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Vertical characteristics of anaerobic oxidation of ammonium (anammox) in a coastal saline-alkali field
Soil & Tillage Research 198 (2020) 104531
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Vertical characteristics of anaerobic oxidation of ammonium (anammox) in a coastal saline-alkali field
T
Maomao Houa,1, Ru Xua,b,1, Zhiyuan Lina, Dan Xid, Yi Wangd, Junling Wena, San’an Niec,*, Fenglin Zhonga,* a
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China Engineering Research Center of Fujian University of Modern Facilities Agriculture, Fuqing, 350300, China c Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China d Forestry College, Fujian Agriculture and Forestry University, Fuzhou, 350002, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anammox N loss Saline-alkali soil Candidatus Scalindua
Anammox contributes significant nitrogen (N) loss in agricultural ecosystems, yet its role in the saline-alkali field is not well predicted. Here, we investigate the activity, functional gene abundance and community composition of anammox bacteria in a coastal saline-alkali soil along depth gradient with isotope tracing technique, quantitative PCR assay, and high-throughput sequencing. The anammox activities ranged from 0.09 to 1.32 nmol N g−1 h−1 but were undetectable below 70 cm. The anammox bacteria were present through the soil with the abundance varying from 2.5 × 104 to 5.3 × 105 copies g−1 soil. The Illumina sequencing targeting anammox 16S rRNA gene identified three anammox genera with Candidatus Scalindua dominating. Anammox contribute 40–87.5% of total N2 at 0−50 cm depth whereas the percentage declined sharply below 50 cm. We conclude that N loss in upper soil is mainly linked to anammox while dominated by denitrification in the deeper coastal salinealkali field.
1. Introduction
(www.fao.org/publications/sofa) thus leads to a massive N loss through ammonia volatilization, N2O emission, runoff and leaching (Nie et al., 2018; Xing and Zhu, 2000). Thus, understanding the process of N loss in the agroecosystem is critical for reducing N loss through agricultural production and horticultural management. It was reported that up to 67% of N2 production was linked to anammox in marine sediments (Dalsgaard et al., 2005). Recent studies suggest that anammox can be an important N loss pathway in agricultural ecosystems (Nie et al., 2015, 2019), which contributed to 5–10 % to soil N2 production (Nie et al., 2019; Yang et al., 2015; Zhu et al., 2015). It is of extremely significance to improve and explore land resources for a growing population and limited agricultural production. Saline-alkali soil may play a crucial role with large-scale and -area of distribution (Cong et al., 2014). The effective way to reclaim and utilize the saline-alkali field may provide much needed information. Anammox reaction occurs in agricultural field (Nie et al., 2019; Shen et al., 2013; Yang et al., 2015), marine ecosystems (Dalsgaard et al., 2012; Kuypers et al., 2005), and land-freshwater interface (Zhu et al., 2013). In this study, a soil developed from alluvial deposits with parent material of both coastal and inland was selected. Based on the previous
Our understanding of microbial N cycling has advanced substantially over the last decades (Kuypers et al., 2018; Thamdrup, 2012). The new processes responsible for N loss involve in ammonium oxidation coupled with nitrite (anammox), sulfate (Sulfammox), ferric iron (Feammox), and natural organic matter (NOM-anammox) under anaerobic condition (Rios-Del Toro and Cervantes, 2019). The discovery of anammox upended our perception of the N loss pathway that has long been considered produced solely by heterotrophic denitrification (Mulder et al., 1995; Vitousek and Howarth, 1991). The anammox process was mediated by bacteria affiliated to the phylum Planctomycetes (Jetten et al., 2010). Anammox bacteria inhabit different natural systems, including marine (Lam and Kuypers, 2011), freshwater (Xue et al., 2017), wetland (Wang et al., 2018), and agricultural soil (Nie et al., 2019). Excess N applied in agricultural ecosystems caused an escalating global problem from both environmental and economic aspects (Guo et al., 2010; Liu et al., 2019; Stark and Richards, 2008; Wei et al., 2017). China consumed the largest amount of chemical N fertilizer
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Corresponding authors. E-mail addresses: [email protected] (S. Nie), [email protected] (F. Zhong). 1 These authors contributed equally to this article. https://doi.org/10.1016/j.still.2019.104531 Received 15 May 2019; Received in revised form 5 November 2019; Accepted 5 December 2019 0167-1987/ © 2019 Elsevier B.V. All rights reserved.
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with 1 ml deionized water (dd H2O). The vials were N2-purged and preincubated for 24 h to deplete NOx− and O2. After pre-incubation, all vials were flushed with N2 once again for further incubation. Three isotope treatments, i.e., (1) negative control (15NH4Cl at 99.09%); (2) positive control (15NH4NO3 at 99.09%); and (3) Na15NO3 at 99.21%, respectively, were performed to estimate the activity of anammox and denitrification. Each vial contained 500 μg of 15N and incubated in a constant-temperature incubator (Queue Systems, WV, USA) at 25 °C. The slurries treated with 15NH4Cl and 15NH4NO3 were solely incubated with 0 h and 24 h, respectively. With the soil treated with Na15NO3, at the time point of 0, 3, 6, 12 and 24 h, 500 μl ZnCl2 (7 M) was added in the vials chronologically to stop the reactions. Before N2 determination, the vials were shaken strongly. The rates of anammox and denitrification were calculated by the content of 29N2 and 30N2, which were determined by a multiflow isotope ratio mass spectrometer system (IRMS) (Isoprime Ltd., Cheadle Hulme, UK). The fractions of 29N2 and 30N2 in soil treated with Na15NO3 were calculated using Eqs. (1) and (2). The mass of headspace N2 (Mtotal) was calculated using Eq (3). The production rates of 29N2 and 30N2 were calculated using Eqs. (4) and (5):
study (Nie et al., 2019; Zhu et al., 2013, 2015), we hypothesis that anammox is detectable in the coastal saline-alkali soil. Currently, the activity, abundance, and role of anammox in the saline-alkali field are poorly characterized. Therefore, the aims of this study were to investigate the activity, contribution to N loss, functional gene abundance and community compositions of anammox bacteria in a coastal saline-alkali field in Eastern China. To achieve these, the 15N tracing technology and quantitative PCR assays were conducted to estimate the rate and abundance of anammox bacteria, respectively. High-throughput sequencing targeting anammox 16S rRNA gene was performed to confirm the community compositions. 2. Materials and methods 2.1. Site description and sampling The study was conducted at Haihan Horticultural Farms (121°09′07″ E, 30°21′15″N) in Cixi, Ningbo, Eastern China. The site was approximately 0.5 km from the East China Sea. The altitude was 6 m above sea level. The climate is humid subtropical, and the mean annual air temperature is 17 °C. The field had been cultivated for three years of rice then planted with Brassica parachinensis. During the overwinter season of pakchoi, the arch shed is used for cold prevention (Fig. S1). The soil was a saline-alkali soil developed from alluvial deposits. The field was only irrigated freshwater occasionally and no fertilizer was applied. In this study, three intact soil cores (5 × 100 cm, diameter × hight) were sampled from the field in August 2018. Then the soil cores were sliced at 10 cm intervals and immediately divided into several parts. A small part was frozen in liquid N2 and stored at −80 °C for molecular analysis; approximately 3.5 g fresh soil was incubated to analysis anammox and denitrification activities; about 5 g soil was incubated for the analysis of nitrification rate; the other part was air dried or stored at 4 °C for determining of physical-chemical analysis.
F 29 =
F 30 =
29N2 = N2total
29N2 28N2 29N2 30N2 1+ + 28N2 28N2
=
30N2 = N2total
30N2 28N2 29N2 30N2 1+ + 28N2 28N2
=
R29 1 + R29 + R30
(1)
R30 1 + R29 + R30
(2)
Mtotal = ρ N2 × Vheadspace
(3)
P29 = (M total × F 29)/(t × Msoil)
(4)
P30 = (M total ×
F30)/(t
× Msoil)
(5)
Dtotal = P30 × FN−2
Atotal = Ra % =
Dtotal × 100% Dtotal + Atotal
2.2. Physicochemical procedures Soil moisture was measured using a MA100 moisture meter (Sartorius Company, Göttingen, Germany). Soil pH was measured using a pH-meter (INESA, Shanghai, China) with a 1:2.5 soil/H2O weight ratio. Soil total C/N was measured using a total C/N analyzer (LECO Corporation, MI, USA). The NH4+ and NO3− were extracted from a 1:5 soil/2 M KCl mixture and quantified by a continuous flow injection analyzer (SYSTEA S.p.A., FR, Italy). Salinity (determined as EC) was analyzed by a Conductivity Meter (INESA, Shanghai, China) in a 1:5 (soil/H2O) suspension.
(6)
FN−1
29
× [P29 + 2 × (1 −
× P30]
(7) (8)
Where F and F denote the fraction of N2 and N2, R and R30 represent the ratio of 29N2 to 28N2 and 30N2 to 28N2, Mtotal is the mass of headspace N2, ρN2 is the density of N2, Vheadspace is the volume of headspace, t is the incubation time and Msoil represents the soil mass in vials, P29 and P30 are the measured production of 29N2 and 30N2 from each vial in the 15NO3− treatment, respectively. The calculation of anammox and denitrification activity was processed according to Dalsgaard and Thamdrup (2002) and Xi et al. (2016). Denitrification and anammox rates were calculated by Eqs. (6) and (7), respectively. The contribution of anammox to total N2 production was assessed by Eq. (8). In slurries treated with negative control, no significant accumulation of 29N2 or 30N2 was detected, suggesting that initial NOx− had been depleted during the pre-incubations. In positive control treatment, 29N2 could be observed while 30N2 was undetectable. The slurries treated with Na15NO3 indicated that anammox and denitrification processes were detectable in the examined soils. Based on each time point, a linear regression of different N2 concentrations was performed to estimate the potential rates of anammox and denitrification.
2.3. Measuring potential nitrification rate The potential nitrification rate was measured as depicted by Kandeler (2002) with some modifications. Fresh soil (3.5 g) was incubated with 20 ml of phosphate-buff ;ered saline (g L−1: NaCl, 8.0; KCl, 0.2; Na2HPO4, 0.2; NaH2PO4, 0.2) and (NH4)2SO4 (final concentration: 1 mM) in a plastic tubes for 24 h. KClO3 (10 mg L−1) was added to the tubes for inhibition of NO2− oxidation. The NO2- was extracted by adding 5 ml of 2 M KCl after incubation prior to centrifugation. The NO2−-N was quantified spectrophotometrically at 545 nm (BioTek Instruments, Inc. VT, USA).
30
FN−1)
29
30
29
2.5. DNA extraction, PCR amplification, and Illumina sequencing 2.4. Measuring anammox and denitrification rate Soil DNA was extracted from 0.5 g (fresh weight, three replicates) samples with the FASTDNA ®SPIN Kit (MoBio Laboratories, CA, USA) for soil according to manufacturer’s protocols. The final DNA concentration and further purification were tested by NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, WI, USA). The DNA quality was examined by 1% agarose gel electrophoresis.
A slurry incubation experiment with different 15N-isotope substrates was conducted to determine anammox and denitrification rates according to Long et al. (2013) and revised by Xi et al. (2016). Briefly, 3.5 g soil (fresh weight, three replicates) were transferred to a volume of 20 ml glass vials (Exetainer, Buckinghamshire, UK) and saturated 2
Soil & Tillage Research 198 (2020) 104531
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The anammox bacterial 16S rRNA gene was amplified with Amx368 F and Amx820R (Schmid et al., 2000) by a GeneAmp 9700 PCR system (ABI, CA, USA). The thermal profile started at 96 °C for 10 min, followed by 35 cycles of 30 s at 96 °C, 1 min at 58 °C, and 1 min at 72 °C. The resulted PCR products were extracted from a 2% agarose gel, purified by the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) and quantified with QuantiFluor™-ST (Promega, WI, USA). Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq platform (Illumina, CA, USA) in accordance with the standardized commercial service. (Majorbio BioPharm Technology Co. Ltd., Shanghai, China). 2.6. Quantitative PCR of hzsB gene Real-time q-PCR was carried out to target the anammox functional gene hzsB with the primer set hzsB_396 F and hzsB_742R (Wang et al., 2012b). Thermal cycling and data operation were conducted using an ABI 7500 system (ABI, CA, USA). The q-PCR reaction was carried out with the following mixture: 9 μL Master Mix (APB, Freiburg, Germany), 1 μL each primer, 1 μL DNA, and 8 μL dd H2O. The standard curve was performed with ten-fold serial dilutions of plasmid DNA with the hzsB gene. Three replicates were carried out for each quantitative assay. The thermal profile was performed with the program: 95 °C for 1 min, followed by 35 cycles of 95 °C for 5 s, 60 °C for 30 s, and 75 °C for 1 min. The amplification efficiencies varied from 90% to 110% and correlation coefficients for linear regression greater than 0.99 were acceptable.
Fig. 2. Vertical distribution of the abundance of anammox bacteria targeting the hzsB gene. Error bars indicate S.E. (n = 3).
539.5–1812.5 μs cm−1. The highest total carbon and N content in the soil was found at the top (0−10 cm) layer and decreased with slightly change from 1.32% to 1.55% and 0.2 to 0.7 g kg−1, respectively. The ferrous in soil cores showed a gradient feature with low concentrations at 0−50 cm (0.02–0.34 cmol kg−1) and high concentrations (1.07–1.88 cmol kg−1) blow 50 cm.
2.7. Statistical analysis 3.2. Abundance of anammox bacteria The initial data were conducted by Microsoft Excel 2016 software and further conducted by SPSS 22.0 for the statistical analysis. The data below the detection limit were recorded as not determined (ND). The figures were completed by Sigmaplot 14.0 software.
To investigate the presence and abundance of anammox bacteria in the soil cores, the hzsB gene was assessed by quantitative PCR assay and the vertical distribution was shown in Fig. 2. The hzsB gene abundance varied greatly and valued from 2.5 × 104 to 5.3 × 105 copies g−1 dry weight soil. The highest abundance of anammox cells in the examined soil was detected at 60−70 cm, while low copy numbers of hzsB gene were recorded at the surface (0−10 cm) and deep layers (80–100 cm). Pearson correlation analysis showed that the hzsB gene copy numbers were not significantly linked with soil properties selected in the present study (p < 0.05) (Table S1).
3. Results 3.1. Soil properties after five years of cultivation The soil physicochemical characteristics along depth gradients at 10 cm intervals are shown in Fig. 1. The soil was slightly alkaline and the pH varied from 8.0-8.55. The soil moisture increased with the depth and ranged from 0.22 to 0.27. The exchangeable NH4+-N was lowest at the top depth and then increased with depth to a concentration of 17.3 mg kg−1. The highest NOx−-N (nitrate and nitrite) concentration was observed at the top 0−10 cm layer and decreased gradually with soil depth. The soil salinity was increased with depth gradients from
3.3. Rates of anammox, denitrification, and nitrification An isotope-tracing experiment was conducted to determine the potential role of anammox as a N2 producer. The potential anammox rates can overestimate the actual in situ rates because some other
Fig. 1. vertical distribution of soil pH (a), moisture (b), electrical conductivity (c), total carbon (d), total nitrogen (e), exchangeable ammonium (f), nitrite + nitrate nitrogen (g) and ferrous in saline-alkali filed soil cores. 3
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Fig. 3. Vertical distribution of anammox rate and ratio of anammox contribution to total N2 production (a) as well as nitrification and denitrification activity (b) at each depth. Error bars indicate S.E. and analyzed with three replicates per site.
processes, like Feammox, can also generate N2 (Ding et al., 2014; RiosDel Toro and Cervantes, 2019). However, in the incubation experiments, no significant accumulation of 30N2 could be observed in the slurries amended with 15NH4+ and 15NH4+ + 14NO3− (Fig. S2). Besides, in 0−70 cm soil layers, where anammox could be detected (Fig. 3a), the in situ concentration of NH4+ was low (Fig. 1f), which may not supply sufficient substrate for Feammox. Hence, the detected anammox rates might not be seriously overestimated. The results showed that active anammox was detected in the soil horizon at 0−70 cm whereas no significant anammox activity could be determined below 70 cm (Fig. 3a). The anammox rates in 0−40 cm soil cores were 0.28–0.94 nmol N g−1 h−1 and increased to the highest value of 1.32 nmol N g−1 h−1 at the 40−50 cm layer. Anammox rates decreased drastically at 50−70 cm (both 0.09 nmol N g−1 h−1). These results indicated that anammox was more likely to occur in the upper layer as well as have a high rate at the surface (0−20 cm) and specific depth (40−50 cm). The contribution to N production via anammox at 0−50 cm depth was up to 40–87.5%, and this percentage is declined drastically to lower than 0.8% below 50 cm, the other attributing to denitrification. Both nitrification and denitrification rates were detected to evaluate the nitrite, which is the substrate for anammox (Fig. 3b). Overall, both nitrification and denitrification rates showed a heterogeneous depth effect. In the soil horizon at 0−50 cm, the denitrification rate was low (0.11 to 0.42 nmol N g−1 h−1) but they increased drastically below 50 cm (5.1–15.5 nmol N g−1 h−1). The nitrification rates of soil cores were high at the surface layer but decreased gradually along with soil depth and the values were very low (0.03–0.36 nmol N g−1 h−1) below 40 cm. These results indicated that nitrification occurred mainly in upper soil, while denitrification dominated in lower soil.
Fig. 4. Vertical distribution of the community compositions and their relative abundance of anammox bacteria in core samples of the saline-alkali field. Table 1 Diversity characteristics of anammox 16S rRNA gene in each depth.
3.4. Community composition and biodiversity of anammox bacteria A total of 4434 effective sequences were obtained from ten layers of soil cores. All sequences were mostly affiliated to the genera of Candidatus Scalindua, Candidatus Brocadia, Candidatus Kuenenia, or unclassified (Fig. 4). Overall, Candidatus Scalindua and Candidatus Kuenenia were present in all soil layers while Candidatus Brocadia could only be identified in particular depth (in the soil horizon at 20−40 cm and 70−100 cm, respectively). Only a small fraction (0.4–3.2%) of anammox bacterial 16S rRNA gene sequences were unidentified thus assigned as unclassified. Candidatus Scalindua was the highest relative abundant anammox bacterial genus (36–97%). The relative abundance of Candidatus Kuenenia showed a variation of percentage from 3% to 53%. Candidatus Brocadia which could only be detected in certain soil layers also showed different fractions (4–53%). The anammox bacterial community compositions had much heterogeneity.
Soil layers (cm)
No. of OTUs
Simpson index
Shannon index
0–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 80–90 90–100
7 3 7 9 7 6 6 7 6 6
0.82 0.93 0.52 0.42 0.49 0.85 0.48 0.35 0.39 0.41
0.36 0.17 0.86 0.97 0.73 0.34 0.77 1.13 1.05 0.85
The clustered OTUs number of anammox 16S rRNA gene in ten representative soil cores with 97% similarity cutoff as well as Simpson and Shannon diversity indices of anammox bacterial communities were shown in Table 1. A total of 10 OTUs were achieved and the OTUs number obtained from each sample varied from 3 to 9. In 0−10 cm, 10−20 cm and 50−60 cm of soil layers, which were dominated the genera of Candidatus Brocadia, the Simpson index accessed dominant
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rate was very low and undetectable below 70 cm (Fig. 3a). The main reason could be the low NOx− concentration (Fig. 1g). In deeper soil, where nitrification was inhibited, denitrifiers didn’t have enough NOx− to utilize for anammox. Consequently, denitrification can’t supply enough substrate for anammox. The anammox bacteria in saline-alkali soil possessed a heterogeneous distribution of activity (Fig. 3a). The functional gene abundance of anammox showed poor correlation with the activity (Table S1), which confirmed what already reported (Bai et al., 2015; Wang et al., 2012a) but is inconsistent with some previous research (Shen et al., 2015, 2017; Yang et al., 2015). The little relation between anammox cell numbers and activity indicates that anammox bacteria can have a different metabolic rate (Hu et al., 2011; Humbert et al., 2010). Although substantial numbers of anammox cell could be quantified below 50 cm, the anammox rates were very low or even undetectable, suggesting those anammox bacteria could be dormant cells thus little or no activity. High-throughput sequencing of anammox 16S rRNA gene indicated that at least three genera, “Scalindua”, “Brocadia” and “Kuenenia” were identified from saline-alkali soil (Fig. 4). Among these, “Scalindua” was the highest relative abundant anammox genus, which differed with other agricultural soils where “Brocadia” and “Kuenenia” usually are dominated genera (Shen et al., 2015, 2017; Wang and Gu, 2013; Zhu et al., 2011). In this study, the salinity (determined as EC) is very high (Fig. 1c). The anammox composition “Scalindua” is more likely to be found in high salinity environments than “Brocadia” and “Kuenenia” bacteria (Dale et al., 2009; Schmid et al., 2007). The presence of “Kuenenia” with a heterogeneous proportion along the ten-soil gradient whereas “Brocadia” could only be measured at particular depth indicates that this anammox bacteria may be better adapted than “Brocadia” bacteria in saline-alkali soil. Taken together, microbial anammox reaction in saline-alkali soil occurred mainly at the upper soil layer (0−50 cm) and showed a high level of contribution to N production (40–87.5%) in comparison to denitrification. The anammox bacteria are more active at the surface (0−20 cm) and specific layer (40−50 cm) and their activities were not correlated with the abundance. Future studies should be addressed on saline-alkali soil with the spatiotemporal variations of the anammox process.
OTUs showed higher values whereas the Shannon index estimated both richness and evenness were lower than that examined from other soil cores. The community diversity in saline-alkali soil differed with soil depth. 4. Discussion In this study, the anammox rates were 0.09–1.32 nmol N g−1 h−1 at 0−70 cm, and this values were comparable to those reported in a vertical vegetable field (0.09–1.43 nmol N g−1 h−1) (Shen et al., 2017) and a paddy soil (0.5–2.9 nmol N g−1 h−1) (Zhu et al., 2011). Based on the incubation results, it is evaluated that approximately 67 g N m-2 yr−1 loss could be attributed by anammox (Table S2). This estimation is higher than that reported in a vegetable field (29.5 g N m−2 yr−1) (Shen et al., 2017), suggesting that anammox could be an important potential source of N loss in saline-alkali soil. On the other side, the denitrification rates were very low (0.11-0.42 nmol N g−1 h−1) at 0−50 cm but increased drastically (5.15–11.76 nmol N g−1 h−1) below 50 cm (Fig. 3b). These results indicate that N loss in upper soil is mainly caused by anammox, while that in deeper soil largely attributes to denitrification. The observed cell abundance of anammox bacteria (2.5 × 104 to 5.3 × 105 copies g−1 soil) in vertical soil layers (Fig. 2) was lower than those found in the vegetable soil (Shen et al., 2015, 2017) and paddy soils (Shen et al., 2014; Wang et al., 2012b; Zhu et al., 2011). The main reason could be attributed to different agronomic management. In agricultural soils, a relatively high amount of N fertilizer may be applied in the field thus may stimulate the growth of anammox bacteria (Nie et al., 2019). However, no fertilizer was applied in the studied soil for five years. The variety of anammox bacterial abundance along the soil gradient has some similarity to paddy soil (Wang et al., 2012b). No significant linear correlation was found between anammox bacterial abundance and soil properties (Table S1). The low abundance of anammox bacteria in the surface saline-alkali soil (Fig. 2) mainly explained by the oxygen pervasion, which could inhibit anammox bacterial growth. A higher detection frequency of hzsB copy number in deeper saline-alkali soil (Fig. 2) indicates that anammox bacteria prefer to special niches, which is similar to the results found in water columns and sediments (Dalsgaard et al., 2003, 2005). The vertical distribution of anammox bacterial cell numbers in saline-alkali soil might involve with both horticulture conditions and physicochemical properties. The anammox rates along surface gradient depth (0−40 cm) were more or less decreased, which have some similarity to the nitrification rate (Fig. 3). In the upper soil, nitrification use NH4+ as substrate to generate NO2− for anammox bacteria. This could be also inferred from the low concentration of NH4+ (Fig. 1f) and high nitrate concentration (Fig. 1g) in soil cores. Other studies also indicate that partial nitrification rather than denitrification can be more likely linked to anammox as nitrification can provide enough nitrite for anammox bacteria (Lam et al., 2007; Zhu et al., 2011). Furthermore, some anammox bacteria can reduce NO3−, which is relatively high in the upper soil layer (Fig. 1g), to NH4+ for anammox (Kartal et al., 2007, 2008). A drastically increase of anammox rate was measured at 40−50 cm (Fig. 3a) indicating anammox bacterial is more active in particular depth. These results have some similarity to that found in the water-soil interaction layer where hotspots of anammox were observed (Zhu et al., 2013). One possible reason could be the high soil moisture and salinity (Fig. 1b and c), which are essential factors for governing anammox activity (Bai et al., 2015; Zhu et al., 2013). Besides, high concentration of ferrous ion (Fig. 1h), which is electron donors thus has a positive effect on anammox reaction (Huang et al., 2014; Strous et al., 2006; Van de Vossenberg et al., 2008; Wei et al., 2019), were detected at 40−50 cm soil depth. Finally, the upper soil layer is subjected to plant growth and water leaching, which may have a negative effect on anammox metabolism. The anammox activity at deeper depth is less susceptible to these factors in comparison with upper soil. The anammox
Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments The research is financially supported by the National Natural Science Foundation of China (4170010194, 41703068), the Postdoctoral Science Foundation of China (2017M622044, 2018M630723), National Science and Technology Major Project of Fujian (2018NZ0002-2), Open Funds by Engineering Research Center of Fujian University of Modern Facilities Agriculture (G2-KF1808), and NDRC Agricultural Five New Project of Fujian (K6017201A). Moreover, the author San’an Nie thanks Lixia Zhao and Xiumei Lei for their kind help. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.still.2019.104531. References Bai, R., Xi, D., He, J.-Z., Hu, H.-W., Fang, Y.-T., Zhang, L.-M., 2015. Activity, abundance and community structure of anammox bacteria along depth profiles in three different paddy soils. Soil Biol. Biochem. 91, 212–221.
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Xue, Y.Y., Yu, Z., Chen, H.H., Yang, J.R., Liu, M., Liu, L.M.A., Huang, B.Q., Yang, J., 2017. Cyanobacterial bloom significantly boosts hypolimnelic anammox bacterial abundance in a subtropical stratified reservoir. FEMS Microbiol. Ecol. 93. Yang, X.-R., Li, H., Nie, S.-A., Su, J.-Q., Weng, B.-S., Zhu, G.-B., Yao, H.-Y., Gilbert, J.A., Zhu, Y.-G., 2015. Potential Contribution of Anammox to Nitrogen Loss from Paddy Soils in Southern China. Appl. Environ. Microb. 81, 938–947. Zhu, G., Wang, S., Wang, W., Wang, Y., Zhou, L., Jiang, B., Op den Camp, H.J.M., Risgaard-Petersen, N., Schwark, L., Peng, Y., Hefting, M.M., Jetten, M.S.M., Yin, C., 2013. Hotspots of anaerobic ammonium oxidation at land-freshwater interfaces. Nat. Geosci. 6, 103–107. Zhu, G., Wang, S., Zhou, L., Wang, Y., Zhao, S., Xia, C., Wang, W., Zhou, R., Wang, C., Jetten, M.S.M., Hefting, M.M., Yin, C., Qu, J., 2015. 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Limnol. Oceanogr. 57, 1331–1346. Ding, L.J., An, X.L., Li, S., Zhang, G.L., Zhu, Y.G., 2014. Nitrogen loss through anaerobic ammonium oxidation coupled to Iron reduction from paddy soils in a chronosequence. Environ. Sci. Technol. 48, 10641–10647. Guo, J.H., Liu, X.J., Zhang, Y., Shen, J.L., Han, W.X., Zhang, W.F., Christie, P., Goulding, K.W.T., Vitousek, P.M., Zhang, F.S., 2010. Significant acidification in major chinese croplands. Science 327, 1008–1010. Hu, B.L., Shen, L.D., Xu, X.Y., Zheng, P., 2011. Anaerobic ammonium oxidation (anammox) in different natural ecosystems. Biochem. Soc. T 39, 1811–1816. Huang, X.L., Gao, D.W., Peng, S., Tao, Y., 2014. Effects of ferrous and manganese ions on anammox process in sequencing batch biofilm reactors. J. Environ. Sci. China 26, 1034–1039. Humbert, S., Tarnawski, S., Fromin, N., Mallet, M.P., Aragno, M., Zopfi, J., 2010. Molecular detection of anammox bacteria in terrestrial ecosystems: distribution and diversity. ISME J. 4, 450–454. Jetten, M., Camp, H., Kuenen, J., Strous, M., Krieg, N., Ludwig, W., Whitman, W., Hedlund, B., Paster, B., Staley, J., 2010. Description of the order brocadiales. In: Krieg, N.R., Staley, J.T., Hedlund, B.P., Paster, B.J., Ward, N., Ludwig, W., WB, W. (Eds.), Bergey’s Manual of Systematic Bacteriology. Springer, Heidelberg, pp. 596–603. Kandeler, D.E., 2002. Potential nitrification test. In: Schinner, F., Öhlinger, R., Kandeler, D.E., Margesin, R. (Eds.), Methods in Soil Biology. Springer, Berlin, Heidelberg. Kartal, B., Kuypers, M.M.M., Lavik, G., Schalk, J., den Camp, H.J.M.O., Jetten, M.S.M., Strous, M., 2007. Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environ. Microbiol. 9, 635–642. Kartal, B., van Niftrik, L., Rattray, J., van de Vossenberg, J.L.C.M., Schmid, M.C., Sinninghe Damsté, J., Jetten, M.S.M., Strous, M., 2008. Candidatus ‘Brocadia fulgida’: an autofluorescent anaerobic ammonium oxidizing bacterium. FEMS Microbiol. Ecol. 63, 46–55. Kuypers, M.M.M., Lavik, G., Woebken, D., Schmid, M., Fuchs, B.M., Amann, R., Jørgensen, B.B., Jetten, M.S.M., 2005. Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc. Natl. Acad. Sci. U. S. A. 102, 6478. Kuypers, M.M.M., Marchant, H.K., Kartal, B., 2018. The microbial nitrogen-cycling network. Nat. Rev. Microbiol. 16, 263–276. Lam, P., Jensen, M.M., Lavik, G., McGinnis, D.F., Muller, B., Schubert, C.J., Amann, R., Thamdrup, B., Kuypers, M.M.M., 2007. Linking crenarchaeal and bacterial nitrification to anammox in the Black Sea. Proc. Natl. Acad. Sci. U. S. A. 104, 7104–7109. Lam, P., Kuypers, M.M.M., 2011. Microbial nitrogen cycling processes in oxygen minimum zones. Annu. Rev. Mar. Sci. 3, 317–345. Liu, Y., Ge, T., Zhu, Z., Liu, S., Luo, Y., Li, Y., Wang, P., Gavrichkova, O., Xu, X., Xu, J., Wu, J., Guggenberger, G., Kuzyakov, Y., 2019. Carbon input and allocation by rice into paddy soils: a review. Soil Biol. Biochem. 133, 97–107. Long, A., Heitman, J., Tobias, C., Philips, R., Song, B., 2013. Co-occurring anammox, denitrification, and codenitrification in agricultural soils. Appl. Environ. Microb. 79, 168–176. Mulder, A., van de Graaf, A.A., Kuenen, J.G., Robertson, L.A., 1995. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16, 177–183. Nie, S.A., Li, H., Yang, X.R., Zhang, Z.J., Weng, B.S., Huang, F.Y., Zhu, G.B., Zhu, Y.G., 2015. Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere. ISME J. 9, 2059–2067. Nie, S.A., Zhao, L.X., Lei, X.M., Sarfraz, R., Xing, S.H., 2018. Dissolved organic nitrogen distribution in differently fertilized paddy soil profiles: implications for its potential loss. Agric. Ecosyst. Environ. Appl. Soil Ecol. 262, 58–64. Nie, S.A., Zhu, G.B., Singh, B., Zhu, Y.G., 2019. Anaerobic ammonium oxidation in agricultural soils-synthesis and prospective. Environ. Pollut. 244, 127–134. 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Vertical characteristics of anaerobic oxidation of ammonium (anammox) in a coastal saline-alkali field
T
Maomao Houa,1, Ru Xua,b,1, Zhiyuan Lina, Dan Xid, Yi Wangd, Junling Wena, San’an Niec,*, Fenglin Zhonga,* a
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, 350002, China Engineering Research Center of Fujian University of Modern Facilities Agriculture, Fuqing, 350300, China c Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China d Forestry College, Fujian Agriculture and Forestry University, Fuzhou, 350002, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Anammox N loss Saline-alkali soil Candidatus Scalindua
Anammox contributes significant nitrogen (N) loss in agricultural ecosystems, yet its role in the saline-alkali field is not well predicted. Here, we investigate the activity, functional gene abundance and community composition of anammox bacteria in a coastal saline-alkali soil along depth gradient with isotope tracing technique, quantitative PCR assay, and high-throughput sequencing. The anammox activities ranged from 0.09 to 1.32 nmol N g−1 h−1 but were undetectable below 70 cm. The anammox bacteria were present through the soil with the abundance varying from 2.5 × 104 to 5.3 × 105 copies g−1 soil. The Illumina sequencing targeting anammox 16S rRNA gene identified three anammox genera with Candidatus Scalindua dominating. Anammox contribute 40–87.5% of total N2 at 0−50 cm depth whereas the percentage declined sharply below 50 cm. We conclude that N loss in upper soil is mainly linked to anammox while dominated by denitrification in the deeper coastal salinealkali field.
1. Introduction
(www.fao.org/publications/sofa) thus leads to a massive N loss through ammonia volatilization, N2O emission, runoff and leaching (Nie et al., 2018; Xing and Zhu, 2000). Thus, understanding the process of N loss in the agroecosystem is critical for reducing N loss through agricultural production and horticultural management. It was reported that up to 67% of N2 production was linked to anammox in marine sediments (Dalsgaard et al., 2005). Recent studies suggest that anammox can be an important N loss pathway in agricultural ecosystems (Nie et al., 2015, 2019), which contributed to 5–10 % to soil N2 production (Nie et al., 2019; Yang et al., 2015; Zhu et al., 2015). It is of extremely significance to improve and explore land resources for a growing population and limited agricultural production. Saline-alkali soil may play a crucial role with large-scale and -area of distribution (Cong et al., 2014). The effective way to reclaim and utilize the saline-alkali field may provide much needed information. Anammox reaction occurs in agricultural field (Nie et al., 2019; Shen et al., 2013; Yang et al., 2015), marine ecosystems (Dalsgaard et al., 2012; Kuypers et al., 2005), and land-freshwater interface (Zhu et al., 2013). In this study, a soil developed from alluvial deposits with parent material of both coastal and inland was selected. Based on the previous
Our understanding of microbial N cycling has advanced substantially over the last decades (Kuypers et al., 2018; Thamdrup, 2012). The new processes responsible for N loss involve in ammonium oxidation coupled with nitrite (anammox), sulfate (Sulfammox), ferric iron (Feammox), and natural organic matter (NOM-anammox) under anaerobic condition (Rios-Del Toro and Cervantes, 2019). The discovery of anammox upended our perception of the N loss pathway that has long been considered produced solely by heterotrophic denitrification (Mulder et al., 1995; Vitousek and Howarth, 1991). The anammox process was mediated by bacteria affiliated to the phylum Planctomycetes (Jetten et al., 2010). Anammox bacteria inhabit different natural systems, including marine (Lam and Kuypers, 2011), freshwater (Xue et al., 2017), wetland (Wang et al., 2018), and agricultural soil (Nie et al., 2019). Excess N applied in agricultural ecosystems caused an escalating global problem from both environmental and economic aspects (Guo et al., 2010; Liu et al., 2019; Stark and Richards, 2008; Wei et al., 2017). China consumed the largest amount of chemical N fertilizer
⁎
Corresponding authors. E-mail addresses: [email protected] (S. Nie), [email protected] (F. Zhong). 1 These authors contributed equally to this article. https://doi.org/10.1016/j.still.2019.104531 Received 15 May 2019; Received in revised form 5 November 2019; Accepted 5 December 2019 0167-1987/ © 2019 Elsevier B.V. All rights reserved.
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with 1 ml deionized water (dd H2O). The vials were N2-purged and preincubated for 24 h to deplete NOx− and O2. After pre-incubation, all vials were flushed with N2 once again for further incubation. Three isotope treatments, i.e., (1) negative control (15NH4Cl at 99.09%); (2) positive control (15NH4NO3 at 99.09%); and (3) Na15NO3 at 99.21%, respectively, were performed to estimate the activity of anammox and denitrification. Each vial contained 500 μg of 15N and incubated in a constant-temperature incubator (Queue Systems, WV, USA) at 25 °C. The slurries treated with 15NH4Cl and 15NH4NO3 were solely incubated with 0 h and 24 h, respectively. With the soil treated with Na15NO3, at the time point of 0, 3, 6, 12 and 24 h, 500 μl ZnCl2 (7 M) was added in the vials chronologically to stop the reactions. Before N2 determination, the vials were shaken strongly. The rates of anammox and denitrification were calculated by the content of 29N2 and 30N2, which were determined by a multiflow isotope ratio mass spectrometer system (IRMS) (Isoprime Ltd., Cheadle Hulme, UK). The fractions of 29N2 and 30N2 in soil treated with Na15NO3 were calculated using Eqs. (1) and (2). The mass of headspace N2 (Mtotal) was calculated using Eq (3). The production rates of 29N2 and 30N2 were calculated using Eqs. (4) and (5):
study (Nie et al., 2019; Zhu et al., 2013, 2015), we hypothesis that anammox is detectable in the coastal saline-alkali soil. Currently, the activity, abundance, and role of anammox in the saline-alkali field are poorly characterized. Therefore, the aims of this study were to investigate the activity, contribution to N loss, functional gene abundance and community compositions of anammox bacteria in a coastal saline-alkali field in Eastern China. To achieve these, the 15N tracing technology and quantitative PCR assays were conducted to estimate the rate and abundance of anammox bacteria, respectively. High-throughput sequencing targeting anammox 16S rRNA gene was performed to confirm the community compositions. 2. Materials and methods 2.1. Site description and sampling The study was conducted at Haihan Horticultural Farms (121°09′07″ E, 30°21′15″N) in Cixi, Ningbo, Eastern China. The site was approximately 0.5 km from the East China Sea. The altitude was 6 m above sea level. The climate is humid subtropical, and the mean annual air temperature is 17 °C. The field had been cultivated for three years of rice then planted with Brassica parachinensis. During the overwinter season of pakchoi, the arch shed is used for cold prevention (Fig. S1). The soil was a saline-alkali soil developed from alluvial deposits. The field was only irrigated freshwater occasionally and no fertilizer was applied. In this study, three intact soil cores (5 × 100 cm, diameter × hight) were sampled from the field in August 2018. Then the soil cores were sliced at 10 cm intervals and immediately divided into several parts. A small part was frozen in liquid N2 and stored at −80 °C for molecular analysis; approximately 3.5 g fresh soil was incubated to analysis anammox and denitrification activities; about 5 g soil was incubated for the analysis of nitrification rate; the other part was air dried or stored at 4 °C for determining of physical-chemical analysis.
F 29 =
F 30 =
29N2 = N2total
29N2 28N2 29N2 30N2 1+ + 28N2 28N2
=
30N2 = N2total
30N2 28N2 29N2 30N2 1+ + 28N2 28N2
=
R29 1 + R29 + R30
(1)
R30 1 + R29 + R30
(2)
Mtotal = ρ N2 × Vheadspace
(3)
P29 = (M total × F 29)/(t × Msoil)
(4)
P30 = (M total ×
F30)/(t
× Msoil)
(5)
Dtotal = P30 × FN−2
Atotal = Ra % =
Dtotal × 100% Dtotal + Atotal
2.2. Physicochemical procedures Soil moisture was measured using a MA100 moisture meter (Sartorius Company, Göttingen, Germany). Soil pH was measured using a pH-meter (INESA, Shanghai, China) with a 1:2.5 soil/H2O weight ratio. Soil total C/N was measured using a total C/N analyzer (LECO Corporation, MI, USA). The NH4+ and NO3− were extracted from a 1:5 soil/2 M KCl mixture and quantified by a continuous flow injection analyzer (SYSTEA S.p.A., FR, Italy). Salinity (determined as EC) was analyzed by a Conductivity Meter (INESA, Shanghai, China) in a 1:5 (soil/H2O) suspension.
(6)
FN−1
29
× [P29 + 2 × (1 −
× P30]
(7) (8)
Where F and F denote the fraction of N2 and N2, R and R30 represent the ratio of 29N2 to 28N2 and 30N2 to 28N2, Mtotal is the mass of headspace N2, ρN2 is the density of N2, Vheadspace is the volume of headspace, t is the incubation time and Msoil represents the soil mass in vials, P29 and P30 are the measured production of 29N2 and 30N2 from each vial in the 15NO3− treatment, respectively. The calculation of anammox and denitrification activity was processed according to Dalsgaard and Thamdrup (2002) and Xi et al. (2016). Denitrification and anammox rates were calculated by Eqs. (6) and (7), respectively. The contribution of anammox to total N2 production was assessed by Eq. (8). In slurries treated with negative control, no significant accumulation of 29N2 or 30N2 was detected, suggesting that initial NOx− had been depleted during the pre-incubations. In positive control treatment, 29N2 could be observed while 30N2 was undetectable. The slurries treated with Na15NO3 indicated that anammox and denitrification processes were detectable in the examined soils. Based on each time point, a linear regression of different N2 concentrations was performed to estimate the potential rates of anammox and denitrification.
2.3. Measuring potential nitrification rate The potential nitrification rate was measured as depicted by Kandeler (2002) with some modifications. Fresh soil (3.5 g) was incubated with 20 ml of phosphate-buff ;ered saline (g L−1: NaCl, 8.0; KCl, 0.2; Na2HPO4, 0.2; NaH2PO4, 0.2) and (NH4)2SO4 (final concentration: 1 mM) in a plastic tubes for 24 h. KClO3 (10 mg L−1) was added to the tubes for inhibition of NO2− oxidation. The NO2- was extracted by adding 5 ml of 2 M KCl after incubation prior to centrifugation. The NO2−-N was quantified spectrophotometrically at 545 nm (BioTek Instruments, Inc. VT, USA).
30
FN−1)
29
30
29
2.5. DNA extraction, PCR amplification, and Illumina sequencing 2.4. Measuring anammox and denitrification rate Soil DNA was extracted from 0.5 g (fresh weight, three replicates) samples with the FASTDNA ®SPIN Kit (MoBio Laboratories, CA, USA) for soil according to manufacturer’s protocols. The final DNA concentration and further purification were tested by NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, WI, USA). The DNA quality was examined by 1% agarose gel electrophoresis.
A slurry incubation experiment with different 15N-isotope substrates was conducted to determine anammox and denitrification rates according to Long et al. (2013) and revised by Xi et al. (2016). Briefly, 3.5 g soil (fresh weight, three replicates) were transferred to a volume of 20 ml glass vials (Exetainer, Buckinghamshire, UK) and saturated 2
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The anammox bacterial 16S rRNA gene was amplified with Amx368 F and Amx820R (Schmid et al., 2000) by a GeneAmp 9700 PCR system (ABI, CA, USA). The thermal profile started at 96 °C for 10 min, followed by 35 cycles of 30 s at 96 °C, 1 min at 58 °C, and 1 min at 72 °C. The resulted PCR products were extracted from a 2% agarose gel, purified by the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) and quantified with QuantiFluor™-ST (Promega, WI, USA). Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq platform (Illumina, CA, USA) in accordance with the standardized commercial service. (Majorbio BioPharm Technology Co. Ltd., Shanghai, China). 2.6. Quantitative PCR of hzsB gene Real-time q-PCR was carried out to target the anammox functional gene hzsB with the primer set hzsB_396 F and hzsB_742R (Wang et al., 2012b). Thermal cycling and data operation were conducted using an ABI 7500 system (ABI, CA, USA). The q-PCR reaction was carried out with the following mixture: 9 μL Master Mix (APB, Freiburg, Germany), 1 μL each primer, 1 μL DNA, and 8 μL dd H2O. The standard curve was performed with ten-fold serial dilutions of plasmid DNA with the hzsB gene. Three replicates were carried out for each quantitative assay. The thermal profile was performed with the program: 95 °C for 1 min, followed by 35 cycles of 95 °C for 5 s, 60 °C for 30 s, and 75 °C for 1 min. The amplification efficiencies varied from 90% to 110% and correlation coefficients for linear regression greater than 0.99 were acceptable.
Fig. 2. Vertical distribution of the abundance of anammox bacteria targeting the hzsB gene. Error bars indicate S.E. (n = 3).
539.5–1812.5 μs cm−1. The highest total carbon and N content in the soil was found at the top (0−10 cm) layer and decreased with slightly change from 1.32% to 1.55% and 0.2 to 0.7 g kg−1, respectively. The ferrous in soil cores showed a gradient feature with low concentrations at 0−50 cm (0.02–0.34 cmol kg−1) and high concentrations (1.07–1.88 cmol kg−1) blow 50 cm.
2.7. Statistical analysis 3.2. Abundance of anammox bacteria The initial data were conducted by Microsoft Excel 2016 software and further conducted by SPSS 22.0 for the statistical analysis. The data below the detection limit were recorded as not determined (ND). The figures were completed by Sigmaplot 14.0 software.
To investigate the presence and abundance of anammox bacteria in the soil cores, the hzsB gene was assessed by quantitative PCR assay and the vertical distribution was shown in Fig. 2. The hzsB gene abundance varied greatly and valued from 2.5 × 104 to 5.3 × 105 copies g−1 dry weight soil. The highest abundance of anammox cells in the examined soil was detected at 60−70 cm, while low copy numbers of hzsB gene were recorded at the surface (0−10 cm) and deep layers (80–100 cm). Pearson correlation analysis showed that the hzsB gene copy numbers were not significantly linked with soil properties selected in the present study (p < 0.05) (Table S1).
3. Results 3.1. Soil properties after five years of cultivation The soil physicochemical characteristics along depth gradients at 10 cm intervals are shown in Fig. 1. The soil was slightly alkaline and the pH varied from 8.0-8.55. The soil moisture increased with the depth and ranged from 0.22 to 0.27. The exchangeable NH4+-N was lowest at the top depth and then increased with depth to a concentration of 17.3 mg kg−1. The highest NOx−-N (nitrate and nitrite) concentration was observed at the top 0−10 cm layer and decreased gradually with soil depth. The soil salinity was increased with depth gradients from
3.3. Rates of anammox, denitrification, and nitrification An isotope-tracing experiment was conducted to determine the potential role of anammox as a N2 producer. The potential anammox rates can overestimate the actual in situ rates because some other
Fig. 1. vertical distribution of soil pH (a), moisture (b), electrical conductivity (c), total carbon (d), total nitrogen (e), exchangeable ammonium (f), nitrite + nitrate nitrogen (g) and ferrous in saline-alkali filed soil cores. 3
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Fig. 3. Vertical distribution of anammox rate and ratio of anammox contribution to total N2 production (a) as well as nitrification and denitrification activity (b) at each depth. Error bars indicate S.E. and analyzed with three replicates per site.
processes, like Feammox, can also generate N2 (Ding et al., 2014; RiosDel Toro and Cervantes, 2019). However, in the incubation experiments, no significant accumulation of 30N2 could be observed in the slurries amended with 15NH4+ and 15NH4+ + 14NO3− (Fig. S2). Besides, in 0−70 cm soil layers, where anammox could be detected (Fig. 3a), the in situ concentration of NH4+ was low (Fig. 1f), which may not supply sufficient substrate for Feammox. Hence, the detected anammox rates might not be seriously overestimated. The results showed that active anammox was detected in the soil horizon at 0−70 cm whereas no significant anammox activity could be determined below 70 cm (Fig. 3a). The anammox rates in 0−40 cm soil cores were 0.28–0.94 nmol N g−1 h−1 and increased to the highest value of 1.32 nmol N g−1 h−1 at the 40−50 cm layer. Anammox rates decreased drastically at 50−70 cm (both 0.09 nmol N g−1 h−1). These results indicated that anammox was more likely to occur in the upper layer as well as have a high rate at the surface (0−20 cm) and specific depth (40−50 cm). The contribution to N production via anammox at 0−50 cm depth was up to 40–87.5%, and this percentage is declined drastically to lower than 0.8% below 50 cm, the other attributing to denitrification. Both nitrification and denitrification rates were detected to evaluate the nitrite, which is the substrate for anammox (Fig. 3b). Overall, both nitrification and denitrification rates showed a heterogeneous depth effect. In the soil horizon at 0−50 cm, the denitrification rate was low (0.11 to 0.42 nmol N g−1 h−1) but they increased drastically below 50 cm (5.1–15.5 nmol N g−1 h−1). The nitrification rates of soil cores were high at the surface layer but decreased gradually along with soil depth and the values were very low (0.03–0.36 nmol N g−1 h−1) below 40 cm. These results indicated that nitrification occurred mainly in upper soil, while denitrification dominated in lower soil.
Fig. 4. Vertical distribution of the community compositions and their relative abundance of anammox bacteria in core samples of the saline-alkali field. Table 1 Diversity characteristics of anammox 16S rRNA gene in each depth.
3.4. Community composition and biodiversity of anammox bacteria A total of 4434 effective sequences were obtained from ten layers of soil cores. All sequences were mostly affiliated to the genera of Candidatus Scalindua, Candidatus Brocadia, Candidatus Kuenenia, or unclassified (Fig. 4). Overall, Candidatus Scalindua and Candidatus Kuenenia were present in all soil layers while Candidatus Brocadia could only be identified in particular depth (in the soil horizon at 20−40 cm and 70−100 cm, respectively). Only a small fraction (0.4–3.2%) of anammox bacterial 16S rRNA gene sequences were unidentified thus assigned as unclassified. Candidatus Scalindua was the highest relative abundant anammox bacterial genus (36–97%). The relative abundance of Candidatus Kuenenia showed a variation of percentage from 3% to 53%. Candidatus Brocadia which could only be detected in certain soil layers also showed different fractions (4–53%). The anammox bacterial community compositions had much heterogeneity.
Soil layers (cm)
No. of OTUs
Simpson index
Shannon index
0–10 10–20 20–30 30–40 40–50 50–60 60–70 70–80 80–90 90–100
7 3 7 9 7 6 6 7 6 6
0.82 0.93 0.52 0.42 0.49 0.85 0.48 0.35 0.39 0.41
0.36 0.17 0.86 0.97 0.73 0.34 0.77 1.13 1.05 0.85
The clustered OTUs number of anammox 16S rRNA gene in ten representative soil cores with 97% similarity cutoff as well as Simpson and Shannon diversity indices of anammox bacterial communities were shown in Table 1. A total of 10 OTUs were achieved and the OTUs number obtained from each sample varied from 3 to 9. In 0−10 cm, 10−20 cm and 50−60 cm of soil layers, which were dominated the genera of Candidatus Brocadia, the Simpson index accessed dominant
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rate was very low and undetectable below 70 cm (Fig. 3a). The main reason could be the low NOx− concentration (Fig. 1g). In deeper soil, where nitrification was inhibited, denitrifiers didn’t have enough NOx− to utilize for anammox. Consequently, denitrification can’t supply enough substrate for anammox. The anammox bacteria in saline-alkali soil possessed a heterogeneous distribution of activity (Fig. 3a). The functional gene abundance of anammox showed poor correlation with the activity (Table S1), which confirmed what already reported (Bai et al., 2015; Wang et al., 2012a) but is inconsistent with some previous research (Shen et al., 2015, 2017; Yang et al., 2015). The little relation between anammox cell numbers and activity indicates that anammox bacteria can have a different metabolic rate (Hu et al., 2011; Humbert et al., 2010). Although substantial numbers of anammox cell could be quantified below 50 cm, the anammox rates were very low or even undetectable, suggesting those anammox bacteria could be dormant cells thus little or no activity. High-throughput sequencing of anammox 16S rRNA gene indicated that at least three genera, “Scalindua”, “Brocadia” and “Kuenenia” were identified from saline-alkali soil (Fig. 4). Among these, “Scalindua” was the highest relative abundant anammox genus, which differed with other agricultural soils where “Brocadia” and “Kuenenia” usually are dominated genera (Shen et al., 2015, 2017; Wang and Gu, 2013; Zhu et al., 2011). In this study, the salinity (determined as EC) is very high (Fig. 1c). The anammox composition “Scalindua” is more likely to be found in high salinity environments than “Brocadia” and “Kuenenia” bacteria (Dale et al., 2009; Schmid et al., 2007). The presence of “Kuenenia” with a heterogeneous proportion along the ten-soil gradient whereas “Brocadia” could only be measured at particular depth indicates that this anammox bacteria may be better adapted than “Brocadia” bacteria in saline-alkali soil. Taken together, microbial anammox reaction in saline-alkali soil occurred mainly at the upper soil layer (0−50 cm) and showed a high level of contribution to N production (40–87.5%) in comparison to denitrification. The anammox bacteria are more active at the surface (0−20 cm) and specific layer (40−50 cm) and their activities were not correlated with the abundance. Future studies should be addressed on saline-alkali soil with the spatiotemporal variations of the anammox process.
OTUs showed higher values whereas the Shannon index estimated both richness and evenness were lower than that examined from other soil cores. The community diversity in saline-alkali soil differed with soil depth. 4. Discussion In this study, the anammox rates were 0.09–1.32 nmol N g−1 h−1 at 0−70 cm, and this values were comparable to those reported in a vertical vegetable field (0.09–1.43 nmol N g−1 h−1) (Shen et al., 2017) and a paddy soil (0.5–2.9 nmol N g−1 h−1) (Zhu et al., 2011). Based on the incubation results, it is evaluated that approximately 67 g N m-2 yr−1 loss could be attributed by anammox (Table S2). This estimation is higher than that reported in a vegetable field (29.5 g N m−2 yr−1) (Shen et al., 2017), suggesting that anammox could be an important potential source of N loss in saline-alkali soil. On the other side, the denitrification rates were very low (0.11-0.42 nmol N g−1 h−1) at 0−50 cm but increased drastically (5.15–11.76 nmol N g−1 h−1) below 50 cm (Fig. 3b). These results indicate that N loss in upper soil is mainly caused by anammox, while that in deeper soil largely attributes to denitrification. The observed cell abundance of anammox bacteria (2.5 × 104 to 5.3 × 105 copies g−1 soil) in vertical soil layers (Fig. 2) was lower than those found in the vegetable soil (Shen et al., 2015, 2017) and paddy soils (Shen et al., 2014; Wang et al., 2012b; Zhu et al., 2011). The main reason could be attributed to different agronomic management. In agricultural soils, a relatively high amount of N fertilizer may be applied in the field thus may stimulate the growth of anammox bacteria (Nie et al., 2019). However, no fertilizer was applied in the studied soil for five years. The variety of anammox bacterial abundance along the soil gradient has some similarity to paddy soil (Wang et al., 2012b). No significant linear correlation was found between anammox bacterial abundance and soil properties (Table S1). The low abundance of anammox bacteria in the surface saline-alkali soil (Fig. 2) mainly explained by the oxygen pervasion, which could inhibit anammox bacterial growth. A higher detection frequency of hzsB copy number in deeper saline-alkali soil (Fig. 2) indicates that anammox bacteria prefer to special niches, which is similar to the results found in water columns and sediments (Dalsgaard et al., 2003, 2005). The vertical distribution of anammox bacterial cell numbers in saline-alkali soil might involve with both horticulture conditions and physicochemical properties. The anammox rates along surface gradient depth (0−40 cm) were more or less decreased, which have some similarity to the nitrification rate (Fig. 3). In the upper soil, nitrification use NH4+ as substrate to generate NO2− for anammox bacteria. This could be also inferred from the low concentration of NH4+ (Fig. 1f) and high nitrate concentration (Fig. 1g) in soil cores. Other studies also indicate that partial nitrification rather than denitrification can be more likely linked to anammox as nitrification can provide enough nitrite for anammox bacteria (Lam et al., 2007; Zhu et al., 2011). Furthermore, some anammox bacteria can reduce NO3−, which is relatively high in the upper soil layer (Fig. 1g), to NH4+ for anammox (Kartal et al., 2007, 2008). A drastically increase of anammox rate was measured at 40−50 cm (Fig. 3a) indicating anammox bacterial is more active in particular depth. These results have some similarity to that found in the water-soil interaction layer where hotspots of anammox were observed (Zhu et al., 2013). One possible reason could be the high soil moisture and salinity (Fig. 1b and c), which are essential factors for governing anammox activity (Bai et al., 2015; Zhu et al., 2013). Besides, high concentration of ferrous ion (Fig. 1h), which is electron donors thus has a positive effect on anammox reaction (Huang et al., 2014; Strous et al., 2006; Van de Vossenberg et al., 2008; Wei et al., 2019), were detected at 40−50 cm soil depth. Finally, the upper soil layer is subjected to plant growth and water leaching, which may have a negative effect on anammox metabolism. The anammox activity at deeper depth is less susceptible to these factors in comparison with upper soil. The anammox
Declaration of Competing Interest The authors declare that they have no conflict of interest. Acknowledgments The research is financially supported by the National Natural Science Foundation of China (4170010194, 41703068), the Postdoctoral Science Foundation of China (2017M622044, 2018M630723), National Science and Technology Major Project of Fujian (2018NZ0002-2), Open Funds by Engineering Research Center of Fujian University of Modern Facilities Agriculture (G2-KF1808), and NDRC Agricultural Five New Project of Fujian (K6017201A). Moreover, the author San’an Nie thanks Lixia Zhao and Xiumei Lei for their kind help. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.still.2019.104531. References Bai, R., Xi, D., He, J.-Z., Hu, H.-W., Fang, Y.-T., Zhang, L.-M., 2015. Activity, abundance and community structure of anammox bacteria along depth profiles in three different paddy soils. Soil Biol. Biochem. 91, 212–221.
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