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Autotrophic and Heterotrophic Nitrification in a Highly Acidic Subtropical Pine Forest Soil
Abstract
The occurrence of nitrification in some acidic forest soils is still a subject of debate. Identification of main nitrification pathways in acidic forest soils is still largely unknown. Acidic yellow soil (Oxisol) samples were selected to test whether nitrification can occur or not in acidic subtropical pine forest ecosystems. Relative contributions of autotrophs and heterotrophs to nitrification were studied by adding selective nitrification inhibitor nitrapyrin. Soil NH⁺4-N concentrations decreased, but NO⁻3-N concentrations increased significantly for the no-nitrapyrin control during the first week of incubation, indicating that nitrification did occur in the acidic subtropical soil. The calculated net nitrification rate was 0.49 mg N kg⁻¹ d⁻¹ for the no-nitrapyrin control during the first week of incubation. Nitrapyrin amendment resulted in a significant reduction of NO⁻3-N concentration. Autotrophic nitrification rate averaged 0.28 mg N kg⁻¹ d⁻¹ and the heterotrophic nitrification rate was 0.21 mg N kg⁻¹ d⁻¹ in the first week. Ammonia-oxidizing bacteria (AOB) abundance increased slightly during incubation, but nitrapyrin amendment significantly decreased AOB amoA gene copy numbers by about 80%. However, the ammonia-oxidizing archaea (AOA) abundance showed significant increases only in the last 2 weeks of incubation and it was also decreased by nitrapyrin amendment. Our results indicated that nitrification did occur in the present acidic subtropical pine forest soil, and autotrophic nitrification was the main nitrification pathway. Both AOA and AOB were the active biotic agents responsible for autotrophic nitrification in the acidic subtropical pine forest soil.
... Nitrification inhibitors including nitrapyrin have been shown to impact the target gene, amoA, differently in AOA and AOB (Cui et al., 2013;Duan et al., 2019;Duncan et al., 2017;Faeflen et al., 2016;Fisk et al., 2015;Gu et al., 2018;Xi et al., 2017). In our study the abundance of AOB amoA was reduced when nitrapyrin was co-applied with UAN compared to UAN alone in three of the four soils (Vertosol, Tenosol and Sodosol) ( Table 3; Fig. S3) and in all four soils when gene abundance was considered as amoA copies ng −1 DNA (Fig. S3). ...
... In our study the abundance of AOB amoA was reduced when nitrapyrin was co-applied with UAN compared to UAN alone in three of the four soils (Vertosol, Tenosol and Sodosol) ( Table 3; Fig. S3) and in all four soils when gene abundance was considered as amoA copies ng −1 DNA (Fig. S3). This result suggests nitrapyrin co-applied with fertiliser is inhibiting ammonia oxidation by AOB rather than AOA for a range of soil types, supporting several previous studies (Cui et al., 2013;Duan et al., 2019;Duncan et al., 2017;Faeflen et al., 2016;Xi et al., 2017). Four of these studies were microcosm experiments with constant environmental conditions and soil sourced from temperate or Mediterranean climates (Cui et al., 2013;Duncan et al., 2017;Xi et al., 2017) and a subtropical climate (Faeflen et al., 2016), while the fifth was a field study of an Anthrosol in the humid subtropical monsoon region of China (Duan et al., 2019). ...
... This result suggests nitrapyrin co-applied with fertiliser is inhibiting ammonia oxidation by AOB rather than AOA for a range of soil types, supporting several previous studies (Cui et al., 2013;Duan et al., 2019;Duncan et al., 2017;Faeflen et al., 2016;Xi et al., 2017). Four of these studies were microcosm experiments with constant environmental conditions and soil sourced from temperate or Mediterranean climates (Cui et al., 2013;Duncan et al., 2017;Xi et al., 2017) and a subtropical climate (Faeflen et al., 2016), while the fifth was a field study of an Anthrosol in the humid subtropical monsoon region of China (Duan et al., 2019). An exception was presented by Gu et al. (2018) who showed that letter are not significantly different at P < 0.05. ...
The compound nitrapyrin can act as a potential nitrification inhibitor (NI) when applied with ammonium (NH4+)-based N fertiliser. The efficacy of nitrapyrin applications may be soil dependent however, being influenced by soil texture, organic carbon (C), and moisture. The objective of this study was to assess the effects of nitrapyrin on the nitrification process in soils of different physicochemical characteristics and resident microbial
communities. Four agricultural soils were incubated in microcosms for five weeks, after which nitrapyrin effects were assessed for soil NH4+, nitrate (NO3−), the microbial functional genes ammonia-monooxygenase (amoA) and nitrite oxidoreductase (nxr), and bacterial and archaeal nitrifier genera using 16S rRNA sequencing.
Nitrapyrin co-applied with urea ammonium nitrate (UAN) fertiliser inhibited nitrification, shown by increased retention of NH4+ in all four soils and a lower accumulation of NO3− in three of the four soils compared to UAN treated soil. These results are most likely due to the inhibition of growth of ammonia oxidising bacteria (AOB) as nitrapyrin plus UAN reduced bacterial amoA abundance in three of the four soils compared to the UAN
treatment.
This study is the first to report increased archaeal amoA abundance in response to the co-application of nitrapyrin and UAN in four soils, possibly due to reduced competition from AOB. The bacterial nitrifier genera Nitrosospira and Nitrospira and archaeal nitrifier genus Nitrososphaera were detected in all four soils using 16S rRNA sequencing. The effects of nitrapyrin on downstream N transformations were assessed based on nxr and Nitrospira abundance though no conclusive treatment effects could be discerned for the four soils. Our results provide evidence of the interplay of AOB, ammonia oxidising archaea (AOA) and nitrite-oxidising bacteria (NOB) in nitrapyrin and fertiliser treated soils and will be useful in developing strategies to improve the efficacy of nitrification inhibitors in future studies.
... Moreover, a range of environmental factors, such as soil moisture, redox potential, pH and nutrient availability, have been proved to be able to affect soil nitrifiers and denitrifiers (Bárta et al., 2010;Hu et al., 2013;Zhang et al., 2014;Zhang et al., 2015;Tang et al., 2016). Although a number of studies have been devoted to understanding the relationships among soil properties, functional microbes and N 2 O fluxes (Fang et al., 2008a;Yu et al., 2014;Zhang et al., 2014;Faeflen et al., 2016;Chen et al., 2017), our knowledge for the responses of microbialregulated N 2 O emission to elevated N deposition in subtropical forest soils is still limited (Avrahami et al., 2002;Zhong et al., 2015;Cui et al., 2016;Gao et al., 2016). ...
... Previous study exhibited higher abundance of ammonia-oxidizing archaea (AOA) in the same subtropical forest which is consistent with our present finding (Isobe et al., 2012). Moreover, several studies have highlighted a predominant role of AOA in nitrification (Gubry-Rangin et al., 2010;Verhamme et al., 2011;Zhang et al., 2012;Faeflen et al., 2016). So the dominant autotrophic nitrification in our current results (Table S4) might be explained by the more abundant AOA than AOB in the strong acidic soil (Zhang et al., 2012;Faeflen et al., 2016). ...
... Moreover, several studies have highlighted a predominant role of AOA in nitrification (Gubry-Rangin et al., 2010;Verhamme et al., 2011;Zhang et al., 2012;Faeflen et al., 2016). So the dominant autotrophic nitrification in our current results (Table S4) might be explained by the more abundant AOA than AOB in the strong acidic soil (Zhang et al., 2012;Faeflen et al., 2016). For a better defining the relative importance of AOA and AOB in the nitrification processes, future explorations of the archaeal and bacterial ammonia oxidizer communities need to be considered (Gubry-Rangin et al., 2010). ...
Atmospherically-deposited nitrogen (N) can stimulate complex soil N metabolisms and accumulations over time. Whether long-term (decadal) N deposition effects on soil N transformations and functional microbes differ from the short-term (annual) effects has rarely been assessed. Here we conducted a laboratory ¹⁵N tracing study with soil samples from a short-term (one year) N addition site and a long-term (12 years) site in a subtropical forest. The effects of simulated N deposition on soil N2O emissions, N transformation rates and microbial nitrifying and denitrifying genes were determined. Our results showed that: (1) long-term N addition did not change soil N2O fluxes significantly in comparison to the short-term N addition. Denitrification, heterotrophic nitrification and autotrophic nitrification contributed 53%, 28% and 18% to total N2O emissions, respectively. (2) Autotrophic nitrification was the dominant N transformation process, except for the high-N treatment at the long-term site. The magnitude of soil N transformation rates was significantly different among N addition treatments but not between short- and long-term N addition sites. However, long-term N addition changed the responses of specific N transformation rates to N addition markedly, especially for the rates of nitrification, organic N mineralization to NH4⁺, NO3⁻ immobilization and dissimilatory NO3⁻ reduction to NH4⁺ (DNRA). (3) Responses of ammonia oxidizing archaea and bacteria (AOA and AOB) were more variable than those of denitrifying N2O-producers (nirK) and denitrifying N2O-reducers (nosZ), particularly at the long-term site. (4) The close correlations among N2O flux, functional genes and soil properties observed at the short-term site were weakened at the long-term site, posing a decreased risk for N losses in the acid subtropical forest soil. There is evidence for an adaptation of functional microbial communities to the prevailing soil conditions and in response to long-term natural and anthropogenic N depositions.
... Each soil had two treatments: N (N fertilisation) and NI (N fertilisation and nitrapyrin amendment). To avoid the bias of urea hydrolysis rate among different soils, (NH 4 ) 2 SO 4 , rather than urea, was applied as the N fertiliser at a rate of 100 mg N kg − 1 dry soil (Faeflen et al., 2016;Li et al., 2019a), and nitrapyrin was applied as nitrification inhibitor at a rate of 1 mg kg − 1 dry soil based on the recommended dose in previous literatures (Burzaco et al., 2014;Xi et al., 2017;Gu et al., 2018) and our preliminary experiments (data were not shown). Before the microcosm experiment started, all soil samples were preincubated for 1 week at a unified moisture condition at about 30% water-filled pore space (WFPS) by adding sterilized water or air drying in a well-ventilated environment at room temperature (~25 • C). ...
... However, nitrapyrin amendment also significantly reduced AOA abundances and N 2 O emissions in an acid soil (LY) in our study (Fig. 1d). Considering that AOA generally dominated nitrification over AOB in acid soils and their sensitive response to NIs amendment has been frequently recorded in soils where AOA or both AOA and AOB are active (Zhang et al., 2012;Faeflen et al., 2016;Gu et al., 2018). These results suggested that the inhibitory effect of NIs, such as nitrapyrin, is not specific for AOB, AOA or comammox Nitrospira, but rather more dependent on the dominant active nitrifier groups. ...
... The community of ammonia oxidizers is intensely influenced by anthropogenic disturbances in terrestrial ecosystems (Di et al., 2009;Faeflen et al., 2016). In this study, we found a rapid increase of AOB gene abundance in the urea alone or urea plus manure threated soil. ...
... Similarly, Xi et al. (2017) noted that nitrapyrin inhibited AOB activity rather than AOA under vegetable soil. On the other hand, Faeflen et al. (2016) reported that both AOA and AOB gene abundance were reduced by nitripyrin treated acidic forest soil, by contrast a study showed that either AOA or AOB community structure was not affected by nitrapyrin addition . Fisk et al. (2015) pointed out that nitrapyrin inhibited both AOA and AOB activities but inhibitory effects were sharply attenuated when soil temperature increased. ...
Synthetic inhibitors and organic amendment have been proposed for mitigating greenhouse gas N2O emissions. However, their combined effect on the N2O emissions and ammonia-oxidizer (ammonia-oxidizing bacteria and archaea, AOB and AOA) communities remains unclear in calcareous soils under climate warming. We conducted two incubation experiments (25 and 35 °C) to examine how N2O emissions and AOA and AOB communities responded to organic amendment (urea plus cattle manure, UCM), and in combination with urease (N-(n-butyl) thiophosphoric triamide, NBPT) and nitrification inhibitor (nitrapyrin). The treatments of UCM + nitrapyrin and UCM + nitrapyrin + NBPT significantly lowered total N2O emissions by average 64.5 and 71.05% at 25 and 35 °C, respectively, compared with UCM treatment. AOB gene abundance and α-diversity (Chao1 and Shannon indices) were significantly increased by the application of urea and manure (P < 0.05). However, relative to UCM treatment, nitrapyrin addition treatments decreased AOB gene abundance and Chao 1 index by average 115.4 and 30.4% at 25 and 35 °C, respectively. PCA analysis showed that UCM or UCM plus nitrapyrin notably shifted AOB structure at both temperatures. However, fertilization had little effects on AOA community (P > 0.05). Potential nitrification rate (PNR) was greatly decreased by nitrapyrin addition, and PNR significantly positively correlated with AOB gene abundance (P = 0.0179 at 25 °C and P = 0.0029 at 35 °C) rather than AOA (P > 0.05). Structural equation model analysis showed that temperature directly increased AOA abundance but decrease AOB abundance, while fertilization indirectly influenced AOB community by altering soil NH4⁺, pH and SOC. In conclusion, the combined application of organic amendment, NBPT and nitrapyrin significantly lowered N2O emissions via reducing AOB community in calcareous soil even at high temperature. Our findings provide a solid theoretical basis in mitigating N2O emissions from calcareous soil under climate warming.
... The net nitrification rates were calculated by subtracting the concentration of NO 3 − -N at the beginning from that at the end of the incubation divided by the length of incubation (Faeflen et al. 2016;Nugroho et al. 2005). We determined net nitrification rates at six incubation intervals (between days 0 and 1, 1 and 7, 7and 14, 14 and 21, 21 and 28, 28 and 35). ...
... The net nitrification rates in the no inhibitor addition treatment represent the net (autotrophic plus heterotrophic) nitrification rates. The net nitrification rates in the treatment with inhibitor addition represent the net heterotrophic nitrification rates (Faeflen et al. 2016). Net autotrophic nitrification rates were calculated by subtracting the net heterotrophic from the net nitrification rate and then divided by the incubation intervals. ...
How cattle urine and dung addition, and their interaction with the mineral soil regulate cumulative autotrophic and heterotrophic nitrifications and greenhouse gas (GHG) emission is poorly understood. We investigated the effect of urine (applied at 425 mg N kg⁻¹ soil) and dung (applied at 200 mg total N kg⁻¹ soil) addition on cumulative autotrophic and heterotrophic nitrifications, and GHG emission in a grassland soil in a 35-day laboratory incubation experiment under six treatments: CK, unamended control; U, urine addition; Ds, dung added on the soil surface; U-Ds, urine addition + dung added on the soil surface; Dm, dung mixed into the soil; and U-Dm, urine addition + dung mixed into the soil. Compared with the CK, the U, U-Ds, and U-Dm treatments increased cumulative net nitrification (autotrophic + heterotrophic) by 548, 587, and 505% and cumulative autotrophic nitrification by 593, 618, and 508%, but the Dm treatment decreased cumulative net and autotrophic nitrifications by 77 and 83%, respectively. However, cumulative heterotrophic nitrification was less than 1% of the cumulative autotrophic nitrification in the CK and it was decreased by Ds and Dm as compared with the CK at the end of the incubation. Cumulative emissions of carbon dioxide (CO2) and nitrous oxide (N2O) were in the order of CK < U < Ds < Dm < U-Ds < U-Dm. In addition, all urine and dung treatments reduced cumulative methane (CH4) uptake (negative values of CH4 emission) as compared with the CK. Overall, soil cumulative net and autotrophic nitrifications were higher, but GHG emission was lower, with dung added on the soil surface than with it mixed into the soil. We conclude that urine addition increases but dung addition decreases cumulative autotrophic nitrification; dung addition also decreases heterotrophic nitrification, and how dung is added to the soil is important in regulating the nitrification processes and GHG emission. Therefore, urine and dung management can alter soil N transformations and has implications for mitigating climate change.
... In our study, amoA-AOA but not amoA-AOB were significantly positively correlated with N 2 O emissions, implying that amoA-AOA was the primary gene family dominating soil nitrification in the investigated site of the temperate forest. This is consistent with the results that N 2 O production from nitrification in terrestrial ecosystems is dominated by AOA, which are more abundant than AOB (Faeflen et al., 2016). ...
Increases in soil available nitrogen (N) influence N-cycle gene abundances and emission of nitrous oxide (N2O), which is primarily due to N-induced soil acidification in forest. Moreover, the extent of microbial-N saturation could control microbial activity and N2O emission. The contributions of N-induced alterations of microbial-N saturation and N-cycle gene abundances to N2O emission have rarely been quantified. Here, the mechanism underlying N2O emission under N additions (three chemical forms of N, i.e., NO3--N, NH4+-N and NH4NO3-N, and each at two rates, 50 and 150 kg N ha-1 year-1, respectively) spanning 2011-2021 was investigated in a temperate forest in Beijing. Results showed N2O emissions increased at both low and high N rates of all the three forms compared with control during the whole experiment. However, N2O emissions were lower in high rate of NH4NO3-N and NH4+-N treatments than the corresponding low N rates in the recent three years. Effects of N on microbial-N saturation and abundances of N-cycle genes were dependent on the N rate and form as well as experimental time. Specifically, negative effects of N on N-cycle gene abundances and positive effects of N on microbial-N saturation were demonstrated in high N rate treatments, particularly with NH4+ addition during 2019-2021. Such effects were associated with soil acidification. A hump-backed trend between microbial-N saturation and N2O emissions was observed, suggesting N2O emissions decreased with increase of the microbial-N saturation. Furthermore, N-induced decreases in N-cycle gene abundances restrained N2O emissions. In particular, the nitrification process, dominated by ammonia-oxidize archaea, is critical to determination of N2O emissions in response to the N addition in the temperate forest. We confirmed N addition promoted soil microbial-N saturation and reduced N-cycle gene abundances, which restrained the continuous increase in N2O emissions. It is important for understanding the forest-N-microbe nexus under climate change.
... The soil samples analyzed do not present a significant variation in bacterial density. The isolated strains belong to a single physiological group detected, lithoautotrophic nitrifying bacteria; these results agreed with analyses carried out in this type of ecosystem, where it is evident that autotrophic nitrification is the main nitrification pathway in acid soils of a pine forest [38]. It has been reported that parameters such as pH, temperature, and altitude have influenced the structure of macro-and micro-communities in Andean ecosystems [13]. ...
Nitrification is part of the nitrogen cycle that occurs naturally in ecosystems. It is related to the presence of microorganisms and their metabolism, especially bacteria, which are involved in oxidizing compounds such as NH4+ and NO2− to NO3−. In this study, we evaluated the nitrification potential in 12 bacteria strains that belong to the genera Aeromonas, Bacillus, Buttiauxella, Mycobacterium, Paenibacillus, Serratia, and Yersenia, which are part of the cultivable microbial community from soil in a native forest and pine forest in The Labrado area within the Machangara micro-watershed in the Andes located in the south of Ecuador. This investigation aims to identify heterotrophic and lithoautotrophic strains using specific culture media for ammonium oxidative (AOL-AOH) and nitrate oxidation bacteria (ONL-ONH). The formation of nitrifying halos in the culture media allowed the identification of 10 strains with nitrifying potential. Five strains were from the pine forest, four were isolated from the native forest, and one strain was shared between both forests. The Serratia and Yersinia genera have a high NO2− oxidation capacity. Their inoculation in synthetic water rich in nitrogenous products allowed us to determine 40% and 94% nitrite reduction percentages and cell retention times of 20 to 40 days. Our results are promising for their possible potential use in environmental bioremediation processes through inoculation in wastewater for the biological removal of nitrogenous compounds.
... n = 54; Table S6), indicating that NH 4 + is an important substrate for heterotrophic nitrifiers in these biomes . Our study was inconsistent with studies on regional scales that indicated the dominance of GHN over GAN in tropical and subtropical forests (Huygens et al., 2008;Zhang et al., 2013), but was in line with other studies that suggested that GAN was the main nitrification pathway in these forests (Faeflen et al., 2016). Given that NO 3 − is more likely to be lost to the environment, there is a need to understand the relative magnitude of NO 3 − consumption processes (i.e., I NO 3 and DNRA) in tropical and subtropical forests. ...
Tropical and subtropical forest biomes are a main hotspot for the global nitrogen (N) cycle. Yet, our understanding of global soil N cycle patterns and drivers and their response to N deposition in these biomes remains elusive. By a meta-analysis of 2426-single and 161-paired observations from 89 published 15 N pool dilution and tracing studies, we found that gross N mineralization (GNM), immobilization of ammonium (INH4 ) and nitrate (INO3 ), and dissimilatory nitrate reduction to ammonium (DNRA) were significantly higher in tropical forests than in subtropical forests. Soil N cycle was conservative in tropical forests with ratios of gross nitrification (GN) to INH4 (GN/INH4 ) and of soil nitrate to ammonium (NO3 - /NH4 + ) less than one, but was leaky in subtropical forests with GN/INH4 and NO3 - /NH4 + higher than one. Soil NH4 + dynamics were mainly controlled by soil substrate (e.g., total N), but climatic factors (e.g., precipitation and/or temperature) were more important in controlling soil NO3 - dynamics. Soil texture played a role, as GNM and INH4 were positively correlated with silt and clay contents, while INO3 and DNRA were positively correlated with sand and clay contents, respectively. The soil N cycle was more sensitive to N deposition in tropical forests than in subtropical forests. Nitrogen deposition leads to a leaky N cycle in tropical forests, as evidenced by the increase in GN/INH4 , NO3 - /NH4 + and nitrous oxide emissions and the decrease in INO3 and DNRA, mainly due to the decrease in soil microbial biomass and pH. Dominant tree species can also influence soil N cycle pattern, which has changed from conservative in deciduous forests to leaky in coniferous forests. We provide global evidence that tropical, but not subtropical, forests are characterized by soil N dynamics sustaining N availability and that N deposition inhibits soil N retention and stimulates N losses in these biomes.
... We included as many nitrification inhibitor studies as possible in this synthesis [42][43][44][45][46][47][48][49][50]. However, several nitrification inhibitor studies were not included because of the following reasons: 1) measurements of net nitrification [51][52][53], 2) no flux data on O NH4 and O Norg [54], 3) nitrification rate expressed as milligrams of N per kilogram (mg N kg − 1 soil), not milligrams of N per kilogram per day (mg N kg − 1 soil d − 1 ) [55], and 4) incubation temperature not within 20-25 • C range [40,56]. ...
The major mechanisms and drivers of autotrophic (O NH4) and heterotrophic (O Nrec) nitrification in terrestrial soils are still not well understood. A synthetic analysis of the factors influencing O NH4 and O Nrec in cropland, forest, and grassland soils was thus conducted to establish an evidence-based prediction after compiling data from 71 reports. This synthesis applied single-factor regression approach to highlight major relationships, with variable importance measures provided using random forest (RF) models. Empirical evidence suggests that N mineralization (M tot) modulates O NH4 fluxes in croplands and grasslands. pH has more of an effect than M tot on O NH4 fluxes in acidic cropland and forest soils. Fungal biomass can be predictive of O Nrec fluxes in strongly acidic forest soils. The ratio of heterotrophic to gross nitrification (R hn) might increase with forest soil C:N ratios. RF models revealed that soil C:N ratio, soil N (TN), O NH4 , M tot and N mineralization-immobilization turnover (MIT) are important regulators of O Nrec fluxes in forests. pH is a significant factor affecting R hn in forest soils. However, its influence is less than that of soil C:N ratio, TN and M tot. There is little control from soil C-N status (e.g., TN and soil C:N ratio), microbial C-N status (e.g., M tot and MIT), and soil pH status over O Nrec and R hn in croplands and grasslands. In summary, this study advances our understanding of determinants to O NH4 and O Nrec in various soil habitats. It highlights that acidity-based mechanisms outweigh substrate-based mechanisms in mediating O NH4 in acidic cropland and forest soils. Soil and microbial N status are central to heterotrophic nitrifying activities in forest ecosystems.
... Nitrification is also considered as an indirect driver of N loss during the flooded rice-growing season because the denitrification rate in flooded soil is controlled by the nitrification rate instead of the activities of denitrification enzymes (Lan et al., 2017). In environments unfavorable to autotrophic nitrifying bacteria, nitrification may result from the activity of heterotrophic microorganisms (Dolinšek et al., 2013) since ammonia-oxidizing bacteria are weaker competitors for ammonium than ammonia-assimilating heterotrophs when ammonium is limited (Faeflen et al., 2016). ...
p>Nitrification, or the process of oxidation of ammonium to nitrate in the soil, needs to be inhibited because it reduces the efficiency of nitrogen fertilizers. Vertisols have 2:1 minerals and have high negative charge, so ammonium is more absorbed by soil particles, whereas nitrate is free to move in the soil and diffuses into the plant tissue or is leached with gravity water. This study aimed to determine the litter treatment that can inhibit the nitrification process in Vertisols on sweet corn plants. This research was conducted in June until November 2019 in the Plastic House of Plesungan, Gondangrejo, Karanganyar, Indonesia. This study used a basic completely randomized design with a single factor (litter type) as an immobilizer. The types of litter used in this study were Gliricidia maculata , Albizia falcataria , Senna siamea , and Tithonia diversifolia . The parameters observed were ammonium content, nitrification potential, average nitrate content, actual nitrification, plant height, number of leaves, and dry crown plant. Tithonia diversifolia gave the highest actual nitrification of 23.26%. Senna siamea has the lowest actual nitrification of 12.36%, followed by Gliricidia maculata with 17.39% and Albizia falcataria with 17.67%. This shows that the Tithonia diversifolia litter has the highest value in inhibiting nitrification. Maize plants treated with the Tithonia diversifolia litter had the best plant growth compared to those applied with other treatments. Therefore, among the treatments used, the Tithonia diversifolia litter was most optimal in inhibiting nitrification in Vertisols.</p
... In general, there is little knowledge about which processes are responsible for nitrification in acidic subtropical forest soils. Zhang et al. (2013) found heterotrophic nitrification to be the main pathway for NO 3 − production, whereas Faeflen et al. (2016) (Kirkham and Bartholomew, 1954;Huygens et al., 2008) and by the Ntrace tool . 3) Microbial N cycling prevails over physiochemical N retention in the HS soil because of N saturation; by contrast, in colluvial GDZ soil, abiotic processes play a more important role for N retention. ...
N leaching and gaseous N emissions from forested catchments are controlled by soils differing in nitrogen (N) status and turnover depending on landscape position. To understand the impact of topography on N retention and dissipation in forested catchments suffering from high atmospheric N deposition, we carried out an ex-situ ¹⁵N-tracing study with soils from a hillslope (HS) and a hydrologically connected groundwater discharge zone (GDZ) of an N-saturated subtropical forest in South China. Despite being severely N-saturated, soil from HS incorporated a substantial amount of added ¹⁵N-NH4⁺ instantly into recalcitrant organic N. The remaining NH4⁺ was cycled via a microbial loop of fast N immobilization and re-mineralization, slowly releasing NH4⁺ for autotrophic nitrification. Heterotrophic nitrification was only observed right after tracer application. Added ¹⁵N-NO3⁻ cycled between soil microbial biomass and dissolved organic N without being stored in the recalcitrant organic N pool, explaining the strong propensity of HS soils for NO3⁻ leaching. By contrast, the soil from GDZ acted as a sink for added N by incorporating ¹⁵N-NH4⁺ into recalcitrant organic N and denitrifying ¹⁵N-NO3⁻ to gaseous N. Here, N immobilization exceeded N mineralization, suggesting N limitation. Heterotrophic nitrification was the main pathway of NH3 oxidation in the GDZ soil, and N2O-N contributed substantially to N removal. Abiotic processes played a role in NO3⁻ incorporation into organic N but not in N2O production, while DNRA was negligible in either soil. Overall, our findings suggest strong topographic control on N cycling, which might explain the unexpectedly high N retention and removal from N-saturated forests in subtropical China.
... This difference in the decline of the 15 N enrichment of the NO − 3 pool is arguably related to the different nitrification processes (autotrophic vs. heterotrophic). For example, Faeflen et al. (2016) compared the contributions of autotrophs and heterotrophs to nitrification in Oxisol (organic matter 22 g kg −1 , pH 5.0) and reported that autotrophic nitrification was the main nitrification pathway. In the Andosol from grassland, the main pathway of NO − 3 production was possibly heterotrophic nitrification, which is not effectively inhibited by nitrapyrin. ...
In the tropics, frequent nitrogen (N) fertilization of grazing areas can potentially increase nitrous oxide (N2O) emissions. The application of nitrification inhibitors has been reported as an effective management practice for potentially reducing N loss from the soil-plant system and improving N use efficiency (NUE). The aim of this study was to determine the effect of the co-application of nitrapyrin (a nitrification inhibitor, NI) and urea in a tropical Andosol on the behavior of N and the emissions of N2O from autotrophic and heterotrophic nitrification. A greenhouse experiment was performed using a soil (pH 5.9, organic matter content 78 g kg–1, and N 5.6 g kg–1) sown with Cynodon nlemfuensis at 60% water-filled pore space to quantify total N2O emissions, N2O derived from fertilizer, soil ammonium (NH4⁺) and nitrate (NO3–), and NUE. The study included treatments that received deionized water only (control, CK) and two doses of ¹⁵N-enriched urea (65 (UR) and 129 mg N kg–1 (UD)) without or with 350 g nitrapyrin for each 100 kg N (UR + NI and UD + NI). No significant differences were observed in soil NH+ content between the UR and UR + NI treatments, probably because of soil mineralization and immobilization (influenced by high soil organic matter content). Nitrapyrin application failed to maintain a stable pool of labeled NO3⁻ due to the additional NO⁻ produced by heterotrophic nitrification, which is not effectively inhibited by nitrapyrin. After 56 d, N2O emissions in UR (0.51 ± 0.12 mg N2O-N kg–1) and UR + NI (0.45 ± 0.13 mg N2O-N kg–1) were not significantly different; by contrast, emissions were 36.3% lower in UD + NI than in UD. It was concluded that the soil organic N mineralization and heterotrophic nitrification are the main processes of NH4⁺ and NO3– production. Additionally, it was found that N2O emissions were partially a consequence of the direct oxidation of the soil's organic N via heterotrophic nitrification coupled to denitrification. Finally, the results suggest that nitrapyrin would likely exert significant mitigation on N2O emissions only if a substantial N surplus exists in soils with high organic matter content.
... The AOA-amoA gene was abundant in the soils that received both natural (Ct) and simulated N depositions (LN, MN and HN) (Figure 3b), which explains the prevailing autotrophic nitrification activity (Faeflen et al., 2016;Zhang et al., 2012) in subtropical forest soils. Moreover, the AOA-amoA abundance was higher than the AOB-amoA abundance (Figures 3a, 3b). ...
Elevated nitrogen (N) deposition has induced substantial impacts on the emissions of nitrous oxide (N2O) from forest ecosystems, but how soil microbes regulate the production/consumption of N2O under elevated N deposition remains poorly understood, particularly in high N deposition subtropical forests that are characterized by distinct wet‐dry seasonality. We established a field N addition experiment in a subtropical forest in southern China to explore the influences of low, medium and high (35, 70, and 105 kg N ha‐1 yr‐1, respectively) N addition on N2O efflux and its associated microbial functional genes [amoA for nitrifiers (ammonia‐oxidizing bacteria (AOB) and ammonia‐oxidizing archaea (AOA)) and nirK and nosZ for denitrifiers]. The results showed the following: (1) The N2O emissions were stimulated by N addition in the dry season but were depressed in the wet season. (2) The nirK and nosZ abundances were generally stimulated by N addition, whereas the AOB‐amoA and AOA‐amoA abundances showed divergent responses to N addition. (3) Based on the results of principal component and Pearson correlation analyses, N2O effluxes were associated with microbial biomass in the wet season but with nirK and nosZ abundances in the dry season. Structural equation modeling analyses further indicated that both nitrifiers and denitrifiers under N addition contributed to the generation of N2O in the dry season, whereas the decreased production of N2O in the wet season was primarily caused by denitrifiers. Therefore, seasonally specific strategies should be developed to mitigate the emissions of N2O from subtropical forests with distinct seasonal precipitation patterns.
... However, some studies have also reported conflicting results. Faeflen et al. (2016) found that nitrapyrin decreased AOB amoA gene copy numbers by approximately 80% and significantly decreased AOA abundance in acidic forest soils (Oxisol). Moreover, nitrapyrin diminished both the growth and activity of the AOA Nitrosotalea devanaterra in liquid culture and soil (Lehtovirta-Morley et al. 2013). ...
PurposeYellow clay paddy soil (Oxisols) is a low-yield soil with low nitrogen use efficiency (NUE) in southern China. The nitrification inhibitor nitrapyrin (2-chloro-6- (tricholoromethyl)-pyridine, CP) has been applied to improve NUE and reduce environmental pollution in paddy soil. However, the effects of nitrapyrin combined with nitrogen fertilizers on ammonia oxidizers in yellow clay paddy soil have not been examined. Materials and methodsA randomized complete block design was set with three treatments: (1) without nitrogen fertilizer (CK), (2) common prilled urea (PU), and (3) prilled urea with nitrapyrin (NPU). Soil samples were collected from three treatments where CK, PU, and NPU had been repeatedly applied over 5 years. Soil samples were analyzed by quantitative PCR and 454 high-throughput pyrosequencing of the amoA gene to investigate the influence of nitrapyrin combined with nitrogen on the abundance and community structure of ammonia oxidizers in yellow clay paddy soil. Results and discussionThe potential nitrification rate (PNR) of the soil was significantly correlated with the abundances of both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Application of urea significantly stimulated AOA and AOB growth, whereas nitrapyrin exhibited inhibitory effects on AOA. Phylogenetic analysis showed that the most dominant operational taxonomic units (OTUs) of AOA and AOB were affiliated with the Nitrosotalea cluster and Nitrosospira cluster 12, respectively. AOA and AOB community structures were not altered by urea and nitrapyrin application. Conclusions
Nitrogen fertilization stimulated nitrification and increased the population sizes of AOA and AOB. Nitrapyrin affected the abundance, but not community structure of ammonia oxidizers in yellow clay soil. Our results suggested that nitrapyrin improving NUE and inhibiting PNR was attributable to the inhibition of AOA growth.
... Nitrification by heterotrophic microbes is now ignored in many studies regarding nitrifiers. However, large contribution of heterotrophic nitrifying microbes to nitrification in acidic forest soil is still proposed or debated (Faeflen et al., 2016;Yu et al., 2017;Zhang et al., 2015Zhang et al., , 2014Zhu et al., 2015). ...
In southern China, high levels of atmospheric nitrogen (N) are being deposited in forests. Soil acidification and high rates of soil nitrification and subsequent NO3⁻ leaching have been observed in the N-saturated forests. We previously did not detect NH3-oxidizing bacteria in non-N-saturated or N-saturated forest soils. However, NH3-oxidizing archaea were present in both soils, more in the saturated ones. The purpose of this study was to investigate the roles of autotrophic NH3-oxidizing archaea and heterotrophic nitrifiers in soil N transformations in N-saturated and non-N-saturated forests in southern China. We investigated the contribution of heterotrophic nitrifiers in the soils by determining the gross nitrification rates with and without an inhibitor of autotrophic nitrification, acetylene (C2H2). We also reevaluated nitrification by NH3-oxidizing archaea by correlating the C2H2-inhibited gross nitrification rates with the abundance of the amoA transcripts of NH3-oxidizing archaea. We further measured the gross NH4⁺ production rates and analyzed the community composition of the NH3-oxidizing archaea. The results suggest that NH3-oxidizing archaea, rather than heterotrophic nitrifiers and NH3-oxidizing bacteria, are responsible for the nitrification in the N-saturated forest soils. NH3-oxidizing archaea in the soils could be acidophilic, having low amoA diversity, indicating their strong adaptation to the highly acidified soils. The gross NH4⁺ production rate did not differ between N-saturated and non-N-saturated forests; however, the gross nitrification rate was higher in N-saturated forests. Consequently, the high abundance and NH3 oxidation activity of NH3-oxidizing archaea caused the high rates of nitrification and subsequent leaching of NO3⁻ in the N-saturated forest. This study suggests that acidophilic NH3-oxidizing archaea have a great impact on soil N cycling in N-saturated forests.
Studies on nitrification, a crucial process of biogeochemical N cycling, have traditionally focused on autotrophic microorganisms. Recent discoveries, however, highlight the importance of heterotrophic nitrification as a key to N cycling, particularly in acidic soils. While molecular approaches have advanced our understanding of the key players in autotrophic nitrification, the biochemical mechanisms and corresponding genes of heterotrophic nitrification are nearly unknown. First, we reviewed the advantages and limitations of existing approaches to analyze heterotrophic nitrification in soils. 15N labeling of organic compounds (e.g. amino acids) allows to determine solely the nitrification of organic N. Because many bacteria have similar autotrophic nitrification enzymes that oxidize inorganic N, it is necessary to inhibit autotrophic nitrification to determine the heterotrophic N nitrification activity by 15N techniques. The use of existing inhibitors, however, can mislead the conclusions because not all inhibitors stop autotrophic nitrification completely, and some can decrease heterotrophic nitrification. Their effects strongly depend on the composition of the nitrifier community and soil properties. The use of modern molecular approaches is limited by suitable genetic biomarkers. Second, we propose the following methods to investigate heterotrophic nitrification processes: i) isolation and purification of heterotrophic nitrification enzymes, followed by determination of the amino acid sequence of proteins to design genetic markers; ii) use of DNA-based stable isotopes (13C, 15N); iii) combining fluorescence in situ hybridization with microautoradiography (14C) to determine the composition of heterotrophic nitrifier communities; and iv) scheme to select autotrophic nitrification inhibitors. We suggest to improve the existing approaches to shed new light on the processes of heterotrophic nitrification, which can reach 99% of total nitrification in forest soils and strongly affect N stocks and fluxes in terrestrial ecosystems.
Rates of nitrogen transformations support quantitative descriptions and predictive understanding of the complex nitrogen cycle, but measuring these rates is expensive and not readily available to researchers. Here, we compiled a dataset of gross nitrogen transformation rates (GNTR) of mineralization, nitrification, ammonium immobilization, nitrate immobilization, and dissimilatory nitrate reduction to ammonium in terrestrial ecosystems. Data were extracted from 331 studies published from 1984–2022, covering 581 sites. Globally, 1552 observations were appended with standardized soil, vegetation, and climate data (49 variables in total) potentially contributing to the observed variations of GNTR. We used machine learning-based data imputation to fill in partially missing GNTR, which improved statistical relationships between theoretically correlated processes. The dataset is currently the most comprehensive overview of terrestrial ecosystem GNTR and serves as a global synthesis of the extent and variability of GNTR across a wide range of environmental conditions. Future research can utilize the dataset to identify measurement gaps with respect to climate, soil, and ecosystem types, delineate GNTR for certain ecoregions, and help validate process-based models.
Increasing nitrogen (N) and phosphorus (P) deposition influences primary forest soil properties related to C and N dynamics, which may significantly affect greenhouse gas (GHG) emissions. We examined how the fertilization pattern and variation in soil in forest types can affect GHG emissions. We conducted a global systematic review of 66 publications on GHG emissions, pH, and C and N soil properties to examine the mechanisms underlying GHG emissions under N, P, and N×P additions in diverse forest ecosystems. The results of our meta-analysis showed that N and N×P addition considerably promote nitrous oxide (N2O) emissions in tropical forests, and P addition insignificantly decreased N2O emissions. N addition and P addition inhibit CO2 emissions in subtropical forests, which contributes to C storage, although the latter effect was nonsignificant, and P addition increases C dioxide emissions in tropical forests. Moreover, additions of N and N×P promote and inhibit overall methane uptake in the variety of forests studied, respectively. Additionally, the results indicated that the form, rate, duration, and N: P ratio of fertilization and the mean annual precipitation and mean annual temperature are influential variables affecting GHG emissions from forests under the various fertilizer additions. Our results highlight that when accurately predicting the effect of N and P deposition on soil GHG emissions, the characteristics of different forest types should be synthetically considered, such as experimental conditions, environmental variables, and soil properties. These results advance the understanding of the responding mechanism of soil GHG emissions in forests to different N and P addition models.
Wildfire has several environmental and ecological effects on soil biogeochemistry, including the nitrogen (N) cycle in forest ecosystems. In this study with 30 days of incubation, we examined the effect of a fire event on changes in soil properties, especially gross N mineralization and nitrification processes in boreal forest soils. We also tested how the addition and removal of fire-derived natural pyrogenic carbon (PyC, 1.5%) affected N transformation processes. After incubation, the concentrations of NH4⁺-N, NO3⁻-N, total N, and dissolved organic N were largely enhanced in the burnt soils compared to the unburnt soils. Fire significantly decreased gross N mineralization rates from 6.88 μg N g⁻¹ d⁻¹ to 0.87 μg N g⁻¹ d⁻¹ and NH4⁺ immobilization rates from 6.04 μg N g⁻¹ d⁻¹ to 0.55 μg N g⁻¹ d⁻¹ but did not significantly affect gross N nitrification rates or NO3⁻ immobilization rates on day 30. The removal from the burnt soil or addition to the unburnt soil of PyC had no significant effect on N transformation. The abundances of nirK, nosZ I, narG, and nifH in the burnt soils were the highest across the different treatments. Our study revealed that fire decreased gross N mineralization processes, which was associated with enhanced abundances of N-cycling genes (nirK, nosZ I and narG) in the boreal forest soils.
Despite the fact that heterotrophic nitrification was identified more than 100 years ago, the biochemistry of heterotrophic nitrifiers is poorly known and their contribution to nitrification in soil is still speculative. Heterotrophic nitrifiers need organic compounds as their energy source in contrast to the chemolithotrophic nitrifiers. Most of the potential pathways for nitrite/nitrate production by heterotrophs can be considered as secondary metabolism. Only nitrification and simultaneous denitrification by some heterotrophic bacteria is known to have connection to the energy metabolism. Evidently, the nitrification pathways of bacteria and fungi differ. Some heterotrophic bacteria oxidizing ammonia have ammonia monooxygenase (AMO), but there are also heterotrophic bacteria oxidizing ammonia without AMO. The structure of AMO of chemolithotrophic ammonia oxidizers and heterotrophs differs. AMO has not been found in nitrifying fungi. The conditions for heterotrophic nitrification in soil highly differ from those in waste waters where heterotrophic nitrification activity can be high. Heterotrophic nitrification in soil is limited by the low availability of easily decomposable organic substrates. Possible nitrification by heterotrophs in the rhizosphere and endophytic root microbes gaining a good supply of substrates from plants is not known. Fungi are of special interest in heterotrophic nitrification in soil because fungi can evidently nitrify not only easily decomposable substrates but also when decomposing recalcitrant organic compounds (like lignin) which are abundant in soil. Nitrite/nitrate production from easily decomposable nitrogenous organic compounds, such as amino acids, is common among heterotrophic bacteria and fungi. The general conclusion that heterotrophs use solely an “organic pathway” in their nitrification is, however, not valid because also ammonia can be oxidized. Owing to the diverse, poorly known biochemistry of heterotrophic nitrification and methodological difficulties to differentiate heterotrophic and chemolithotrophic nitrification in soil, the role of heterotrophic nitrification in soil nitrogen cycle remains uncertain.
The aim of this study was to investigate the combined effects of organic manure and nitrification inhibitor on nitrogen (N) transformation, nitrous oxide (N2O) emissions and the gene abundance of ammonia oxidizers in acidic and alkaline soils. Therefore, an incubation experiment was conducted with red (pH 5.51) and calcareous soils (pH 8.12) collected from Hunan Province and Xinjiang Autonomous Region of China, respectively, including four different treatments as follows: CK (no fertilizer), U (only urea), UM (60% urea-N plus cattle manure-N), UMCP (UM + nitrapyrin). The results showed that UMCP treatment significantly increased retention time of ammonium nitrogen (NH4⁺-N) in both red and calcareous soils (P < 0.05), compared with the treatments of CK, U and UM. The inhibitory efficiency of nitrapyrin, based on the ratio of NH4⁺-N/NO3⁻-N, was nearly twice higher in calcareous soil (71 days) than that in red soil (36 days). The potential nitrification rates (PNR) were significantly increased by the urea or urea plus manure application, but were greatly reduced by nitrapyrin addition. Nitrapyrin addition significantly inhibited the gene abundance of ammonia-oxidizing bacteria (AOB) in calcareous soil, whereas inhibited the ammonia-oxidizing archaea (AOA) growth in red soil. UMCP treatment lowered N2O emissions and increased N recovery compared with UM. Linear regression analysis showed that PNR was positively correlated with AOB (P = 0.048) and AOA abundance (P = 0.0014) in red soil, but only correlated with AOB (P = 0.0032) in calcareous soil. Random forest and structural equation model (SEM) analyses revealed that pH, NH4⁺-N and organic carbon (SOC) were the key factors affecting the abundance of AOA and AOB, and AOA contributes more to N2O emissions than AOB from red soil, whilst only AOB is highly responsible for the N2O production from calcareous soil. Our result suggests that nitrapyrin plus organic amendment could be serving as an effective fertilization scenario on mitigating N2O emissions and enhancing N recovery and utilization in acidic and alkaline soils.
We conducted a 456-d laboratory incubation of an old-growth coniferous forest soil to aid in the elucidation of C controls on N cycling processes in forest soils. Gross rates of N mineralization, immobilization, and nitrification were measured by ^1^5N isotope dilution, and net rates N mineralization and nitrification were calculated from changes in KCl-extractable inorganic N and NO"3^-"@ON pool sizes, respectively. Changes in the availability of C were assessed by monitoring rates of CO"2 evolution and the sizes of extractable organic C and microbial biomass pools. Net and gross rates of N mineralization (r^2 = 0.038, P = .676) and nitrification (r^2 = 0.403, P = .125) were not significantly correlated over the course of the incubation, suggesting that the factors controlling N consumptive and productive processes do not equally affect these processes. A significant increase in the NO"3^- pool size (net nitrification) only occurred after 140 d, when the NO"3^- pool size increased suddenly and massively. However, gross nitrification rates were substantial throughout the entire incubation and were poorly correlated with these changes in NO"3^- pool sizes. Concurrent decreases in the microbial biomass suggest that large increases in NO"3^- pool sizes after prolonged incubation of coniferous forest soil may arise from reductions in the rate of microbial immobilization of NO"3^-, rather than from one of the mechanisms proposed previously (e.g., sequestering of NH"4^+ by microbial heterotrophs, the deactivation of allelopathic compounds, or large increases in autotrophic nitrifier populations). Strong correlations were found between rates of CO"2 evolution and gross N mineralization (r^2 = 0.974, P
In this study, we investigated the impact of soil pH on the diversity and abundance of archaeal ammonia oxidizers in 27 different forest soils across Germany. DNA was extracted from topsoil samples, the amoA gene, encoding ammonia monooxygenase, was amplified; and the amplicons were sequenced using a 454-based pyrosequencing approach. As expected, the ratio of archaeal (AOA) to bacterial (AOB) ammonia oxidizers’ amoA genes increased sharply with decreasing soil pH. The diversity of AOA differed significantly between sites with ultra-acidic soil pH (4.5, regardless of geographic position and vegetation. These OTUs could be related to the Nitrosotalea group 1.1 and the Nitrososphaera subcluster 7.2, respectively, and showed significant similarities to OTUs described from other acidic environments. Conversely, none of the major OTUs typical of sites with a soil pH >4.6 could be found in the ultra- and extreme acidic soils. Based on a comparison with the amoA gene sequence data from a previous study performed on agricultural soils, we could clearly show that the development of AOA communities in soils with ultra-acidic pH (
Purpose
Acidic red soils account for 21% of land area in China and contain low ammonia concentration due to ionization to ammonium. The unusual high affinity for ammonia of marine Nitrosopumilus maritimus and acidophilic soil Nitrosotalea devanaterra has suggested that ammonia-oxidizing archaea (AOA) may have greater selective advantage over ammonia-oxidizing bacteria (AOB) in ammonia-limited environment because ammonia rather than ammonium is thought to be the actual substrate for oxidation. The aim of this study was to assess whether nitrification activity can be attributed to AOA and/or AOB by relating community structures of AOA and AOB to nitrification activity in acidic red soils in southern China.
Materials and methods
In this study, the composition and abundance of AOA community were investigated in acidic red soils of coniferous Pinus forest, broad-leaf Cinnamomum forest, bush forest (BF), and a 30-year agricultural field converted from bush forest (BFA). The composition of AOA based on archaeal amoA genes were analyzed by denaturant gradient gel electrophoresis, and the abundances of AOA communities were determined by real-time quantitative polymerase chain reaction, while soil nitrification activity was measured using 15N pool enrichment technique.
Results and discussion
15N pool enrichment technique indicated nitrification activity in acidic red soils, but AOB were not detected. The absence of AOB in acidic red soils could be well explained by the low ammonia concentration ranging from 17.8 to 34.3 nM, which is far below the known threshold values required to support the growth of AOB in culture. Nitrification activity change coupled well with abundance and composition changes of archaeal amoA genes, particularly for acidic BF and BFA soils. Phylogenetic analysis demonstrated that the putatively active AOA were related to amoA transcripts in a hot spring within the soil Crenarchaeota group 1.1b lineage.
Conclusions
These results suggest that AOA play important roles in ammonia oxidation in acidic red soils tested in this study.
Aims
Low gas diffusivity of the litter layer is held responsible for high seasonal nitrous oxide (N2O) and low nitric oxide (NO) emissions from acid beech forest soils with moder type humus. The objectives were (i) to evaluate whether these beech forest soils generally exhibit high seasonal N2O emissions and (ii) to assess the influence of gas diffusivity and nitrogen (N) mineralisation on N oxide fluxes.
Methods
We measured N2O and NOx (NO + NO2) fluxes in six German beech stands and determined net N turnover rates and gas diffusivity of soil samples taken at each chamber.
Results
High N2O emissions (up to 113 μg N m−2 h−1) were only observed at one beech stand. Net nitrification of the organic layer and soil gas diffusivity explained 77 % of the variation in N2O fluxes (P = 0.001). Fluxes of NOx were low (−6.3 to 12.3 μg N m−2 h−1) and appeared to be controlled by NOx concentrations in the forest air.
Conclusions
Low soil gas diffusivity and high N turnover rates promoted high N2O losses in times of high soil respiration but were not necessarily associated with moder type humus. High seasonal emissions are probably less common in German beech forests than previously assumed.
To date, occurrence and stimulation of different nitrification pathways in acidic soils remains unclear. Laboratory incubation experiments, using the acetylene inhibition and 15N tracing methods, were conducted to study the relative importance of heterotrophic and autotrophic nitrification in two acid soils (arable (AR) and coniferous forest) in subtropical China, and to verify the reliability of the 15N tracing model. The gross rate of autotrophic nitrification was 2.28 mg kg−1 day−1, while that of the heterotrophic nitrification (0.01 mg kg−1 day−1) was negligible in the AR soil. On the contrary, the gross rate of autotrophic nitrification was very low (0.05 mg kg−1 day−1) and the heterotrophic nitrification (0.98 mg kg−1 day−1) was the predominant NO3− production pathway accounting for more than 95 % of the total nitrification in the coniferous forest soil. Our results showed that the 15N tracing model was reliable when used to study soil N transformation in acid subtropical soils.
Nitrification, the microbial oxidation of NH^+_4-N, plays a key role in the cycling of N in forested and other terrestrial ecosystems. Solution losses of nitrate and gaseous losses of N_2 and nitrous oxides are important vectors of N loss from many forested systems and are directly or indirectly controlled by the activity of the nitrifiers. These losses can also have important consequences for downstream ecosystems, groundwater quality, and atmospheric concentrations of ozone. Relative nitrification (the proportion of the total mineral N that is nitrate at the end of an incubation period) provides an independent means of evaluating the general importance of site factors thought to regulate nitrification in situ. Regression of relative nitrification against soil pH, C:N, and percentage N, with the use of data from previously published studies, suggest that although these factor may be important regulators of nitrification in particular sites, they are not good predictors of nitrification across a wide range of sites. Reasons for their low predictive ability may include limitations of current measurement techniques or the capacity of nitrifiers to adapt to relatively extreme conditions.
The oxidation of ammonia to nitrate, nitrification, is a key process in the nitrogen cycle. Ammonia-oxidizing archaea are present in large numbers in the ocean and soils, suggesting a potential role for archaea, in addition to bacteria, in the global nitrogen cycle. However, the importance of archaea to nitrification in agricultural soils is not well understood. Here, we examine the contribution of ammonia-oxidizing archaea and bacteria to nitrification in six grassland soils in New Zealand using a quantitative polymerase chain reaction. We show that although ammonia-oxidizing archaea are present in large numbers in these soils, neither their abundance nor their activity increased with the application of an ammonia substrate, suggesting that their abundance was not related to the rate of nitrification. In contrast, the number of ammonia-oxidizing bacteria increased 3.2-10.4-fold and their activity increased 177-fold, in response to ammonia additions. Indeed, we find a significant relationship between the abundance of ammonia-oxidizing bacteria and the rate of nitrification. We suggest that nitrification is driven by bacteria rather than archaea in these nitrogen-rich grassland soils.
Purpose Boreal forests are considered to be more sensitive to global climate change compared with other terrestrial ecosystems, but the long-term impact of climate change and forest management on soil microbial functional diversi-ty is not well understood. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are the most important players in nitrogen (N) cycling-associated processes in terrestrial ecosystems. This study investigated the separate and com-bined impacts of long-term soil warming and fertilization on soil AOB and AOA community structures and abundances in a Norway spruce stand in northern Sweden. Materials and methods The soil-warming experiment was established in the buffer zones of two irrigated plots (I) and complete nutrient solution plots (IL) since 1995. The warm-ing treatment started in April each year by maintaining soil temperature on warmed plots at 5°C above the temperature in unwarmed plots using heating cables. In August 2006, soil samples were collected from eight subplots for molec-ular analysis. The abundance of bacterial and archaeal amoA genes was determined by quantitative polymerase chain reaction. Similarly, total bacterial and archaeal population sizes have also been determined. The diversity of AOB and AOA was assessed by constructing amoA gene clone librar-ies, and different genotypes were screened with restriction fragment length polymorphism. Results and discussion Results showed that fertilization did not significantly affect the abundance of the bacterial amoA gene under either warming or non-warming conditions; however, warming decreased the abundance under fertiliza-tion treatments. No significant effects of fertilization and soil warming were observed on the number of thaumarch-aeal amoA gene copies across all treatments. In this study, amoA gene abundance of AOB was significantly higher than that of AOA across all treatments. The community structure of both AOB and AOA was strongly influenced by fertil-ization. For bacterial amoA genes, Nitrosospira cluster 2 was present across all treatments, but the only genotype was observed in the fertilization treatments while, for thau-marchaeal amoA genes, the relative abundance of soil clus-ter 5 increased in fertilization treatments. By comparison, soil-warming effects on AOB and AOA community struc-ture were not significant. Canonical correspondence analy-sis showed a positive correlation between fertilization and both dominant genotypes of AOB and AOA. Conclusions These results indicated that the abundance of AOA and AOB was not affected by fertilization or warming alone, but the interaction of fertilization and warming re-duced the abundance of AOB. The community composition of ammonia-oxidizers was more affected by the nutrient-optimized fertilization than the soil warming.
The competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea, the heterotrophic species Arthrobacter globiformis and roots of Plantago lanceolata (Ribwort plantain) was studied in a series of model systems of increasing complexity, i.e. energy-limited continuous cultures, non-water-saturated continuously percolated soil columns and pots with γ-sterilized soil planted with axetic P. lanceolata seedlings. The effects of bacterial grazing by the flagellate species Adriamonas peritocrescens on the competition for ammonium were also investigated in the three model systems.
It was found that N. europaea was a weaker competitor for ammonium than either A. globiformis or plant roots of P. lanceolata. It is assumed that the heterotrophic bacteria have a higher affinity for ammonium than the nitrifying bacteria, whereas growing plant roots have a greater capacity to exploit the soil for ammonium than the immobile nitrifying bacteria. It is not very likely that allelochemicals were involved in suppressing the nitrification process. Four reasons are given for this assumption.
Presence of the flagellates strongly stimulated the potential nitrification rate in all model systems. It is assumed that there is a more even distribution over the soil of either nitrifying bacteria or their substrate ammonium in the presence of flagellates. In addition to the distribution effect, there is a stimulation of the potential ammonium oxidation rate. The results are discussed in the light of the function of nitrate as nitrogen sink in the biogeochemical nitrogen cycle.
A study was carried out to investigate the potential gross nitrogen (N) transformations in natural secondary coniferous and
evergreen broad-leaf forest soils in subtropical China. The simultaneously occurring gross N transformations in soil were
quantified by a 15N tracing study. The results showed that N dynamics were dominated by NH4+ turnover in both soils. The total mineralization (from labile and recalcitrant organic N) in the broad-leaf forest was more
than twice the rate in the coniferous forest soil. The total rate of mineral N production (NH4+ + NO3−) from the large recalcitrant organic N pool was similar in the two forest soils. However, appreciable NO3− production was only observed in the coniferous forest soil due to heterotrophic nitrification (i.e. direct oxidation of organic
N to NO3−), whereas nitrification in broad-leaf forest was little (or negligible). Thus, a distinct shift occurred from predominantly
NH4+ production in the broad-leaf forest soil to a balanced production of NH4+ and NO3− in the coniferous forest soil. This may be a mechanism to ensure an adequate supply of available mineral N in the coniferous
forest soil and most likely reflects differences in microbial community patterns (possibly saprophytic, fungal, activities
in coniferous soils). We show for the first time that the high nitrification rate in these soils may be of heterotrophic rather
than autotrophic nature. Furthermore, high NO3− production was only apparent in the coniferous but not in broad-leaf forest soil. This highlights the association of vegetation
type with the size and the activity of the SOM pools that ultimately determines whether only NH4+ or also a high NO3− turnover is present.
Keywords
15N tracing technique–Model–Labile and recalcitrant organic N–Gross N transformation–Heterotrophic nitrification
Forest soils show a great degree of temporal and spatial variation of nitrogen mineralization. The aim of the present study was to explain temporal variation in nitrate leaching from a nitrogen-saturated coniferous forest soil by potential nitrification, mineralization rates and nitrate uptake by roots. Variation in nitrate production in time and space, between the different organic horizons, has been related to temperature, moisture content, substrate availability and pH. Temporal variation in concentrations of nitrate and ammonium in the forest floor was significant during a one-year cycle, when randomly taken samples were pooled. Nitrogen concentrations differed between the different organic horizons with highest concentrations found in the litter layer, decreasing with increasing depth. Ammonium concentrations always exceeded nitrate concentrations by a factor ten, indicating that ammonium was not limiting nitrification. Nitrification potential, the nitrate production at field moisture at 25°C, was highest in the litter layer, lower in the fragmentation layer and hardly measurable in the mineral soil. Uptake of nitrate by roots and changes in mineralization rates turned out to be unimportant to explain variation in time, as seasonal fluctuations seem to be less important than spatial variation. We found that horizontal spatial variation in potential nitrate production, leaching of nitrate and nitrogen concentrations from non-pooled field samples was higher than variation in time. All this reflects the actual spatial variation in the field, which is not explained by differences in moisture content or temperature. Overall neither pH nor substrate availability could explain this observed variation, however, local variation in microsites may be responsible for small-scale spatial variation. Allelopathic compounds and/or the composition of the microbial community are suggested as factors possibly affecting nitrate production.
A method was developed to determine the contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to the nitrification potentials (NPs) of soils taken from forest, pasture, cropped, and fallowed (19 years) lands. Soil slurries were exposed to acetylene to irreversibly inactivate ammonia monooxygenase, and upon the removal of acetylene, the recovery of nitrification potential (RNP) was monitored in the presence and absence of bacterial or eukaryotic protein synthesis inhibitors. For unknown reasons, and despite measureable NPs, RNP did not occur consistently in forest soil samples; however, pasture, cropped, and fallowed soil RNPs commenced after lags that ranged from 12 to 30 h after acetylene removal. Cropped soil RNP was completely prevented by the bacterial protein synthesis inhibitor kanamycin (800 μg/ml), whereas a combination of kanamycin plus gentamicin (800 μg/ml each) only partially prevented the RNP (60%) of fallowed soils. Pasture soil RNP was completely insensitive to either kanamycin, gentamicin, or a combination of the two. Unlike cropped soil, pasture and fallowed soil RNPs occurred at both 30°C and 40°C and without supplemental NH(4)(+) (≤ 10 μM NH(4)(+) in solution), and pasture soil RNP demonstrated ∼ 50% insensitivity to 100 μM allyl thiourea (ATU). In addition, fallowed and pasture soil RNPs were insensitive to the fungal inhibitors nystatin and azoxystrobin. This combination of properties suggests that neither fungi nor AOB contributed to pasture soil RNP and that AOA were responsible for the RNP of the pasture soils. Both AOA and AOB may contribute to RNP in fallowed soil, while RNP in cropped soils was dominated by AOB.
Nitrification plays a central role in the global nitrogen cycle and is responsible for significant losses of nitrogen fertilizer, atmospheric pollution by the greenhouse gas nitrous oxide, and nitrate pollution of groundwaters. Ammonia oxidation, the first step in nitrification, was thought to be performed by autotrophic bacteria until the recent discovery of archaeal ammonia oxidizers. Autotrophic archaeal ammonia oxidizers have been cultivated from marine and thermal spring environments, but the relative importance of bacteria and archaea in soil nitrification is unclear and it is believed that soil archaeal ammonia oxidizers may use organic carbon, rather than growing autotrophically. In this soil microcosm study, stable isotope probing was used to demonstrate incorporation of (13)C-enriched carbon dioxide into the genomes of thaumarchaea possessing two functional genes: amoA, encoding a subunit of ammonia monooxygenase that catalyses the first step in ammonia oxidation; and hcd, a key gene in the autotrophic 3-hydroxypropionate/4-hydroxybutyrate cycle, which has been found so far only in archaea. Nitrification was accompanied by increases in archaeal amoA gene abundance and changes in amoA gene diversity, but no change was observed in bacterial amoA genes. Archaeal, but not bacterial, amoA genes were also detected in (13)C-labeled DNA, demonstrating inorganic CO(2) fixation by archaeal, but not bacterial, ammonia oxidizers. Autotrophic archaeal ammonia oxidation was further supported by coordinate increases in amoA and hcd gene abundance in (13)C-labeled DNA. The results therefore provide direct evidence for a role for archaea in soil ammonia oxidation and demonstrate autotrophic growth of ammonia oxidizing archaea in soil.
Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus "Nitrosopumilus maritimus" strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.
The discovery of ammonia oxidation by mesophilic and thermophilic Crenarchaeota and the widespread distribution of these organisms in marine and terrestrial environments indicated an important role for them in the global nitrogen cycle. However, very little is known about their physiology or their contribution to nitrification. Here we report oligotrophic ammonia oxidation kinetics and cellular characteristics of the mesophilic crenarchaeon 'Candidatus Nitrosopumilus maritimus' strain SCM1. Unlike characterized ammonia-oxidizing bacteria, SCM1 is adapted to life under extreme nutrient limitation, sustaining high specific oxidation rates at ammonium concentrations found in open oceans. Its half-saturation constant (K(m) = 133 nM total ammonium) and substrate threshold (<or=10 nM) closely resemble kinetics of in situ nitrification in marine systems and directly link ammonia-oxidizing Archaea to oligotrophic nitrification. The remarkably high specific affinity for reduced nitrogen (68,700 l per g cells per h) of SCM1 suggests that Nitrosopumilus-like ammonia-oxidizing Archaea could successfully compete with heterotrophic bacterioplankton and phytoplankton. Together these findings support the hypothesis that nitrification is more prevalent in the marine nitrogen cycle than accounted for in current biogeochemical models.
In recent years, China has conducted considerable research focusing on the emission and effects of sulphur (S) on human health and ecosystems. By contrast, there has been little emphasis on anthropogenic nitrogen (N) so far, even though studies conducted abroad indicate that long-range atmospheric transport of N and ecological effects (e.g. acidification of soil and water) may be significant. The Sino-Norwegian project IMPACTS, launched in 1999, has established monitoring sites at five forest ecosystems in the southern part of PR China to collect comprehensive data on air quality, acidification status and ecological effects. Here we present initial results about N dynamics at two of the IMPACTS sites located near Chongqing and Changsha, including estimation of atmospheric deposition fluxes of NOx and NHx and soil N transformations. Nitrogen deposition is high at both sites when compared with values from Europe and North America (25-38 kg ha(-1) yr(-1)). About 70% of the deposited N comes as NH4, probably derived from agriculture. Leaching of N from soils is high and nearly all as NO3-. Transformation of N to NO3- in soils results in acidification rates that are high compared to rates found elsewhere. Despite considerable leaching of NO3- from the root zone of the soils, little NO3- appears in streamwater. This indicates that N retention or denitrification, both causing acid neutralization, may be important and probably occur in the groundwater and groundwater discharge zones. The soil flux density of mineral N, which is the sum of N deposition and N mineralization, and which is dominated by the N mineralization flux, may be a good indicator for leaching of NO3- in soils. However, this indicator seems site specific probably due to differences in land-use history and current N requirement.
Nitrification, the microbial oxidation of ammonia to nitrite and nitrate, occurs in a wide variety of environments and plays a central role in the global nitrogen cycle. Catalyzed by the enzyme ammonia monooxygenase, the ability to oxidize ammonia was previously thought to be restricted to a few groups within the β- and γ-Proteobacteria. However, recent metagenomic studies have revealed the existence of unique ammonia monooxygenase α-subunit (amoA) genes derived from uncultivated, nonextremophilic Crenarchaeota. Here, we report molecular evidence for the widespread presence of ammonia-oxidizing archaea (AOA) in marine water columns and sediments. Using PCR primers designed to specifically target archaeal amoA, we find AOA to be pervasive in areas of the ocean that are critical for the global nitrogen cycle, including the base of the euphotic zone, suboxic water columns, and estuarine and coastal sediments. Diverse and distinct AOA communities are associated with each of these habitats, with little overlap between water columns and sediments. Within marine sediments, most AOA sequences are unique to individual sampling locations, whereas a small number of sequences are evidently cosmopolitan in distribution. Considering the abundance of nonextremophilic archaea in the ocean, our results suggest that AOA may play a significant, but previously unrecognized, role in the global nitrogen cycle.
• Crenarchaeota
• nitrification
• ammonia monooxygenase
The relation between environmental factors and the presence of ammonia-oxidising bacteria (AOB), and its consequences for the N transformation rates were investigated in nine Scots pine (Pinus sylvestris L.) forest soils. In general, the diversity in AOB appears to be strikingly low compared to other ecosystems. Nitrosospira cluster 2, as determined by temporal temperature gradient electrophoresis and sequencing, was the only sequence cluster detected in the five soils with high nitrification rates. In the four soils with low nitrification rates, AOB-like sequences could not be detected. Differences in nitrification rates between the forest soils correlated to soil C/N ratio (or total N) and atmospheric N deposition.
In a previous study, ammonia-oxidizing bacteria (AOB)-like sequences were detected in the fragmentation layer of acid Scots pine (Pinus sylvestris L.) forest soils (pH 2.9-3.4) with high nitrification rates (>11.0 microg g-1 dry soil week-1), but were not detected in soils with low nitrification rates (<0.5 microg g-1 dry soil week-1). In the present study, we investigated whether this low nitrification rate has a biotic cause (complete absence of AOB) or an abiotic cause (unfavorable environmental conditions). Therefore, two soils strongly differing in net nitrification were compared: one soil with a low nitrification rate (location Schoorl) and another soil with a high nitrification rate (location Wekerom) were subjected to liming and/or ammonium amendment treatments. Nitrification was assessed by analysis of dynamics in NH4+-N and NO3- -N concentrations, whereas the presence and composition of AOB communities were assessed by polymerase chain reaction-denaturing gradient gel electrophoresis and sequencing of the ammonia monooxygenase (amoA) gene. Liming, rather than ammonium amendment, stimulated the growth of AOB and their nitrifying activity in Schoorl soil. The retrieved amoA sequences from limed (without and with N amendment) Schoorl and Wekerom soils exclusively belong to Nitrosospira cluster 2. Our study suggests that low nitrification rates in acidic Scots pine forest soils are due to pH-related factors. Nitrosospira cluster 2 detected in these soils is presumably a urease-positive cluster type of AOB.
This chapter examines the effects of nitrification inhibitors on nitrogen transformations, other than nitrification, in soils. Retardation of nitrification may result in less movement and transport of mineral nitrogen because of higher NH4/NO3 ratios in soils caused by retardation of nitrification. Keeping nitrogen in the ammonium form by retarding nitrification reduces movement of mineral nitrogen because ammonium is retained by soil particles and is thus less mobile. It is found that nitrapyrin inhibited nitrification of ammonium in a loamy sand soil and also reduced the amounts of nitrate leached in soil columns over 2–5 weeks. Nitrification inhibitors may effect mineralization of soil nitrogen in some situations. They can also influence immobilization of nitrogen because of persistence of ammonium, which is preferentially immobilized over nitrate by soil microorganism. Nitrification inhibitors can also influence mineralization of soil nitrogen. It is observed that nitrification inhibitors increase immobilization of N by increasing the persistence of NH4. Nitrification inhibitors also check NO2 accumulation in soils and thus block fixation of NO2 into organic matter.
Nitrogen cycling is an important aspect of forest ecosystem functioning. Pristine temperate rainforests have been shown to produce large amounts of bioavailable nitrogen, but despite high nitrogen turnover rates, loss of bioavailable nitrogen is minimal in these ecosystems. This tight nitrogen coupling is achieved through fierce competition for bioavailable nitrogen by abiotic processes, soil microbes and plant roots, all of which transfer bioavailable nitrogen to stable nitrogen sinks, such as soil organic matter and above-ground forest vegetation. Here, we use a combination of in situ 15N isotope dilution and 15N tracer techniques in volcanic soils of a temperate evergreen rainforest in southern Chile to further unravel retention mechanisms for bioavailable nitrogen. We find three processes that contribute significantly to nitrogen bioavailability in rainforest soils: heterotrophic nitrate production, nitrate turnover into ammonium and into a pool of dissolved organic nitrogen that is not prone to leaching loss, and finally, the decoupling of dissolved inorganic nitrogen turnover and leaching losses of dissolved organic nitrogen. Identification of these biogeochemical processes helps explain the retention of bioavailable nitrogen in pristine temperate rainforests.
Soil surface electrochemical properties may have a strong influence on nitrifying microorganisms, H+ and NH4+ activities, and therefore on the nitrification process. A gradient of surface electrochemical parameters was obtained by amendment of a subtropical acid pine soil (Oxisol) with 0% (control), 3%, 5%, 8%, 10% and 12% pure Ca-Montmorillonite by weight. The H+ and NH4+ activities, the abundance of the ammonia-oxidizing bacterial (AOB) and archaeal (AOA) amoA gene copies, and time-dependent kinetics of net nitrification were investigated. Soil particle surface specific area ranged from 53 to 103 m2 g−1 and increased with increasing montmorillonite application rate. Similar to specific area, surface charge quantity, surface charge density, electric field strength and surface potential increased after montmorillonite amendment. The H+ and NH4+ activities decreased linearly after montmorillonite addition. AOB amoA gene copy number was 1.82 × 105 copies g−1 for unamended soil, and the highest AOB amoA gene copy numbers were found for the 10% montmorillonite amendment (3.11 × 107 g−1 soil), which was more than 150 times higher than unamended soil. AOA amoA gene copy numbers were 9.19 × 103 copies g−1 dry unamended soil, and the highest AOA amoA gene copy numbers were found in the 8% montmorillonite amendment (1.22 × 105 g−1 soil). Although pH significantly decreased during the first three weeks of incubation, no significant difference was observed between the unamended control and different rates of montmorillonite addition treatments during the whole incubation. The largest net nitrification (103 mg N kg−1) was observed in the 10% montmorillonite amendment and the lowest in unamended soil (62 mg N kg−1). While montmorillonite did not change the kinetic patterns of net nitrification, the highest nitrification potential (275 mg N kg−1) for the 10% montmorillonite treatment was more than 3 times higher than unamended soil from simulation of time-dependent kinetics. Nitrification was significantly stimulated after montmorillonite amendment in acid soil mainly due to an increase in the quantity and activity of AOB and AOA. We concluded that soil particle surface parameters can significantly influence nitrification, especially in acid soils.
In Ahvenisto esker, southern Finland, artificial recharging of groundwater has been done by sprinkling infiltration, i.e. by sprinkling lake water directly onto forest soil. Due to infiltration, the pH of the humus layer rose from about 5 to 6.5, nitrification was initiated and the fluxes of N2O and leaching of nitrate from the soil increased. Our aim was to study nitrogen transformations in different soil layers and to determine the response of nitrification to pH. Nitrification in ammonium-enriched soil suspensions was pH-dependant in a gradient from 4.7 to 6.7. In the soils subjected to infiltration, the production of (NO2+NO3)-N was inhibited by decreasing the pH to 5.3 or lower. Low pH also led to decreased numbers of nitrifiers. In the soils not subjected to infiltration (control soils), (NO2+NO3)-N production initiated at pH 6.7 and the numbers of nitrifiers increased. In incubation experiments, with no added ammonium, the adjustment of pH to 6.7 also initiated nitrification in the control soils. Thus, increase in soil pH was the main reason for initiation of nitrification at this site. During infiltration, N2O was produced mainly by denitrification and approximately 75% of the denitrification products was N2. In the samples from the humus layer, the concentrations of (NO2+NO3)-N, the net production of mineral N and net nitrification were in general less, whereas denitrification enzyme activity and denitrification potential were higher than in the samples from the mineral soil layer. The mineral soil may therefore contribute substantially to the leaching of nitrate.
Forty-five soil samples were collected from rice paddy land (R), tea garden land (T), forestland (F), brush land (B), and upland (U) in Jiangxi province, a subtropical region of China. These soils were derived from Quaternary red earth (Q), Tertiary red sandstone (S), and granite (G). Their denitrification capacities were determined after treatment with 200 mg NO3−-N kg−1 soil by measuring changes in NO3−-N content during a 28-day anaerobic incubation under N2 gas in the headspace, at 30°C. The subtropical soils studied here were characterized by generally small denitrification capacities, ranging from no denitrification capacity to complete disappearance of added NO3−-N within 11 days of incubation. With few exceptions, NO3−-N reduction with incubation time followed a first-order relationship with reaction constants of 0 – 0.271 day−1, but the data could be simulated better by a logarithmic relationship. Thus, denitrification capacity was determined by the reaction constant of the first-order reaction, the slope of the logarithmic relationship, and the averaged NO3−-N reduction rate in the first 7 days of anaerobic incubation (ranging from 0 to 28.5 mg kg−1day−1), and was significantly larger in the soils derived from G than from Q and S for all land uses except for rice paddy land. Soil organic carbon and nitrogen availability are the key factors that determine differences in denitrification capacity among the three soil parent materials. Rice cultivation significantly promoted denitrification capacity compared with the other four land uses and masked the effect of soil parent materials on denitrification capacity. This is most likely due to increases in organic carbon and total N content in the soil, which promoted the population and biological activities of microorganisms which are able to respire anaerobically when the rice soil is flooded. Neither the increased pH of upland soil caused by the addition of lime for upland crop production, nor the decreased pH of the tea garden soil by the acidification effect of tea plants altered soil denitrification capacity. Our results suggest that land use and management practices favour soil carbon and/or nitrogen accumulation and anaerobic microorganism activities enhance soil denitrification capacity.
Ammonia-oxidizers play a key role in nitrification, which is important for nitrogen cycling and soil function. However, little is known about how vegetation successions and agricultural practices caused by human activities impact the ammonia-oxidizers and nitrification process. Putative ammonia-oxidizing bacteria (AOB) and archaea (AOA) communities under different land utilization patterns of restoration (forest), degradation (pasture), cropland and pine plantation were analysed in an acidic red soil based on bacterial and archaeal amoA genes together with archaeal 16S rRNA gene. Real-time PCR, terminal restriction fragment length polymorphism (T-RFLP) and sequencing of clone libraries were conducted to study their abundance and community structure. Land utilization pattern showed significant effects on the copy numbers of all these genes, but only the bacterial amoA gene correlated significantly with potential nitrification rates (PNR). The cropland plot possessed the highest bacterial amoA gene copies and PNR, while the degradation plot was opposite to that. There were no significant variations in the bacterial amoA gene structure, which was dominated by Clusters 10 and 11 in Nitrosospira. However, archaeal amoA gene structure varied among different land utilization patterns especially for the cropland. The degradation plot was dominated by Crenarchaea 1.1c-related groups from which the amoA gene could not been amplified in this study, while other plots were dominated by Crenarchaea 1.1a/b group based on archaeal 16S rRNA gene analysis. These results indicated significant effects of land utilization patterns on putative ammonia oxidizers, which were especially obvious in the degradation and cropland plots where frequent human disturbance occurred.
The impacts of ammonium-based N (NH4+ - N) addition on soil nitrification and acidification were investigated in terms of kinetic mechanisms and major factors controlling these soil processes for terrestrial ecosystems in subtropical China. Soil samples were collected from an upland soil derived from a sandstone parent (SU), a brush-land soil from a granite (GB), and a forest soil from a quaternary red earth (QF) in a typical subtropical region of China. The samples were incubated at 30°C with soil moisture content of 60% water holding capacity (WHC) for 35 days, after adding ammonium sulphate, urea, and ammonium bicarbonate at rates of 0, 100, and 250 mg N kg-1, respectively. Nitrification in SU soil (pH 6.27) followed a first-order reaction model (P < 0.001). Addition of ammonium sulphate, urea and ammonium bicarbonate significantly (P < 0.05) stimulated nitrification. As a result, the soil was significantly acidified (P < 0.05) and the soil pH at the end of incubation decreased with increasing N addition. In contrast, nitrification in QF (pH 4.46) and GB (pH 4.82) soils followed a zero-order reaction model (P < 0.001) and hence the addition of NH4+ did not directly affect soil nitrification. However, the chemical input directly changed initial pH of GB and QF soils, resulting in either a decrease or an increase in NO3- production, dependent on the impact of the chemicals applied. At the end of incubation, the pH of QF and GB soils was significantly higher (P < 0.05) in treatments with NH 4+-input than without NH4+-input. These results indicated that for some acid soils nitrification was not controlled by available NH4+-N and that NH 4+-N-input was not necessary to stimulate soil nitrification. And so no acceleration of soil acidification occurred. In order to characterize nitrification intensity in these humid soils and its effect on acidification, nitrification without N-amendment is a better indicator than with N-amendment.
Net nitrification rates tend to be low or negligible in the forest floor of many coniferous forests of North-East Scotland. The most likely process controls are substrate availability, pH, allelopathy, water potential, nutrient status and temperature. These are discussed in relation to field and laboratory studies of net and potential rates of nitrification.
Fungi make up by far the largest part of the nitrifier community in the coniferous forest floor. Very little is known about the distribution and activity of autotrophs in these systems, although it is certain that in vitro evidence suggesting autotrophs cannot nitrify at pH levels characteristic of coniferous forest soils is unrealistic.
Because of the metabolic diversity of nitrifying fungi, a variety of organic and inorganic nitrification pathways may exist in coniferous forests. The possible involvement of free radicles in fungal nitrification in coniferous forest soils is also suggested.
A complete understanding of nitrification in coniferous forest soils can only result from field characterisation of N flux such as through the use of 15N. This must be combined with ecophysiological characterisation of the organisms involved in order that the complexity of nitrification in coniferous forest soils can be resolved.
A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.
N transformation rates in soil from a riparian wetland that receives runoff from adjacent pastoral land were investigated in a short-term (250min), anaerobic laboratory incubation (20C). A joint 15N tracing-isotope dilution technique was employed that used paired incubations of labelled (99atom % 15N) NO3–-unlabelledNH4+ and unlabelled NO3–-labelled (99 atom % 15N) NH4+ at three N input levels (0.4, 4 and 24g N g–1 soil). At each N input level NO3– and NH4+ were added in equal proportions (0.2, 2 and 12g N g–1 soil). Soil and gas samples were analysed after 10, 70 and 250min, and the fate of 15N and N transformation rates were determined for each time period; 0–10min (phase 1), 10–70min (phase 2) and 70–250min (phase 3). N transformation rates for all processes except gross NH4+ mineralisation were very high during phase 1. Processes favoured by aerobic conditions, NO3– immobilisation (0–17% NO3– removal, 0–8.2g N g–1 soil h–1), autotrophic nitrification (~2% NH4+ removal, 0.58–0.88g N g–1 soil h–1) and heterotrophic nitrification (11–35g N g–1 soil h–1) increased with increased N input while the anaerobic dissimilatory NO3– reduction to NH4+ process (1–6% NO3– removal, 0.48–0.62g N g–1 soil h–1) decreased, presumably due to the oxidising effect of higher NO3– inputs. Denitrification (8–78% NO3– removal, 3.8–9.6g N g–1 soil h–1) exhibited no clear trend related to N input levels. NH4+ immobilisation (39–72% NH4+ removal, 15–19g N g–1 soil h–1) was higher than NO3– immobilisation. Gross NH4+mineralisation (0.27–0.80g N g–1 soil h–1) was the only process not detected in phase 1 and one of few processes measurable in phases 2 or 3.
N-SERVE, 2-chloro, 6-(trichloromethyl)pyridine, shown to be a potent inhibitor of the ammonia oxidase system ofNitrosomonas was not effective at all in theNitrobacter nitrite oxidizing system at concentrations which had caused complete inhibition of ammonia oxidation, i.e. 1 – 1.5 ppm of the chemical. Very little inhibition of nitrite oxidation by intact cells was observed even in the presence of 50 ppm N-SERVE. Concentrations of 80 to 175 ppm, however, inhibited the nitrite oxidation by 60 to 75 percent. The cell-free nitrite oxidizing system of the chemoautotroph was found to be virtually unaffected by these unusually high concentrations of the inhibitor and 175 ppm concentration caused only a 10 percent inhibition. As in theNitrosomonas systems, N-SERVE proved to be inhibitory for the cytochrome oxidase present in the cell-free extracts ofNitrobacter but, again, levels from 80 – 175 ppm of the inhibitor were required for a 50 percent inhibition of the enzyme. The nitrite activating enzyme “nitrite-cytochromec reductase” was scarcely affected by these levels of N-SERVE. As a matter of fact, a slight stimulation of enzyme activity was observed. Copper ions, as in the case ofNitrosomonas, effectively reversed the N-SERVE inhibition of theNitrobacter cytochrome oxidase system.
Alcaligenes faecalis suppressed the growth of 11 strains of fungal plant pathogens in vitro. When it was cultivated on a synthetic medium containing (NH4)2 SO4 as the sole nitrogen source, NH2OH, NO2- and NO3- were produced, indicating that heterotrophic nitrification was occurring. The suppressive effect of A. faecalis on plant pathogens was due to its NH2OH produced. Rapid Science Ltd. 1998
Floristic species composition and differences in litter quality between species are the primary factors controlling N mineralization in forest ecosystems. Generalizations about species effects on N cycling are based on measurements of net rates of mineralization and nitrification. However, there have been few tests on the ability of these measurements to reflect the mechanistic complexity underlying the species effects. The objectives of this study are to: (1) determine whether differences in net mineralization and net nitrification rates between stands of different species composition are due to differences in gross rates of mineralization, nitrification, and microbial consumption; (2) determine whether field and laboratory assays of net mineralization and nitrification are useful indicators of internal N dynamics; and (3) test the hypothesis that microbial immobilization increases with rates of mineralization and nitrification. We measured net rates of mineralization and nitrification in the field and in the laboratory, and gross rates of mineralization, nitrification and microbial consumption in different stands at two sites in eastern New York State. The results indicated that vegetation type was not always a robust indicator of N cycling differences between ecosystems. At one site there was no difference in net mineralization (P<0.05) between oak and maple stands, and no nitrification in either forest type. We attributed this lack of conformity to expected patterns to either differences in soil moisture regimes resulting from landscape position, forest floor disturbance by earthworms, or influences of previous land-use. At the second site, both beech and maple stands showed significantly greater rates of net nitrification than oak stands (P<0.05) and beech had significantly greater (P<0.05) rates of net mineralization than both maple and oak. Gross rates of mineralization, nitrification and microbial consumption were very high and often exceeded net rates by an order of magnitude. Gross rates were not good indicators of differences between forest types and in most cases we did not find differences in gross rates between stands where we found differences in net rates. We found a strong relationship between microbial consumption of NH4+ or NO3− and gross rates of mineralization or nitrification (R2=0.83 and R2=0.52, respectively).
The relationship between pH and nitrification in different layers of a nitrogen-saturated acid Douglas fir forest soil was studied. Nitrification potentials (on basis of dry weight) of overground degrading needles, litter and fermentation layers were much higher than those of the humus and upper mineral layers. In all layers, nitrate production was probably due to chemolithotrophic bacteria as it was inhibited by acetylene. The litter layer contained relatively high numbers (105 g dry soil−1) of acid-sensitive ammonium-oxidizing bacteria, whereas the numbers in the other layers were just above or lower than the detection limit (103 g dry soil−1) of the MPN method used. Measurements of nitrate production in soil suspensions indicated that a pH increase (pH 6 vs pH 4) stimulated ammonium oxidation in the litter layer but not or to a lesser extent in the fermentation and humus layers. It is argued that both acid-sensitive and acid-tolerant ammonium-oxidizing bacteria are contributing to nitrification in the litter layer. In the fermentation and humus layers acid-tolerant or even acidophilic bacteria are thought to be responsible for ammonium oxidation.
Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) co-exist in soil, but their relative distribution may vary depending on the environmental conditions. Effects of changes in soil organic matter and nutrient content on the AOB and AOA are poorly understood. Our aim was to compare effects of long-term soil organic matter depletion and amendments with labile (straw) and more recalcitrant (peat) organic matter, with and without easily plant-available nitrogen, on the activities, abundances and community structures of AOB and AOA. Soil was sampled from a long-term field site in Sweden that was established in 1956. The potential ammonia oxidation rates, the AOB and AOA amoA gene abundances and the community structures of both groups based on T-RFLP of amoA genes were determined. Straw amendment during 50 years had not altered any of the measured soil parameters, while the addition of peat resulted in a significant increase of soil organic carbon as well as a decrease in pH. Nitrogen fertilization alone resulted in a small decrease in soil pH, organic carbon and total nitrogen, but an increase in primary production. Type and amount of organic matter had an impact on the AOB and AOA community structures and the AOA abundance. Our findings confirmed that AOA are abundant in soil, but showed that under certain conditions the AOB dominate, suggesting niche differentiation between the two groups at the field site. The large differences in potential rates between treatments correlated to the AOA community size, indicating that they were functionally more important in the nitrification process than the AOB. The AOA abundance was positively related to addition of labile organic carbon, which supports the idea that AOA could have alternative growth strategies using organic carbon. The AOB community size varied little in contrast to that of the AOA. This indicates that the bacterial ammonia oxidizers as a group have a greater ecophysiological diversity and potentially cover a broader range of habitats.
Bacteria and fungi were isolated from an acid forest soil in which nitrification occurred via a heterotrophic pathway. Enrichment of soil in a liquid inorganic salts medium, containing β-alanine as the sole source of C and N, led to formation of NO2− and NO3−. In pure culture, β-alanine was not a suitable substrate for nitrification by any of the isolates, but did support nitrification when one bacterium (BD) was co-cultured with a fungus. This bacterium was gram-positive and rod-shaped and identified provisionally as an Arthrobacter sp. The bacterium BD was able to nitrify ammonium acetate and, to a lesser extent peptone, in pure culture. Nitrification of ammonium acetate was greater in sterile soil solution than in defined media of inorganic salts. From a less reduced form of N, α-ketoglutaric oxime supported the highest rates of nitrification, but nitrification of pyruvic and ketobutyric oximes was at levels lower than from reduced N. The organism was able to survive and nitrify at pH 3, and despite its isolation from a very acidic environment, at pH≤10. It is proposed that a metabolic product of β-alanine produced by a non-nitrifying microorganism provided a suitable substrate for the nitrifying bacterium.
We studied controls on nitrification in an undisturbed water-limited ecosystem by inhibiting autotrophic nitrifying bacteria in soils with varying levels of vegetative cover. The activity of nitrifying bacteria was disrupted using nitrapyrin, 2-chloro-6-(trichloromethyl)-pyridine, under field conditions in three microenvironments (underneath shrubs, next to grasses and in bare soil). Ammonia-oxidising bacteria were detected by PCR analysis of DNA in soils. The inhibition of nitrification changed the concentrations of NO3− and NH4+ in the soil, while the microenvironment was most important in determining the response of bacteria to the inhibitor. Nitrapyrin application resulted in a significant (p<0.05) reduction in soil NO3− concentration (39%) and a significant increase (p<0.001) in soil NH4+ concentration (41%). Untreated bare-soil microenvironments had the lowest concentrations of NH4+ (1.57 μg/g of dry soil) and NO3− (0.49 μg/g of dry soil) when compared to the other microenvironments, and showed the highest impacts of nitrification inhibition. For example, NH4+ concentrations increased 288% and NO3− concentrations decreased 60% in inhibited bare-soil microenvironments. In contrast, untreated microenvironments underneath shrubs had the highest levels of NH4+ (10.01 μg/g of dry soil) and NO3− (0.69 μg/g of dry soil), but showed no significant effects of inhibition of nitrification on soil nitrogen concentrations.
The techniques of 15N pool dilution and enrichment were used to separate autotrophic and heterotrophic nitrification in an acid woodland soil. Results from laboratory incubations indicated that a maximum of 8% of the observed nitrification could be the result of heterotrophic nitrifiers oxidizing organic nitrogen to nitrate without passing through the exchangeable soil ammonium pool. Autotrophic nitrification rates ranged from 0.96 to 2.64 μg N g−1 d−1, depending on the time of the year the soil was sampled. Heterotrophic nitrification rates ranged from 0.08 to 0.12 μg N g−1 d−1 and appeared unaffected by the nitrification inhibitor N-Serve.
Nitrification is a central component of the global nitrogen cycle. Ammonia oxidation, the first step of nitrification, is performed in terrestrial ecosystems by both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Published studies indicate that soil pH may be a critical factor controlling the relative abundances of AOA and AOB communities. In order to determine the relative contributions of AOA and AOB to ammonia oxidation in two agricultural acidic Scottish soils (pH 4.5 and 6), the influence of acetylene (a nitrification inhibitor) was investigated during incubation of soil microcosms at 20 °C for 1 month. High rates of nitrification were observed in both soils in the absence of acetylene. Quantification of respective amoA genes (a key functional gene for ammonia oxidizers) demonstrated significant growth of AOA, but not AOB. A significant positive relationship was found between nitrification rate and AOA, but not AOB growth. AOA growth was inhibited in the acetylene-containing microcosms. Moreover, AOA transcriptional activity decreased significantly in the acetylene-containing microcosms compared with the control, whereas no difference was observed for the AOB transcriptional activity. Consequently, growth and activity of only archaeal but not bacterial ammonia oxidizer communities strongly suggest that AOA, but not AOB, control nitrification in these two acidic soils.
Mixed cultures of a heterotrophic nitrifier/aerobic denitrifier, Thiosphaera pantotropha, and an autotrophic nitrifier, Nitrosomonas europaea, were grown in chemostats under dual ammonia-and acetate limitation. Because of simultaneous nitrification and denitrification by T. pantotropha, the activity of the cultures was evaluated from nitrogen balances as complete as possible. Under most conditions studied, no interaction took place between the two bacteria. Only above a critical C/N ratio of 10.4, T. pantotropha was able to outcompete N. europaea for ammonia (dilution rate = 0.04 h⁻¹). At dissolved oxygen concentrations below 10 μM, the autotroph became oxygen-limited and the heterotroph dominated in the culture. Moreover, when the dilution rate was increased to 0.065 h⁻¹, N. europaea could not maintain itself successfully in the chemostat, even when the C/N ratio was as low as 2.2. Nitrification by T. pantotropha was equivalent to that of N. europaea when the cell ratio of heterotrophs/autotrophs was 250. The relevance of these observations to the nitrogen cycle in natural environments is discussed.
Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 degrees C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.
For more than 100 years it was believed that bacteria were the only group responsible for the oxidation of ammonia. However, recently, a new strain of archaea bearing a putative ammonia monooxygenase subunit A (amoA) gene and able to oxidize ammonia was isolated from a marine aquarium tank. Ammonia-oxidizing archaea (AOA) were subsequently discovered in many ecosystems of varied characteristics and even found as the predominant causal organisms in some environments. Here, we summarize the current knowledge on the environmental conditions related to the presence of AOA and discuss the possible site-related properties. Considering these data, we deduct the possible niches of AOA based on pH, sulfide and phosphate levels. It is proposed that the AOA might be important actors within the nitrogen cycle in low-nutrient, low-pH, and sulfide-containing environments.
Agricultural ecosystems annually receive approximately 25% of the global nitrogen input, much of which is oxidized at least once by ammonia-oxidizing prokaryotes to complete the nitrogen cycle. Recent discoveries have expanded the known ammonia-oxidizing prokaryotes from the domain Bacteria to Archaea. However, in the complex soil environment it remains unclear whether ammonia oxidation is exclusively or predominantly linked to Archaea as implied by their exceptionally high abundance. Here we show that Bacteria rather than Archaea functionally dominate ammonia oxidation in an agricultural soil, despite the fact that archaeal versus bacterial amoA genes are numerically more dominant. In soil microcosms, in which ammonia oxidation was stimulated by ammonium and inhibited by acetylene, activity change was paralleled by abundance change of bacterial but not of archaeal amoA gene copy numbers. Molecular fingerprinting of amoA genes also coupled ammonia oxidation activity with bacterial but not archaeal amoA gene patterns. DNA-stable isotope probing demonstrated CO(2) assimilation by Bacteria rather than Archaea. Our results indicate that Archaea were not important for ammonia oxidation in the agricultural soil tested.
Traditionally, organisms responsible for major biogeochemical cycling processes have been determined by physiological characterization of environmental isolates in laboratory culture. Molecular techniques have, however, confirmed the widespread occurrence of abundant bacterial and archaeal groups with no cultivated representative, making it difficult to determine their ecosystem function. Until recently, ammonia oxidation, the first step in the globally important process of nitrification, was thought to be performed almost exclusively by bacteria. Metagenome studies, followed by laboratory isolation, then demonstrated the potential for significant ammonia oxidation by mesophilic crenarchaea, whose ecosystem function was previously unknown. Re-assessment of the role of bacteria in ammonia oxidation is now required and this article reviews the current evidence for the relative importance of bacteria and archaea. Much of this evidence is based on metagenomic analysis and molecular techniques for estimation of gene and gene transcript abundance, changes in ammonia oxidizer community structure during active nitrification and phylogeny of natural communities. These studies have been complemented by physiological characterization of a laboratory isolate and by incorporation of labelled substrates. Data from these studies provide increasingly convincing evidence for the importance of archaeal ammonia oxidizers in the global nitrogen cycle. They also highlight the need to re-assess the importance of ammonia-oxidizing bacteria, the requirement and limitations of molecular techniques in linking specific microbial groups to ecosystem function and the limitations of reliance on laboratory cultures.
An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea. Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin. When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity. The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O2 uptake and NO2- production rates. The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase. These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways. We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants.
N-SERVE, 2-chloro, 6-(trichloromethyl)pyridine, shown to be a potent inhibitor of the ammonia oxidase system of Nitrosomonas was not effective at all in the Nitrobacter nitrite oxidizing system at concentrations which had caused complete inhibition of ammonia oxidation, i.e. 1 - 1.5 ppm of the chemical. Very little inhibition of nitrite oxidation by intact cells was observed even in the presence of 50 ppm N-SERVE. Concentrations of 80 to 175 ppm, however, inhibited the nitrite oxidation by 60 to 75 percent. The cell-free nitrite oxidizing system of the chemoautotroph was found to be virtually unaffected by these unusually high concentrations of the inhibitor and 175 ppm concentration caused only a 10 percent inhibition. As in the Nitrosomonas systems, N-SERVE proved to be inhibitory for the cytochrome oxidase present in the cell-free extracts of Nitrobacter but, again, levels from 80 - 175 ppm of the inhibitor were required for a 50 percent inhibition of the enzyme. The nitrite activating enzyme "nitrite-cytochrome c reductase" was scarcely affected by these levels of N-SERVE. As a matter of fact, a slight stimulation of enzyme activity was observed. Copper ions, as in the case of Nitrosomonas, effectively reversed the N-SERVE inhibition of the Nitrobacter cytochrome oxidase system.
High levels of wet N and acidic deposition were measured in southeast Brazil. In this study we addressed the sensitivity of water bodies and soils to acidification and N deposition in the Piracicaba River basin (12,400 km2). Average acid neutralization capacity (ANC) at 23 river sampling sites varied from 350 to 1800 microeq l(-1). Therefore, rivers and streams in the Piracicaba basin are well buffered, if the lower limit of 200 microeq l(-1) is assumed as an indication of poorly buffered waters. ANC is increased by untreated wastewaters discarded into rivers and streams of the region. Average NO3 concentrations varied from 20 to 70 microeq l(-1). At the most polluted river sites, NO3 concentration is not highest, however, probably due to NO3 reduction and denitrification. Most of the nitrogen in streams is also provided by wastewaters and not by wet deposition. The majority of the soils in the basin, however, are acidic with a low base cation content and high aluminum concentration. Therefore, soils in this basin are poorly buffered and, in areas of forest over sandy soils, acidification may be a problem.