhibition of autotrophic nitrification by acetylene was observed during short term incubations (Killham, 1987; Hart et al., 1997; Pedersen et al., 1999). The main reason could be that a fraction of autotrophic nitrifiers could not be exposed to acetylene during the short term incubation (De Boer and Kowalchuk, 2001). To make sure that autotrophic nitrification can be inhibited completely, soil samples for the acetylene treatment was exposed to 1 KPa (1%) acetylene for one day in the present study. Therefore, the demonstration of heterotrophic nitrification could be more convincing in the present investigation (De Boer and Kowalchuk, 2001). Based on this assumption, the importance of heterotrophic nitrification in the N dynamics of the studied acid soils was assessed.
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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Fig. 7. The average contribution of heterotrophic nitrification to total nitrification for the different substrates in the whole incubation: The contribution in the Cinnamomum chekiangense (CI) soil (a) and the Castanopsis fargesii (CA) soil (b), respectively. L-g: Lglutamic acid; G: Glycine; M: maize straw; and A: (NH4)2SO4. Identical letters indicate no statistical differences for the average values between the treatments.
The results, in the present investigation, indicated that NO 3 production via heterotrophic nitrification process convincingly occurred in the studied acidic soils. The heterotrophic nitrifiers could use both ammonium and organic N compounds substrates for nitrification, in line with some previous studies (Focht and Verstraete, 1977; Rho, 1986; Stroo et al., 1986; Honda et al., 1998; De Boer and Kowalchuk, 2001). The heterotrophic nitrifying bacteria or fungal, which possess ammonia and hydroxylamine oxidizing enzymes, could be responsible for the oxidation of ammonium by heterotrophs (Moir et al., 1996; Daum et al., 1998; Nishio et al., 1998). The results, in the present study, in which accumulation of 15N-NO 3 significantly increased with the incubation time for the (NH4)2SO4 treatment in the presence of acetylene (1 KPa), testified the existence of inorganic heterotrophic nitrification pathway. However, some investigations reported that NHþ 4 was not an important substrate for heterotrophic nitrification, while, unidentified organic N compounds were the substrate in acidic soils (Barraclough and Puri, 1995; Pedersen et al., 1999). Therefore, many previous studies used the term of heterotrophic nitrification to indicate the oxidation of organic N to NO 3 (Barraclough and Puri, 1995; Pedersen et al., 1999; Müller et al., 2007; Zhang et al., 2011). If the oxidation of ammonium by heterotrophs was not taken into account, the potentially underestimating heterotrophic nitrification, while, overestimating autotrophic nitrification could occur. The organic N substrates heterotrophic nitrification pathway, mainly driving by fungi, was the process that the organic aminonitrogen was oxidized to NO 3 (Killham, 1990; De Boer and Kowalchuk, 2001), which involved the processes that amine or amide was oxidized to a substituted hydroxylamine, which was subsequently oxidized to a nitroso-compound and then to a nitrocompound (Killham, 1990). In the present study, 15N-NO 3 production in the presence of acetylene for the Glycine and L-glutamic acid treatments was clearly observed and was obviously higher than that in the (NH4)2SO4 treatment, confirmed the occurrence of organic N heterotrophic nitrification pathway. Based on the change 15 of 15N-NO N-NHþ 3 and 4 concentration in the Glycine, L-glutamic acid and (NH4)2SO4 treatments for the studied soils, we speculated that the oxidation of amino organic N compounds (e.g. Glycine and L-glutamic acid used in this study) to NO3 by heterotrophic nitrification process could occur via a combined organic and inorganic pathway. Most of amino organic N was mineralized to ammonium firstly (about 63%e78% within 4 days of incubation), then the
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ammonium was oxidized to NO 3 via heterotrophs, i.e. inorganic heterotrophic nitrification pathway. The other part of amino organic N was oxidized directly to NO 3 via organic heterotrophic nitrification pathway. Another possible reason of the results that higher 15N-NO 3 production from amino organic N compounds than from (NH4)2SO4 might be that ammonia derived from amino organic N compounds favored nitrification in acidic soil more than ammonium (Stopnisek et al., 2010; Levi cnik-Höfferle et al., 2012). Further studies were needed to carry out to prove the organic pathway of heterotrophic nitrification and to estimate the relative contribution of organic and inorganic processes to heterotrophic nitrification in the acidic soils. In the present study, based on the assumption that the rate of the inorganic heterotrophic nitrification pathway in the Glycine and L-glutamic acid treatments was equal to the (NH4)2SO4 treatment, the relative contribution of organic and inorganic processes to heterotrophic nitrification in the amino acid treatment could be calculated. The contribution of organic N heterotrophic nitrification process ranged from 42% to 55%, and there was no significant difference between the different amino acid treatments and between the different studied soils. However, for complicated organic N substrate, e.g. maize straw in the present study, almost all 15N-NO 3 was produced by organic N heterotrophic nitrification pathway, due to the low mineralization of added 15 N-maize straw, which was supported by the results that the 15 N-NO 3 production in the presence of acetylene was not obviously different from that in the absence of acetylene in the maize straw treatment during the incubation. The fungal nitrifiers could switch to alternative nitrification pathways depending on substrate availability in the soil (Killham, 1990; Aarnio and Martikainen, 1992). It has been widely accepted that heterotrophic nitrification could make some contribution to total nitrification in acidic soils (Kreitinger et al., 1985; Killham, 1990; Wood, 1990; Huygens et al., 2008; Zhang et al., 2011, 2013a,b), because autotrophic nitrifiers are sensitive to low pH (Weber and Gainey, 1962). However, the relative contribution of heterotrophic nitrification to total nitrification varied largely among the previous investigations (Barraclough and Puri, 1995; Pedersen et al., 1999; Zhang et al., 2013b). Our results showed that the contribution of heterotrophic nitrification to total nitrification varied depending on the substrate treatments as follows: (NH4)2SO4 < amino acid < maize straw, suggested that the substrate may be an important factor in controlling the significance of heterotrophic nitrification, in line with the previous investigations (Killham, 1986; Honda et al., 1998; De Boer and Kowalchuk, 2001). The C/N ratio of N substrate could play important role in affecting the significance of heterotrophic nitrification, since the highest contribution was observed in the addition of maize straw (with the high C/N ratio, 55), while, the lowest was found in the (NH4)2SO4 treatment. Organic N compounds with high C/N ratio increasing bioavailability of C could promote the heterotrophic bacteria growth, in turn, outcompete or inhibit nitrifying bacteria growth and autotrophic nitrification activity (Fauci and Dick, 1994; Stouthamer et al., 1997; Shi and Norton, 2000). In addition, some previous studies suggested that the real significance of nitrification by the fungi could primarily lay in the competitive advantage of fungal growth strategy through this process in the soil (Killham, 1990). The by-products, produced in heterotrophic nitrification process, could well inhibit many microbial competition forms to the fungal nitrifier (Verstraete, 1975). These competition or inhibition effects could be an important reason, which resulted in the contribution of heterotrophic nitrification to total nitrification varied largely depending on substrates in the present study. Thus, the highest the contribution of heterotrophic nitrification to total nitrification was observed in the maize straw treatment with the highest C/N ratio, although soil pH significantly increased
Please cite this article in press as: Zhang, J., et al., The substrate is an important factor in controlling the significance of heterotrophic nitrification in acidic forest soils, Soil Biology & Biochemistry (2014), http://dx.doi.org/10.1016/j.soilbio.2014.05.001
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(pH > 5) in this treatment being more beneficial to autotrophic nitrification than the other treatments. Therefore, the substrate was a more important factor in controlling the significance of heterotrophic nitrification in acidic forest soils than soil pH. The results of 15N pool dilution experiment showed that the gross nitrification rate was 0.55 mg N kg1 d1 and 0.86 mg N kg1 d1 in the CI and CA soils, respectively. Combined the gross nitrification rate and the high contribution of heterotrophic nitrification to total nitrification, we could infer that there were the significant actual heterotrophic nitrification rates and the heterotrophic nitrification was surely an important N transformation process in the studied acidic soils. In this study, high amino organic N concentration (240 mg N kg1) was used to investigate the nitrification. However, to date, there is no information on whether there were sufficient quantities of these N compounds to support the heterotrophic nitrification process in soils (Pedersen et al., 1999). Further study should be carried out to testify heterotrophic nitrification of different N compounds in situ or in intact soil core, using low labeled N concentration. Uncited reference Waid, 1975. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (41222005, 41271255, and 41101209), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (13KJA210002), the Natural Science Foundation of Jiangsu Province (BK2012731), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Aarnio, T., Martikainen, P.J., 1992. Nitrification in forest soil after refertilization with urea or urea and dicyandiamide. Soil Biology and Biochemistry 24, 951e954. Barraclough, D., Puri, G., 1995. The use of 15N pool dilution and enrichment to separate the heterotrophic and autotrophic pathways of nitrification. Soil Biology and Biochemistry 27, 17e22. Daum, M., Zimmer, W., Papen, H., Kloos, K., Nawrath, K., Bothe, H., 1998. Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida. Current Microbiology 37, 281e288. De Boer, W., Kowalchuk, G.A., 2001. Nitrification in acid soils: micro-organisms and mechanisms. Soil Biology and Biochemistry 33, 853e866. Fauci, M.F., Dick, R.P., 1994. Soil microbial dynamics: short-term and long-term effects of inorganic and organic nitrogen. Soil Science Society of America Journal 58, 801e806. Focht, D.D., Verstraete, W., 1977. Biochemical ecology of nitrification and denitrification. Advances in Microbial Ecology 1, 135e214. Hart, S.C., Binkley, D., Perry, D.A., 1997. Influence of red alder on soil nitrogen transformations in two conifer forests of contrasting productivity. Soil Biology and Biochemistry 29, 1111e1123. Honda, N., Hirai, M., Ano, T., Shoda, M., 1998. Antifungal effect of a heterotrophic nitrifier Alcaligenes faecalis. Biotechnology Letters 20, 703e705. Huygens, D., Boeckx, P., Templer, P., Paulino, L., Cleemput, O.V., Oyarzún, C., Müller, C., Godoy, R., 2008. Mechanisms for retention of bioavailable nitrogen in volcanic rainforest soils. Nature Geoscience 1, 543e548. Hyman, M.R., Wood, P.M., 1985. Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene. Biochemistry Journal 227, 719e725.
Islam, A., Chen, D., White, R.E., 2007. Heterotrophic and autotrophic nitrification in two acid pasture soils. Soil Biology and Biochemistry 39, 972e975. Killham, K., 1986. Heterotrophic nitrification. In: Presser, J.I. (Ed.), Nitrification, Special publications for Society for General Microbiology, vol. 20. IRL Press, Oxford, pp. 117e126. Killham, K., 1987. A new perfusion system for measurement and characterization of potential rates of soil nitrification. Plant and Soil 97, 267e272. Killham, K., 1990. Nitrification in coniferous forest soils. Plant and Soil 128, 31e44. Kirkham, D., Bartholomew, W.V., 1954. Equations for following nutrient transformations in soil, utilizing tracer data. Soil Science Society of America Proceedings 18, 33e34. Kreitinger, J.P., Klein, T.M., Novick, N.J., Alexander, M., 1985. Nitrification and characteristics of nitrifying microorganisms in an acid forest soil. Soil Science Society of America Journal 49, 1407e1410. Nicol, G.W., Ausec, L., Mandi Levi cnik-Höfferle, S., c-Mulec, I., Prosser, J.I., 2012. Stimulation of thaumarchaeal ammonia oxidation by ammonia derived from organic nitrogen but not added inorganic nitrogen. FEMS Microbiology Ecology 80, 114e123. Lin, C., Yang, Y., Guo, J., Chen, G., Xie, J., 2011. Fine root decomposition of evergreen broadleaved and coniferous tree species in mid-subtropical China: dynamics of dry mass, nutrient and organic fractions. Plant and Soil 338, 311e327. Lu, L., Jia, Z., 2013. Urease gene-containing Archaea dominate autotrophic ammonia oxidation in two acid soils. Environmental Microbiology 15, 1795e1809. Moir, J.W.B., Crossman, L.C., Spiro, S., Richardson, D.J., 1996. The purification of ammonia mono-oxygenase from Paracoccus denitrificans. FEBS Letters 387, 71e74. Müller, C., Rütting, T., Kattge, J., Laughlin, R.J., Stevens, R.J., 2007. Estimation of parameters in complex 15N tracing models via Monte Carlo sampling. Soil Biology and Biochemistry 39, 715e726. Nishio, T., Yoshikura, T., Mishima, H., Inouye, Z., Itoh, H., 1998. Conditions for nitrification and denitrification by an immobilized heterotrophic bacterium Alcaligenes faecalis OKK17. Journal of Fermentation and Bioengineering 86, 351e356. Pedersen, H., Dunkin, K.A., Firestone, M., 1999. The relative importance of autotrophic and heterotrophic nitrification in a conifer forest soil as measured by 15 N tracer and pool dilution techniques. Biogeochemistry 44, 135e150. Rho, J., 1986. Microbial interactions in heterotrophic nitrification. Canadian Journal of Microbiology 32, 243e247. Schmidt, E.L., 1960. Nitrate formation by Aspergillus flavus in pure and mixed culture environments. In: Transactions of the 7th International Congress of Soil Science, vol. 2, pp. 600e605. Shi, W., Norton, J.M., 2000. Microbial control of nitrate concentrations in an agricultural soil treated with dairy waste compost or ammonium fertilizer. Soil Biology and Biochemistry 32, 1453e1457. Nicol, G.W., Mandi Stopnisek, N., Gubry-Rangin, C., Höfferle, S., c-Mulec, I., Prosser, J.I., 2010. Thaumarchaeal ammonia oxidation in an acidic forest peat soil is not influenced by ammonium amendment. Applied and Environmental Microbiology 76, 7626e7634. Stouthamer, A.H., de Boer, A.P.N., Van der Oost, J., Van Spanning, R.J.M., 1997. Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria. Antonie van Leeuwenhoek 71, 33e41. Stroo, H.F., Klein, M.T., Alexander, M., 1986. Heterotrophic nitrification in an acid forest soil and by an acid-tolerant fungus. Applied and Environmental Microbiology 52, 107e111. Verstraete, W., 1975. Heterotrophic nitrification in soils and aqueous media e a review. Biologu Bulletin of the Academy Sciences of the U.S.S.R. 4, 515e530. Q3 Waid, J.S., 1975. Hydroxamic acids in soil systems. In: Paul, E.A., McLaren, A.D. (Eds.), Soil Biochemistry, vol. 4. Marcel Dekker, New York. Weber, D.F., Gainey, P.L., 1962. Relative sensitivity of nitrifying organisms to hydrogen ions in soils and solutions. Soil Science 94, 138e145. Wood, P.M., 1990. Autotrophic and heterotrophic mechanisms for ammonia oxidation. Soil Use and Management 6, 78e79. Zhang, J., Cai, Z., Zhu, T., Yang, W., Müller, C., 2013a. Mechanisms for the retention of inorganic N in acidic forest soils of southern China. Scientific Reports 3, 2342. http://dx.doi.org/10.1038/srep02342. Zhang, J., Zhu, T., Cai, Z., Müller, C., 2011. Heterotrophic nitrification is the predominant NO 3 production mechanism in coniferous but not broad-leaf acid forest soil in subtropical China. Biology and Fertility of Soils 47, 533e542. Zhang, Y., Zhang, J., Meng, T., Zhu, T., Müller, C., Cai, Z., 2013b. Heterotrophic nitrification is the predominant NO 3 production pathway in acid coniferous forest soil in subtropical China. Biology and Fertility of Soils 49, 955e957.
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