Systematic and Applied Microbiology 37 (2014) 296–304
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Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm
Effects of ammonium on the activity and community of methanotrophs in landfill biocover soils Xuan Zhang, Jiao-Yan Kong, Fang-Fang Xia, Yao Su, Ruo He ∗ Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China
a r t i c l e
i n f o
Article history: Received 3 December 2013 Received in revised form 23 March 2014 Accepted 24 March 2014 Keywords: Methanotroph Waste biocover soil NH4 + -N addition Methane oxidation
a b s t r a c t The influence of NH4 + on microbial CH4 oxidation is still poorly understood in landfill cover soils. In this study, effects of NH4 + addition on the activity and community structure of methanotrophs were investigated in waste biocover soil (WBS) treated by a series of NH4 + -N contents (0, 100, 300, 600 and 1200 mg kg−1 ). The results showed that the addition of NH4 + -N ranging from 100 to 300 mg kg−1 could stimulate CH4 oxidation in the WBS samples at the first stage of activity, while the addition of an NH4 + N content of 600 mg kg−1 had an inhibitory effect on CH4 oxidation in the first 4 days. The decrease of CH4 oxidation rate observed in the last stage of activity could be caused by nitrogen limitation and/or exopolymeric substance accumulation. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. Of these, Methylocaldum was the main methanotroph in the original WBS. With incubation, a higher abundance of Methylobacter was observed in the treatments with NH4 + -N contents greater than 300 mg kg−1 , which suggested that NH4 + -N addition might lead to the dominance of Methylobacter in the WBS samples. Compared to type I methanotrophs, the abundance of type II methanotrophs Methylocystis and/or Methylosinus was lower in the original WBS sample. An increase in the abundance of Methylocystis and/or Methylosinus occurred in the last stage of activity, and was likely due to a nitrogen limitation condition. Redundancy analysis showed that NH4 + -N and the C/N ratio had a significant influence on the methanotrophic community in the WBS sample. © 2014 Elsevier GmbH. All rights reserved.
Introduction Aerobic CH4 oxidation in oxic layers of landfill cover soils plays a significant role in reducing CH4 emissions from landfills. CH4 oxidation in these soils is affected by environmental factors such as soil texture, pH, soil moisture content, CH4 and O2 supply, nutrients and temperature [34]. Nitrogen is an important nutrient for microorganisms that can affect methanotrophic activities, and it subsequently interferes with the capacity of CH4 oxidation in soil. Some studies have shown that higher NH4 + contents in soils tend to inhibit CH4 oxidation, due to substrate competition between NH3 and CH4 for the active site of methane monooxygenase (MMO), which catalyzes the oxidation of CH4 to methanol, and the toxicity of hydroxylamine and nitrite generated from NH3 oxidation [23,24,37–39]. Different amounts of NH4 + might contradict the effects of CH4 oxidation. The CH4 oxidation rate in landfill cover soils was shown to decrease linearly with the initial
∗ Corresponding author. Tel.: +86 571 88982221; fax: +86 571 88982221. E-mail address: [email protected] (R. He). http://dx.doi.org/10.1016/j.syapm.2014.03.003 0723-2020/© 2014 Elsevier GmbH. All rights reserved.
NH4 + content of the soil when the NH4 + -N content reached 25 mgN kg−1 [4]. Similar results were obtained by Scheutz and Kjeldsen [33] who showed that CH4 oxidation rates were unaltered when NH4 + -N amended soil was 14 mg-N kg−1 or below, whereas the oxidation rates decreased at a higher NH4 + -N content. Some studies, on the other hand, indicated that NH4 + -N application could stimulate growth and activity of methanotrophs in landfill cover soils and rice fields [3,10,26,31]. When CH4 flux is high enough to support the growth of methanotrophs, NH4 + can act more as a nutrient than as an inhibitor and strengthen the biological CH4 sink [37]. The stimulative effect of NH4 + on CH4 oxidation is also explained by the competition for nitrogen between the plant and the rhizosphere microbial community, thereby reducing the effective NH4 + concentration below a threshold that is inhibitory to methanotrophic activity [2]. In addition, since aerobic methanotrophs require oxygen for growth, sufficient oxygen generated from high plant density with nitrogen addition can stimulate CH4 oxidation [2]. Aerobic methanotrophs are the major mediator in mitigating CH4 emission from landfills, and most of them belong to Proteobacteria that can be classified into two major groups, type I and type
X. Zhang et al. / Systematic and Applied Microbiology 37 (2014) 296–304
II, based on their cell morphology, ultrastructure, phylogeny and metabolic pathways [14,36]. Nitrogen content has an important effect on the distribution of methanotrophs in the natural environment [34]. The activity and community of methanotrophs in a landfill cover soil with NH4 + addition has been hypothesized to occur in three stages, according to the growth and activity of methanotrophs with relatively high CH4 mixing ratios (>1%) [9]. In the first stage, methanotrophs have a rapid growth rate, with higher rates probably obtained for type I methanotrophs. The second stage represents a decline in the methanotrophic activity, which might be caused by nitrogen limitation conditions for type I methanotrophs. In the third stage, a new growth phase can be observed that might be dominated by N2 -fixing type II methanotrophs. Compared to type I methanotrophs, type II methanotrophs can survive in a nitrogen-limited environment [12,22], which could be due to their nitrogen fixation capacity. Biocover soils, such as compost, waste biocover soil (WBS) and mineralized waste, have high organic matter content, welldistributed particle size and active microorganism activity and have been demonstrated to be good alternative covers for mitigating CH4 emission from landfills [5,17,33]. Ammonium is an important compound generated from the decomposition of deposited waste in landfills, especially in bioreactor landfills with leachate recycling [7,16]. The immediate effect of nitrogen (NH4 + -N and NO3 − -N) addition on CH4 oxidation and N2 O emission in biocover soils has been investigated for short periods (within 150 h) in batch experiments [42,44]. However, there is little information on the responses of methanotrophic communities and their activity to NH4 + -N application in landfill biocover soils. Therefore, the objective of this study was to test the effects of NH4 + -N addition on the activity of the methanotroph community in landfill biocover soils. Batch experiments were conducted with a series of NH4 + -N contents (0, 100, 300, 600 and 1200 mg kg−1 ) in WBS. NH4 + -N, NO3 − -N, total nitrogen (TN), total organic carbon (TOC) contents and the C/N ratio were determined during incubation. Quantitative PCR (Q-PCR), terminal restriction fragment length polymorphism (T-RFLP) and cloning of pmoA (encoding a subunit of the particulate MMO) were applied to analyze the identity and diversity of methanotrophs and their response to NH4 + -N addition.
Materials and methods Landfill cover soil microcosm The WBS used in this study was taken from an organic waste landfill bioreactor (2 m3 ) in a village located in Xindeng town, Zhejiang Province. After removing large particles, the soils were air-dried and sieved through a 4 mm mesh. The particle composition of the experimental WBS samples was 61% of 2–4 mm, 33% of 0.02–2 mm, 5% of 0.002–0.02 mm and 1% of C/N ratio > NO3 − N > TN > ECPS > TOC > ECP. The variance inflation factors of these six parameters were all less than 20, but only NH4 + -N and the C/N ratio had a significant influence on the variance by the Monte Carlo permutation test (P = 0.024 and 0.032, respectively). Discussion In this study, the addition of NH4 + -N ranging from 100 to 300 mg kg−1 could stimulate CH4 oxidation in WBS samples (Fig. 1),
although the NO3 − -N content of the original WBS was high (673 mg kg−1 ) at the first stage. The nitrogen source fixed by pure cultures of methanotrophs has been shown to be NO3 − -N rather than NH4 + -N [27,28]. However, in our previous study, it was also observed that NH4 + -N in WBS samples had a more stimulative effect for methanotroph activity than NO3 − -N at lower concentrations [42]. Further study on the mechanism of the stimulative effect of NH4 + -N on CH4 oxidation in WBS is therefore required. The addition of 600 mg kg−1 of NH4 + -N showed an inhibitory effect on CH4 oxidation in the first 4 days, but a stimulatory effect occurred between days 8 and 15, while the addition of 1200 mg kg−1 of NH4 + -N had a longer (∼8 days) inhibitory effect on CH4 oxidation. This indicated that the stimulatory or inhibitory effect of NH4 + -N addition on CH4 oxidation in WBS was largely dependent on NH4 + -N content. A higher inhibitory content of NH4 + -N on CH4 oxidation was observed in this study compared to other reports in which the NH4 + -N contents of landfill cover soils were amended to 14–40 mg kg−1 [4,33]. This suggested that besides NH4 + -N contents, the effect of NH4 + -N on CH4 oxidation in soils depended on many factors, such as the species of N, CH4 concentration, pH and the methanotrophic community in the soils [34]. Since ammonia is a competitive inhibitor of MMO [2,37,38], a high CH4 concentration can relieve the inhibitory effect of NH4 + -N on CH4 oxidation [42]. The inhibition of NH4 + on CH4 oxidation is also dependent on pH because NH3 is the active substrate for MMO, and it is more toxic than NH4 + [28,37,38]. At the end of the experiment, the pH value of the WBS samples ranging from 7.24 to 7.86 indicated that pH was not the main factor influencing the inhibitory effect of NH4 + -N on CH4 oxidation. Although the C/N ratio and TN content ranged from 28 mg kg−1 to 67 mg kg−1 and 499 mg kg−1 to 1206 mg kg−1 , respectively, on day 15, the CH4 oxidation rate showed a rapid decrease in the experimental treatments after day 22, and remained stable during the period between days 29 and 56. It has been reported that the CH4 oxidation rates have often exhibited a peak followed
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by a decrease to a lower steady state value in long-term laboratory column experiments simulating landfill cover or biofilter environments [20,35,41,43]. It has been hypothesized that the accumulations of EPS caused the decrease in CH4 oxidation rate due to clogging of soil pores, which led to short-circuiting of LFG through the soil material and/or impeded gas diffusion, thus reducing transfer of substrates into the cells [15,41,43]. The increase in the ECPS and ECP contents of the WBS samples from days 15 to 30 might explain the rapid decrease in the CH4 oxidation rate. In the last stage the ECP contents continued to increase, and reached 281–559 mg kg−1 on day 56, while the ECPS content decreased a little in all the experimental treatments, indicating that, compared to ECP, ECPS was more easily used as a carbon source for cell synthesis under conditions of nutrient shortage. EPS accumulation might be an adaptive strategy for methanotrophs and other bacteria for surviving in environments where these nutrients are imbalanced and there is O2 deficiency [32,34]. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. The type I methanotroph Methylocaldum dominated in the original WBS sample, which might be due to the high nutrient condition such as TOC (6.0%, w/w). In previous studies, type I methanotrophs have been reported to outcompete type II methanotrophs in high-nutrient, high-oxygen environments [3,19,40]. After exposure to LFG, the abundance of Methylobacter increased in all the experimental treatments, but a higher abundance of Methylobacter occurred in the WN300, WN600 and WN1200 treatments compared to those in the CK and WN100 treatments. This indicated that the relatively higher content of NH4 + -N addition (300–1200 mg kg−1 ) could lead to Methylobacter predominating in the WBS samples. This result is in agreement with other studies indicating that Methylobacter dominated in Nrich environments [13,30]. The peak abundance of Methylobacter occurred in the WN1200 treatment on day 30, which was later than those in the WN100, WN300 and WN600 treatments on day 15. This is likely to be due to the fact that an NH4 + -N addition of 1200 mg kg−1 inhibited the growth of Methylobacter in the first stage of activity. Compared to type I methanotrophs, the abundance of the type II methanotroph Methylocystis was low (0.2%) in the original WBS sample. However, after day 30, the abundance of Methylocystis showed an increasing trend, and reached 2.7–16.9% in the CK, WN100, WN300 and WN600 treatments, while it was close to 0 in the WN1200 treatment. This might be because, except for the WN1200 treatment, the N-limiting condition (the low NH4 + -N contents of 0.2–70.2 mg kg−1 ) occurred in the experimental treatments between days 30 and 56, leading to the growth of type II methanotrophs. Although the relative abundance of methanotrophs in the clone libraries of pmoA for DNA of the soil samples on day 56 was a little different from that in the T-RFLP profile due to sequences lower than 50, it also confirmed that Methylosinus dominated in the N-limiting condition. A similar result was obtained by De Visscher and Van Cleemput [9], who showed that type I methanotrophs grew faster than type II when inorganic N was not limiting in the first phase, but after a steady-state of a few weeks a new growth phase was probably dominated by N2 -fixing type II methanotrophs. In conclusion, the addition of NH4 + -N ranging from 100 to 300 mg kg−1 could stimulate CH4 oxidation in WBS samples at the first stage, while the addition of NH4 + -N contents of 600 and 1200 mg kg−1 had an inhibitory effect on CH4 oxidation. The community and activity of methanotrophs in the WBS samples varied with NH4 + -N content and incubation time. The NH4 + -N content and C/N ratio had a significant influence on the methanotrophic community in the WBS. These findings will be helpful for understanding the role of biotic and abiotic factors affecting the diversity and activity of methanotrophs in landfill cover soils,
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China with Grant Nos. 41001148, 51178411 and 41371012, as well as the Zhejiang Province Natural Science Foundation for Distinguished Young Scholars (LR13E080002).
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Contents lists available at ScienceDirect
Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm
Effects of ammonium on the activity and community of methanotrophs in landfill biocover soils Xuan Zhang, Jiao-Yan Kong, Fang-Fang Xia, Yao Su, Ruo He ∗ Department of Environmental Engineering, Zhejiang University, Hangzhou 310058, China
a r t i c l e
i n f o
Article history: Received 3 December 2013 Received in revised form 23 March 2014 Accepted 24 March 2014 Keywords: Methanotroph Waste biocover soil NH4 + -N addition Methane oxidation
a b s t r a c t The influence of NH4 + on microbial CH4 oxidation is still poorly understood in landfill cover soils. In this study, effects of NH4 + addition on the activity and community structure of methanotrophs were investigated in waste biocover soil (WBS) treated by a series of NH4 + -N contents (0, 100, 300, 600 and 1200 mg kg−1 ). The results showed that the addition of NH4 + -N ranging from 100 to 300 mg kg−1 could stimulate CH4 oxidation in the WBS samples at the first stage of activity, while the addition of an NH4 + N content of 600 mg kg−1 had an inhibitory effect on CH4 oxidation in the first 4 days. The decrease of CH4 oxidation rate observed in the last stage of activity could be caused by nitrogen limitation and/or exopolymeric substance accumulation. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. Of these, Methylocaldum was the main methanotroph in the original WBS. With incubation, a higher abundance of Methylobacter was observed in the treatments with NH4 + -N contents greater than 300 mg kg−1 , which suggested that NH4 + -N addition might lead to the dominance of Methylobacter in the WBS samples. Compared to type I methanotrophs, the abundance of type II methanotrophs Methylocystis and/or Methylosinus was lower in the original WBS sample. An increase in the abundance of Methylocystis and/or Methylosinus occurred in the last stage of activity, and was likely due to a nitrogen limitation condition. Redundancy analysis showed that NH4 + -N and the C/N ratio had a significant influence on the methanotrophic community in the WBS sample. © 2014 Elsevier GmbH. All rights reserved.
Introduction Aerobic CH4 oxidation in oxic layers of landfill cover soils plays a significant role in reducing CH4 emissions from landfills. CH4 oxidation in these soils is affected by environmental factors such as soil texture, pH, soil moisture content, CH4 and O2 supply, nutrients and temperature [34]. Nitrogen is an important nutrient for microorganisms that can affect methanotrophic activities, and it subsequently interferes with the capacity of CH4 oxidation in soil. Some studies have shown that higher NH4 + contents in soils tend to inhibit CH4 oxidation, due to substrate competition between NH3 and CH4 for the active site of methane monooxygenase (MMO), which catalyzes the oxidation of CH4 to methanol, and the toxicity of hydroxylamine and nitrite generated from NH3 oxidation [23,24,37–39]. Different amounts of NH4 + might contradict the effects of CH4 oxidation. The CH4 oxidation rate in landfill cover soils was shown to decrease linearly with the initial
∗ Corresponding author. Tel.: +86 571 88982221; fax: +86 571 88982221. E-mail address: [email protected] (R. He). http://dx.doi.org/10.1016/j.syapm.2014.03.003 0723-2020/© 2014 Elsevier GmbH. All rights reserved.
NH4 + content of the soil when the NH4 + -N content reached 25 mgN kg−1 [4]. Similar results were obtained by Scheutz and Kjeldsen [33] who showed that CH4 oxidation rates were unaltered when NH4 + -N amended soil was 14 mg-N kg−1 or below, whereas the oxidation rates decreased at a higher NH4 + -N content. Some studies, on the other hand, indicated that NH4 + -N application could stimulate growth and activity of methanotrophs in landfill cover soils and rice fields [3,10,26,31]. When CH4 flux is high enough to support the growth of methanotrophs, NH4 + can act more as a nutrient than as an inhibitor and strengthen the biological CH4 sink [37]. The stimulative effect of NH4 + on CH4 oxidation is also explained by the competition for nitrogen between the plant and the rhizosphere microbial community, thereby reducing the effective NH4 + concentration below a threshold that is inhibitory to methanotrophic activity [2]. In addition, since aerobic methanotrophs require oxygen for growth, sufficient oxygen generated from high plant density with nitrogen addition can stimulate CH4 oxidation [2]. Aerobic methanotrophs are the major mediator in mitigating CH4 emission from landfills, and most of them belong to Proteobacteria that can be classified into two major groups, type I and type
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II, based on their cell morphology, ultrastructure, phylogeny and metabolic pathways [14,36]. Nitrogen content has an important effect on the distribution of methanotrophs in the natural environment [34]. The activity and community of methanotrophs in a landfill cover soil with NH4 + addition has been hypothesized to occur in three stages, according to the growth and activity of methanotrophs with relatively high CH4 mixing ratios (>1%) [9]. In the first stage, methanotrophs have a rapid growth rate, with higher rates probably obtained for type I methanotrophs. The second stage represents a decline in the methanotrophic activity, which might be caused by nitrogen limitation conditions for type I methanotrophs. In the third stage, a new growth phase can be observed that might be dominated by N2 -fixing type II methanotrophs. Compared to type I methanotrophs, type II methanotrophs can survive in a nitrogen-limited environment [12,22], which could be due to their nitrogen fixation capacity. Biocover soils, such as compost, waste biocover soil (WBS) and mineralized waste, have high organic matter content, welldistributed particle size and active microorganism activity and have been demonstrated to be good alternative covers for mitigating CH4 emission from landfills [5,17,33]. Ammonium is an important compound generated from the decomposition of deposited waste in landfills, especially in bioreactor landfills with leachate recycling [7,16]. The immediate effect of nitrogen (NH4 + -N and NO3 − -N) addition on CH4 oxidation and N2 O emission in biocover soils has been investigated for short periods (within 150 h) in batch experiments [42,44]. However, there is little information on the responses of methanotrophic communities and their activity to NH4 + -N application in landfill biocover soils. Therefore, the objective of this study was to test the effects of NH4 + -N addition on the activity of the methanotroph community in landfill biocover soils. Batch experiments were conducted with a series of NH4 + -N contents (0, 100, 300, 600 and 1200 mg kg−1 ) in WBS. NH4 + -N, NO3 − -N, total nitrogen (TN), total organic carbon (TOC) contents and the C/N ratio were determined during incubation. Quantitative PCR (Q-PCR), terminal restriction fragment length polymorphism (T-RFLP) and cloning of pmoA (encoding a subunit of the particulate MMO) were applied to analyze the identity and diversity of methanotrophs and their response to NH4 + -N addition.
Materials and methods Landfill cover soil microcosm The WBS used in this study was taken from an organic waste landfill bioreactor (2 m3 ) in a village located in Xindeng town, Zhejiang Province. After removing large particles, the soils were air-dried and sieved through a 4 mm mesh. The particle composition of the experimental WBS samples was 61% of 2–4 mm, 33% of 0.02–2 mm, 5% of 0.002–0.02 mm and 1% of C/N ratio > NO3 − N > TN > ECPS > TOC > ECP. The variance inflation factors of these six parameters were all less than 20, but only NH4 + -N and the C/N ratio had a significant influence on the variance by the Monte Carlo permutation test (P = 0.024 and 0.032, respectively). Discussion In this study, the addition of NH4 + -N ranging from 100 to 300 mg kg−1 could stimulate CH4 oxidation in WBS samples (Fig. 1),
although the NO3 − -N content of the original WBS was high (673 mg kg−1 ) at the first stage. The nitrogen source fixed by pure cultures of methanotrophs has been shown to be NO3 − -N rather than NH4 + -N [27,28]. However, in our previous study, it was also observed that NH4 + -N in WBS samples had a more stimulative effect for methanotroph activity than NO3 − -N at lower concentrations [42]. Further study on the mechanism of the stimulative effect of NH4 + -N on CH4 oxidation in WBS is therefore required. The addition of 600 mg kg−1 of NH4 + -N showed an inhibitory effect on CH4 oxidation in the first 4 days, but a stimulatory effect occurred between days 8 and 15, while the addition of 1200 mg kg−1 of NH4 + -N had a longer (∼8 days) inhibitory effect on CH4 oxidation. This indicated that the stimulatory or inhibitory effect of NH4 + -N addition on CH4 oxidation in WBS was largely dependent on NH4 + -N content. A higher inhibitory content of NH4 + -N on CH4 oxidation was observed in this study compared to other reports in which the NH4 + -N contents of landfill cover soils were amended to 14–40 mg kg−1 [4,33]. This suggested that besides NH4 + -N contents, the effect of NH4 + -N on CH4 oxidation in soils depended on many factors, such as the species of N, CH4 concentration, pH and the methanotrophic community in the soils [34]. Since ammonia is a competitive inhibitor of MMO [2,37,38], a high CH4 concentration can relieve the inhibitory effect of NH4 + -N on CH4 oxidation [42]. The inhibition of NH4 + on CH4 oxidation is also dependent on pH because NH3 is the active substrate for MMO, and it is more toxic than NH4 + [28,37,38]. At the end of the experiment, the pH value of the WBS samples ranging from 7.24 to 7.86 indicated that pH was not the main factor influencing the inhibitory effect of NH4 + -N on CH4 oxidation. Although the C/N ratio and TN content ranged from 28 mg kg−1 to 67 mg kg−1 and 499 mg kg−1 to 1206 mg kg−1 , respectively, on day 15, the CH4 oxidation rate showed a rapid decrease in the experimental treatments after day 22, and remained stable during the period between days 29 and 56. It has been reported that the CH4 oxidation rates have often exhibited a peak followed
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by a decrease to a lower steady state value in long-term laboratory column experiments simulating landfill cover or biofilter environments [20,35,41,43]. It has been hypothesized that the accumulations of EPS caused the decrease in CH4 oxidation rate due to clogging of soil pores, which led to short-circuiting of LFG through the soil material and/or impeded gas diffusion, thus reducing transfer of substrates into the cells [15,41,43]. The increase in the ECPS and ECP contents of the WBS samples from days 15 to 30 might explain the rapid decrease in the CH4 oxidation rate. In the last stage the ECP contents continued to increase, and reached 281–559 mg kg−1 on day 56, while the ECPS content decreased a little in all the experimental treatments, indicating that, compared to ECP, ECPS was more easily used as a carbon source for cell synthesis under conditions of nutrient shortage. EPS accumulation might be an adaptive strategy for methanotrophs and other bacteria for surviving in environments where these nutrients are imbalanced and there is O2 deficiency [32,34]. Type I methanotrophs Methylocaldum and Methylobacter, and type II methanotrophs (Methylocystis and Methylosinus) were abundant in the WBS samples. The type I methanotroph Methylocaldum dominated in the original WBS sample, which might be due to the high nutrient condition such as TOC (6.0%, w/w). In previous studies, type I methanotrophs have been reported to outcompete type II methanotrophs in high-nutrient, high-oxygen environments [3,19,40]. After exposure to LFG, the abundance of Methylobacter increased in all the experimental treatments, but a higher abundance of Methylobacter occurred in the WN300, WN600 and WN1200 treatments compared to those in the CK and WN100 treatments. This indicated that the relatively higher content of NH4 + -N addition (300–1200 mg kg−1 ) could lead to Methylobacter predominating in the WBS samples. This result is in agreement with other studies indicating that Methylobacter dominated in Nrich environments [13,30]. The peak abundance of Methylobacter occurred in the WN1200 treatment on day 30, which was later than those in the WN100, WN300 and WN600 treatments on day 15. This is likely to be due to the fact that an NH4 + -N addition of 1200 mg kg−1 inhibited the growth of Methylobacter in the first stage of activity. Compared to type I methanotrophs, the abundance of the type II methanotroph Methylocystis was low (0.2%) in the original WBS sample. However, after day 30, the abundance of Methylocystis showed an increasing trend, and reached 2.7–16.9% in the CK, WN100, WN300 and WN600 treatments, while it was close to 0 in the WN1200 treatment. This might be because, except for the WN1200 treatment, the N-limiting condition (the low NH4 + -N contents of 0.2–70.2 mg kg−1 ) occurred in the experimental treatments between days 30 and 56, leading to the growth of type II methanotrophs. Although the relative abundance of methanotrophs in the clone libraries of pmoA for DNA of the soil samples on day 56 was a little different from that in the T-RFLP profile due to sequences lower than 50, it also confirmed that Methylosinus dominated in the N-limiting condition. A similar result was obtained by De Visscher and Van Cleemput [9], who showed that type I methanotrophs grew faster than type II when inorganic N was not limiting in the first phase, but after a steady-state of a few weeks a new growth phase was probably dominated by N2 -fixing type II methanotrophs. In conclusion, the addition of NH4 + -N ranging from 100 to 300 mg kg−1 could stimulate CH4 oxidation in WBS samples at the first stage, while the addition of NH4 + -N contents of 600 and 1200 mg kg−1 had an inhibitory effect on CH4 oxidation. The community and activity of methanotrophs in the WBS samples varied with NH4 + -N content and incubation time. The NH4 + -N content and C/N ratio had a significant influence on the methanotrophic community in the WBS. These findings will be helpful for understanding the role of biotic and abiotic factors affecting the diversity and activity of methanotrophs in landfill cover soils,
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Acknowledgments This work was financially supported by the National Natural Science Foundation of China with Grant Nos. 41001148, 51178411 and 41371012, as well as the Zhejiang Province Natural Science Foundation for Distinguished Young Scholars (LR13E080002).
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