Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.

INTRODUCTION

Anaerobic ammonium oxidation (anammox), which refers to the oxidation of ammonium coupled with the reduction of nitrite under anaerobic conditions, has been recognized as an important pathway for the production of dinitrogen gas in the global nitrogen cycle, which was estimated to be responsible for more than 50% of the nitrogen loss in marine ecosystems (1–3). The anammox process is performed by a monophyletic order of bacteria, the Brocadiales (4). There are five described genera of anammox bacteria to date: “Candidatus Brocadia” (5–7), “Candidatus Kuenenia” (8), “Candidatus Scalindua” (9–12), “Candidatus Anammoxoglobus” (13), and “Candidatus Jettenia” (14, 15). Growing evidence shows that anammox bacteria are broadly distributed in various marine (11, 16, 17) and freshwater habitats (18–20).

The anammox reaction depends on the concomitant presence of both oxidized and reduced inorganic nitrogen compounds under anoxic conditions, and the oxic/anoxic interfaces in terrestrial ecosystems provide suitable habitats for anammox bacteria (21, 22). In terrestrial habitats, ammonium can be released through the process of organic matter mineralization or dissimilatory nitrate reduction to ammonium, and nitrite can be produced by aerobic ammonium oxidizers at oxic/anoxic interfaces or by nitrate reducers under anoxic conditions. Until now, however, there has been very limited evidence for the existence of anammox bacteria in terrestrial ecosystems. Two recent studies reported the presence of diverse anammox bacteria in permafrost soil and a paddy soil (21, 23). Moreover, two soil anammox enrichment cultures were obtained from a nitrogen-loaded peat soil and a paddy soil, respectively (22, 24). These data are too limited to support conclusions about the distribution and diversity of anammox bacteria in terrestrial ecosystems, and it remains unclear whether anammox bacteria are present within a wider range of terrestrial ecosystems. The increasing use of commercial nitrogen-rich fertilizers for promoting crop yield has led to an increased nitrogen load in Chinese agricultural soils, theoretically making them favorable habitats for anammox bacteria (23).

Therefore, the primary objectives of the present study were as follows: (i) to determine the presence of anammox bacteria in an extensive range of agricultural soils collected from 32 different locations in China, (ii) to investigate the diversity and abundance of anammox bacteria in these soils, and (iii) to examine which environmental factors have important impacts on the distribution of anammox communities in the agricultural soils.

MATERIALS AND METHODS

Soil samples.

Soil samples were collected from various agricultural ecosystems in 32 different locations in China (18°27′N to 47°86′N and 88°14′E to 126°54′E) in June 2011. The study area and sampling sites are presented in Fig. S1 in the supplemental material. The dry farmland soil samples were collected and used in this study. In each location, at least three soil cores (5-cm diameter and 10-cm depth) were collected and mixed in the field to form one composite sample. All of the collected samples were sectioned into two subsamples: one was stored at 4°C in sealed containers for about 1 to 2 weeks before the soil physicochemical analysis, and the other was frozen at −80°C for the molecular analysis.

Chemical analysis.

Ammonium and nitrate were extracted from the soil using 2 M KCl. Briefly, the extracts were obtained by shaking (1 h at 200 × g) 50-ml centrifuge tubes containing 10 g soil and 30 ml 2 M KCl. The tubes were then centrifuged at 4,000 × g for 10 min to collect the supernatant. The ammonium in the KCl extracts (supernatant) was determined using a colorimetric method at 623 nm (25), and the nitrate was determined by reduction to nitrite via cadmium reduction and measured spectrophotometrically at 520 nm (26). The soil pH was determined after mixing the soil with water at a ratio (soil/water) of 1:2.5, and the soil organic content was determined by the K₂Cr₂O₇ oxidation method.

DNA extraction and PCR amplification.

The DNA in the soil was extracted from 0.25 g fresh soil using a Power Soil DNA kit (Mo Bio Laboratories, CA) according to the manufacturer's instructions. The extracted DNA was examined by electrophoresis using a 1.0% agarose gel.

A nested PCR approach was conducted to detect the anammox bacterial 16S rRNA genes. In the first round of PCR, the primer set Pla46f-1545r (27, 28) was used to amplify the Planctomycetales 16S rRNA genes. In the second round, the anammox bacterial 16S rRNA genes were amplified using the primer set Amx368f-Amx820r (29). The PCR mixture and thermal cycling programs were conducted as previously described (30). The amplified products were examined by electrophoresis using a 1.0% agarose gel.

Cloning and sequencing.

The PCR products were cloned using the pMD19-T vector (TaKaRa, Bio Inc., Shiga, Japan) according to the manufacturer's instruction. At least 30 positive clones from each sample were selected for sequencing (Shenzhen Huada Genomics Institute, Shenzhen, China). The quality of the recovered sequences was checked using the Chromas LITE (version 2.01) program. The occurrence of chimeric sequences was further examined using the UCHIME algorithm (31). The biological sequences were then aligned using MEGA (version 4.0) software and manually checked and trimmed.

Phylogenetic analysis.

The phylogenetic analysis of the 16S rRNA gene sequences was conducted using MEGA (version 4.0) software and the neighbor-joining method. A bootstrap analysis with 1,000 replicates was applied to estimate the confidence values of the tree nodes.

Statistical analysis.

The number of operational taxonomic units (OTUs) for each clone library was defined using 3% differences in the nucleotide sequences, as determined by using the furthest neighbor algorithm in the DOTUR program (32). DOTUR was also used to generate the Chao1 estimator and the Shannon index. The coverage of the clone libraries was calculated as previously described (33). The distribution of the anammox communities and their correlations with environmental factors were determined by principal components analysis (PCA) and redundancy analysis (RDA), respectively, using the software CANOCO (34). Pearson correlation analyses were used to test the correlations between the anammox bacterial diversity, abundance, and different environmental factors, using the software SPSS (version 18.0; SPSS, Chicago, IL).

qPCR.

Hydrazine synthase (hzs) is a key enzyme in anammox metabolism, consisting of three subunits encoded by the genes hzsA, hzsB, and hzsC, responsible for the synthesis of hydrazine from nitric oxide and ammonium (35, 36). A primer set (hzsA_1597F-hzsA_1857R) (37) targeting the hzsA gene of anammox bacteria was used to quantify the abundance of the anammox bacteria in the collected soils. The specificity of this primer set was confirmed by sequencing the quantitative PCR (qPCR) products from several soil samples (see Fig. S2 in the supplemental material).

The qPCR was performed using an iCycler iQ5 thermocycler (Bio-Rad, CA). Each PCR mixture (25 μl) was composed of 12.5 μl 2× SsoFast EvaGreen supermix (Bio-Rad, CA), 0.5 μl forward and reverse primers, and 1 μl template DNA. The PCR thermal cycle program was performed as previously described (37). The standard curve was constructed from a series of 10-fold dilutions of a known copy number of the plasmid DNA, ranging from 1.28 × 10⁻⁸ to 1.28 × 10⁻² ng of DNA per well. Triplicate qPCRs were performed for each sample and each dilution. The consistency of the qPCR assay makes it a good method for the detection of the copy numbers of the hzsA gene (R² = 0.997) in the soil samples. The linear range of quantification for the qPCR assay was from 9.88 × 10⁷ to 9.88 × 10¹ copies per well.

Nucleotide sequence accession numbers.

The sequences obtained in this study are available in GenBank under accession numbers JQ918892 to JQ919160.

RESULTS

Physicochemical properties of the soil samples.

The physicochemical properties of the collected soil samples, including the soil pH, organic content, ammonium content, nitrate content, and total inorganic nitrogen (TIN) content, are shown in Table 1. Most of the collected samples had similar pH values (near neutral pH), ranging from 6.5 to 7.5. However, samples BH, CS, KM, NC, NJ, NN, and ZJ showed relatively lower pH values (4–6), a finding that may be related to the heavier application of nitrogen-rich fertilization at these sampling sites, which can directly and indirectly acidify the soil (38, 39). The soil organic contents of most of the samples varied from 10 to 30 mg kg⁻¹, and all the soil samples showed relatively high TIN contents, ranging from 20 to 150 mg kg⁻¹. The soil ammonium and nitrate contents varied among the different samples (Table 1): samples ALT, DT, HRB, PG, SJZ, XM, YC, and ZZ contained low ammonium contents (lower than 5 mg kg⁻¹) and high nitrate contents (higher than 30 mg kg⁻¹), whereas samples HZ, NJ, NN, SJ, TL, and TZ had high ammonium contents (higher than 30 mg kg⁻¹) and low nitrate contents (lower than 5 mg kg⁻¹). Such differences may be the result of different types of fertilizers (ammonium fertilizers or nitrate fertilizers) and schedules for fertilization applied at the different sampling locations. In addition, the high nitrate contents of some examined samples could also be the result of high nitrification rates and aerobic soil conditions, which could have adverse impacts on the distribution of anammox communities in these sampling sites.

Table 1.

Physicochemical properties of the collected agricultural soils

Province	City	Sample name	Position	pH	Concn (mg/kg)			Organic carbon content (g/kg)
					NH4+ N	NO3− N	TIN
Xin jiang	A letai	ALT	47°86′N, 88°14′E	7.03	2.00	77.41	82.75	24.91
Hei longjiang	Harbin	HRB	45°82′N, 126°54′E	7.48	5.14	61.58	68.72	32.16
Ji ling	Chang chun	CC	43°84′N, 125°32′E	6.76	6.99	15.78	24.58	9.25
Nei menggu	Chi fen	CF	42°27′N, 118°88′E	7.05	8.48	9.48	20.31	13.41
Liaonin	Shen yang	SY	41°82′N, 123°43′E	7.08	1.61	14.80	19.29	33.62
Beijing	Ping gu	PG	40°39′N, 116°63′E	7.03	4.88	137.53	144.8	22.38
Shan xi	Da tong	DT	40°03′N, 113°19′E	7.48	3.63	29.66	35.45	25.60
Tian jin	Bin hai	BH	39°03′N, 117°70′E	5.92	5.12	25.49	32.87	16.04
Ning xia	Yin Chuan	YC	38°50′N, 106°24′E	7.30	3.11	45.57	51.48	24.19
He bei	Shi jiazhuang	SJZ	38°06′N, 114°52′E	7.55	2.94	33.66	38.33	12.81
Shan dong	Ji nan	JN	36°68′N, 116°99′E	7.25	5.65	47.33	54.90	25.81
Gan su	Lan zhou	LZ	36°08′N, 103°84′E	7.04	11.82	37.45	51.04	20.19
He nan	Zheng zhou	ZZ	34°76′N, 113°62′E	7.19	5.14	39.69	47.04	25.24
Qing hai	Ma qin	MQ	34°47′N, 100°24′E	7.62	7.36	13.72	23.19	16.90
San xi	Xi'an	XA	34°28′N, 108°94′E	6.67	10.52	22.47	34.65	27.74
Si chuan	Guang yuan	GYY	32°44′N, 105°84′E	6.79	17.18	8.00	27.11	26.24
Jiang su	Nan jing	NJ	32°09′N, 118°80′E	3.93	33.50	2.20	37.53	26.89
An hui	He fei	HF	31°83′N, 117°23′E	7.23	16.99	32.96	53.27	31.37
Shang hai	Song jiang	SJ	31°02′N, 121°18′E	7.42	41.40	5.56	49.76	48.50
Hu bei	Jin zhou	JZ	30°36′N, 112°23′E	7.37	7.55	31.35	41.42	20.09
Zhe jiang	Hang zhou	HZ	30°29′N, 120°15′E	7.24	146.10	2.10	149.10	32.00
Chong qing	Tong liang	TL	29°86′N, 106°06′E	7.43	109.23	4.19	117.33	39.94
Xi zang	Lin zhi	LZZ	29°68′N, 94°35′E	7.53	17.08	19.78	38.81	20.36
Jiang xi	Nan chang	NC	28°70′N, 115°86′E	5.02	17.82	25.23	46.35	33.08
Hu nan	Chang sha	CS	28°27′N, 112°94′E	5.42	17.80	44.77	65.51	23.61
Gui zhou	Gui yang	GY	26°66′N, 106°63′E	7.29	6.47	39.55	48.52	3.16
Yun nan	Kun ming	KM	25°05′N, 102°72′E	5.20	32.10	7.06	41.16	26.22
Fu jian	Xia men	XM	24°49′N, 118°09′E	6.29	1.14	79.43	83.70	10.83
Tai wan	Tai zhong	TZ	24°25′N, 120°94′E	7.23	112.61	4.12	127.58	33.40
Guang xi	Nan ning	NN	22°83′N, 108°36′E	3.93	68.79	4.07	75.02	23.61
Guang dong	Zhan jiang	ZJ	21°27′N, 110°35′E	4.85	21.12	38.48	63.05	14.36
Hai nan	San ya	SYY	18°27′N, 109°51′E	6.30	1.61	14.80	19.29	7.80

Phylogenetic analysis of the anammox bacteria.

A total of 1,002 16S rRNA gene sequences were obtained from the soil samples collected from 32 different locations in China. Cloning and sequence analyses confirmed that approximately 96% of the amplified sequences were affiliated with known anammox genera. Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated four separate clusters belonging to “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the samples examined (Fig. 1; also see Fig. S3 in the supplemental material). Sequences of the “Candidatus Brocadia” cluster showed 92.1 to 99.8% and 90.4 to 96.7% identities to the 16S rRNA genes of “Candidatus Brocadia fulgida” and “Candidatus Brocadia anammoxidans,” respectively, and those of the “Candidatus Kuenenia” cluster showed 93.7 to 99.2% identity to the 16S rRNA gene of “Candidatus Kuenenia stuttgartiensis.” Sequences of the “Candidatus Anammoxoglobus” cluster showed 95.4 to 99.2% identity to the 16S rRNA gene of “Candidatus Anammoxoglobus propionicus,” and sequences of the “Candidatus Jettenia” cluster showed 92.5 to 97.1% identity to the 16S rRNA gene of “Candidatus Jettenia asiatica.” On the basis of the anammox 16S rRNA gene clone library, “Candidatus Brocadia” was the most common anammox genus in the agricultural soils examined (Fig. 1). “Candidatus Brocadia”-like sequences were found in 29 of the 32 samples which accounted for approximately 71.3% of all of the retrieved 16S rRNA gene sequences.

Fig 1.

Anammox bacterial genera detected in the agricultural soils examined.

Community composition and diversity of the anammox bacteria.

A heterogeneous distribution of the anammox communities was observed in the collected agricultural soils (Fig. 2a; see Fig. S4 in the supplemental material). Different anammox genera could be simultaneously detected in samples CS, GYY, HZ, JZ, KM, LZ, LZZ, NC, NJ, NN, SJ, SY, TL, XA, XM, and YC, while only a single anammox genus (“Candidatus Brocadia” or “Candidatus Kuenenia”) could be detected in the remaining samples. The uneven distribution of anammox communities may be related to the local soil properties. Indeed, as revealed by the RDA test, the soil organic content and ammonium content were found to be significantly correlated with the distribution of the anammox communities in the samples examined (P < 0.05, 1,000 Monte Carlo permutations) (Fig. 3).

Fig 2.

Distribution (a) and abundance (b) of anammox bacteria in the agricultural soils examined. The base map used is from the National Fundamental Geographic Information System of China.

Fig 3.

RDA ordination plots for the first dimensions to show the relationship between the anammox bacterial community structure and environmental factors in the agricultural soils examined.

The diversity of the anammox 16S rRNA gene sequences in each sample was determined on the basis of the number of OTUs, the Chao1 estimator, and the Shannon index (see Table S1 in the supplemental material). As shown in Table S1 in the supplemental material, the library coverage values (C) varied from 0.93 to 1.00, indicating that the sequencing effort covered a significant amount of the richness of anammox bacteria in the examined soil samples. A total of 14 samples contained the single genus “Candidatus Brocadia,” while only samples CC, HRB, SJZ, SYY, and ZZ exhibited the lowest diversity of anammox bacteria. This is because different OTUs of the “Candidatus Brocadia” genus were detected in these 14 samples (see Table S1 in the supplemental material). A higher diversity of anammox bacteria was found in samples HZ and SY, with 5 and 6 OTUs, respectively, and the same Shannon index of 1.45. The remaining samples showed an intermediate diversity of anammox bacteria, with 2 to 4 OTUs and Shannon index values of 0.14 to 0.98. Pearson correlation analyses showed that the diversity of the anammox bacteria was significantly correlated with the soil ammonium content and the organic content (P < 0.05) (Table 2).

Table 2.

Correlation analyses of the environmental factors and anammox bacterial diversity and abundance in the collected agricultural soils

Environmental factor	Pearson correlation coefficienta
	No. of OTUs	Shannon index	Chao1	Abundance
pH	−0.201	−0.162	−0.195	−0.296
NH4+ N	0.352	0.351	0.325	0.358
NO3− N	−0.186	−0.132	−0.117	−0.270
TIN	0.254	0.326	0.281	0.052
Organic carbon	0.376	0.417	0.416	0.149

^aBoldface type denotes a P value of <0.05, which is typically regarded as significant, as determined by the SPSS (version 18.0) program (SPSS, Chicago, IL).

Abundance of anammox bacteria.

The abundance of the anammox bacteria in the examined soil samples was estimated on the basis of the quantification of the anammox bacterial hzsA genes using the qPCR method described by Harhangi et al. (37). The qPCR results further confirmed the occurrence of anammox bacteria in the collected samples. On the basis of the estimation of only one copy of the hzsA gene per anammox bacterium, as found in the genomes of “Candidatus Kuenenia stuttgartiensis” (35, 36), “Candidatus Brocadia fulgida” (40), “Candidatus Jettenia asiatica” (15), “Candidatus Scalindua profunda” (12), and strain KSU-1 (41), the copy numbers of the anammox bacteria in the collected soil samples ranged from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁴ ± 0.25 × 10⁶ copies per gram of dry weight (Fig. 2b; see Table S1 in the supplemental material). The copy numbers of the anammox bacteria varied greatly among the different samples, showing a heterogeneous distribution of anammox bacterial abundance. Pearson correlation analyses showed that the abundance of anammox bacteria was significantly correlated with the soil ammonium content (P < 0.05) (Table 2).

DISCUSSION

Anammox bacteria were detected in all of the soil samples examined on the basis of the amplification of the anammox bacterial 16S rRNA genes. Quantitative PCR of the hzsA gene further confirmed the presence of anammox bacteria in the agricultural soils examined. The copy number of anammox bacteria found in the soils varied from 6.38 × 10⁴ ± 0.42 × 10⁴ to 3.69 × 10⁶ ± 0.25 × 10⁶ copies per gram of dry weight, which was in the same range as that found in other terrestrial soils (21, 23). These results indicated the broad distribution of anammox bacteria in Chinese agricultural soils, greatly expanding the knowledge of the distribution of anammox bacteria in environments, particularly in terrestrial ecosystems. In contrast, anammox bacteria were detected in only certain soil samples in a previous study (21). Because the soil samples in which no anammox bacteria were found were associated with low nitrogen contents (21), the relatively high nitrogen content in the collected soils (Table 1) may have stimulated the growth of the anammox bacteria to a detectable level in the present study. Similarly, Zhu et al. (23) detected anammox bacteria in all the samples collected from a paddy field that had been subjected to long-term fertilization. Together, these results may indicate that anammox bacteria are broadly distributed in terrestrial ecosystems, mainly those associated with high nitrogen contents.

“Candidatus Scalindua” has been reported to be the single anammox genus in marine ecosystems (11, 16, 42), while four of the five known anammox genera were detected in the collected soil samples: “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia.” Similar results were also observed in other reported terrestrial ecosystems (21–24). It is most likely that separate microniches for different anammox genera existed in the heterogeneous soils (22).

In the present study, the most common anammox species in the collected soil samples were related to the “Candidatus Brocadia” genus, as has been observed in other terrestrial ecosystems (21, 23). These results suggest that “Candidatus Brocadia” may possess better adaptability in terrestrial ecosystems. It was reported that “Candidatus Brocadia” harbors a diverse metabolism (40). This anammox genus could use short-chain organic acids as additional energy sources, which are able to link the oxidation of acetate to carbon dioxide with the reduction of nitrite to dinitrogen gas, instead of ammonia oxidation (6). The use of alternative electron donors leads to the better adaptability of “Candidatus Brocadia” than other anammox genera in heterogeneous soils.

On the basis of the detected anammox 16S rRNA gene sequences and the copy numbers of the anammox hzsA genes, different levels of diversity, compositions, and abundances of the anammox communities were observed in the collected soils, suggesting an uneven distribution of these communities. The uneven distribution of the anammox communities may be influenced by the local soil conditions, though the environmental conditions that determine the distribution of anammox bacteria in ecosystems are currently not well described. Combing the physicochemical properties of the collected samples and the anammox 16S rRNA gene sequences, the soil organic content and the ammonium content were found to be the most important factors influencing the distribution of the anammox communities in the examined soils. Nitrogen-rich fertilizers are the major source of soil ammonium, and cultivation history and cropping system are the important factors influencing the soil organic content in agricultural soils. This indicates that human activities (soil cultivation and soil fertilization) have indirectly influenced the distribution of anammox bacteria in the collected soils.

It has been estimated that the anammox process is responsible for more than 50% of the marine nitrogen loss (10, 43, 44). Furthermore, the anammox process was reported to account for 9 to 40% of the nitrogen loss in freshwater ecosystems (18, 20, 45, 46). At the global scale, however, the overall contribution of anammox to the global nitrogen cycle is still unclear because the role of anammox in terrestrial ecosystems has not been determined due to the limited data available. The anammox process was recently reported to account for 4 to 37% of the soil nitrogen loss in a fertilized paddy soil (23). In the present study, both the anammox bacterial 16S rRNA and hzsA genes (ranging from 10⁴ to 10⁶ copies per gram of dry weight) were detected in the various agricultural soils collected from 32 different locations in China. The broad distribution of anammox bacteria in the Chinese agricultural soils suggests that the anammox process is also a widespread process in terrestrial habitats and that it may play an important role in the nitrogen cycle of terrestrial ecosystems. In future studies, direct determination of the activity of anammox bacteria in Chinese agricultural soils will be required to better understand their quantitative contribution to the terrestrial nitrogen cycle.