Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane-oxidizing bacteria in a paddy soil

Abstract

The anaerobic ammonium-oxidizing (anammox) and nitrite-dependent anaerobic methane-oxidizing (n-damo) bacteria in a paddy soil core (0–100 cm) were investigated with newly designed primers targeting the hydrazine synthase β-subunit (hzsB) of anammox bacteria and the recently published primers targeting the pmoA and 16S rRNA genes of n-damo bacteria. The hzsB gene was identified as a proper biomarker to explore the anammox bacterial biodiversity and abundance in soil. The anammox bacteria were present throughout the soil core with the highest abundance of 2.7 × 10⁶hzsB copies g⁻¹ dry soil at 40–50 cm and were not detectable below 70 cm. Sequences related to at least three species of known anammox bacteria, ‘Brocadia anammoxidans’, ‘Brocadia fulgida’, and ‘Jettenia asiatica’ were detected. By combining the analysis of pmoA and 16S rRNA genes, the n-damo bacteria were observed to be present in 30–70 cm with abundance from 6.5 × 10³ (60–70 cm) to 7.5 × 10⁴ (30–40 cm) copies g⁻¹ dry soil. The pmoA sequences retrieved from different depths closely related to each other and formed a unique clade. Our results showed that anammox and n-damo bacteria co-occurred in the paddy soil. Both of them were abundant in deep layers (30–60 cm) and the community structures changed along depths in the soil core.

Introduction

Ammonium () and methane (CH₄) were previously assumed to be inert under anoxic conditions (Strous & Jetten, 2004; Jetten, 2008). This understanding was gradually changed by the discoveries of anaerobic ammonium oxidation (anammox) (Van de Graaf et al., 1995; Strous et al., 1999) and nitrite-dependent anaerobic methane oxidation (n-damo) (Raghoebarsing et al., 2006; Ettwig et al., 2009, 2010) in which and CH₄ were oxidized anaerobically using nitrite as the electron acceptor.

With the development of molecular biomarkers (Kuypers et al., 2003; Schmid et al., 2005, 2008; Li et al., 2010, 2011; Li & Gu, 2011), anammox bacteria have been detected in many marine ecosystems (Kuypers et al., 2003; Byrne et al., 2009), freshwater ecosystems (Zhang et al., 2007; Zhu et al., 2010), and man-made environments (Quan et al., 2008; Zhu et al., 2011a). Using the isotopic pairing technology, anammox has been identified as an important process in the aquatic nitrogen cycle, accounting for as much as 13% of N₂ production in freshwater Lake Tanganyika (Schubert et al., 2006) and 67% in marine environments (Thamdrup & Dalsgaard, 2002). Although recently anammox bacteria were enriched from a peat soil (Hu et al., 2011), relative little is known about the distribution of anammox bacteria in soil ecosystems because of the lack of suitable primers for quantitative PCR assays and high interfering background in fluorescence in situ hybridization (FISH) analyses by soil matrix components. Hydrazine synthase is a key enzyme in the anammox metabolism, consisting of three subunits encoded by the genes hzsA, hzsB, and hzsC (Strous et al., 2006; Kartal et al., 2011; Harhangi et al., 2012), responsible for the synthesis of hydrazine from nitric oxide and ammonium (Kartal et al., 2011). Previously, the hzsA gene was used as an anammox phylomarker (Harhangi et al., 2012). Here, we developed an additional phylomarker based on the hzsB gene for the biodiversity and quantitative analyses of anammox bacteria in a paddy soil.

Enrichments of n-damo bacteria, members of NC10 phylum, were started from freshwater sediments (Raghoebarsing et al., 2006; Ettwig et al., 2009) and wastewater treatment sludge (Luesken et al., 2011a, c). In 2010, the genome of Methylomirabilis oxyfera, the bacterium responsible for n-damo, was assembled and analyzed (Ettwig et al., 2010). The remarkable presence of genes encoding for particulate methane monooxygenase (pmoCAB) in this anaerobe was explained by its unusual intra aerobic metabolism. Recently published primers specifically targeting the pmoA subunit of n-damo bacteria were used to screen environmental samples, and n-damo bacteria were detected in a wide range of freshwater habitats (Deutzmann & Schink, 2011; Luesken et al., 2011b; Kojima et al., 2012).

Paddy fields are characteristized by cultivation patterns including water logging, which causes anoxic soil conditions. Plant-derived organic substances additionally serve as an important carbon source for CH₄ (Lu & Conrad, 2005). In addition, application of nitrogen-rich fertilizers makes the paddy field a favorable habitat for both anammox and n-damo bacteria. In the present study, we aimed to explore the co-occurrence and vertical distributions of anammox and n-damo bacteria in a paddy soil core with our newly developed anammox primers targeting the β subunit of the hydrazine synthase (hzsB gene) and the primers targeting the pmoA and 16S rRNA genes of n-damo bacteria. Both quantitative and biodiversity analyses are reported.

Materials and methods

Site description and sampling

A paddy field with long-term manure fertilization practice in subtropical China (E120°41′50″ N30°45′50″) was selected for this study. Five soil cores (1m distance) were collected by a stainless steel ring sampler (5cm diameter and 100cm depth) from the field in October 2009 at the growth season. The soil cores were placed in sterile plastic bags, sealed, and transported to the laboratory on ice. Later they were sliced at 10-cm intervals, and slices of the same depth were mixed to form one composite sample. One part was sieved through 2.0-mm sieve for the chemical analysis, and subsamples were used for DNA extraction.

To evaluate the designed primers, biomass from several anammox enrichment cultures in bioreactors from our laboratory (Nijmegen, The Netherlands) were sampled including ‘Candidatus Kuenenia stuttgartiensis’, ‘Candidatus Brocadia fulgida’, ‘Candidatus Brocadia anammoxidans’, ‘Candidatus Scalindua sp.’, and ‘Candidatus Jettenia asiatica’. The cultures were each dominated at 70–95% by single anammox species. The enrichment and cultivation profiles see the previous works (Kartal et al., 2007; Quan et al., 2008; Schmid et al., 2008). Environmental samples from wastewater treatment plants (WWTPs) and sediment were investigated from the Rotterdam and Lichtenvoorde full-scale anammox reactors (Van der Star et al., 2007) and ditches in the Ooijpolder, Olburgen, and Propionaat (The Netherlands), respectively (Hu et al., 2011; Harhangi et al., 2012).

Chemical and statistical analyses

Ammonium, nitrite, and nitrate were extracted from the soil with 2M KCl and measured using a SAN++ Continuous Flow Analyzer (Skalar Analytical, The Netherlands). Total nitrogen, soil organic matter, Mn²⁺, and Mn⁴⁺ were measured according to standard methods (Bao, 2000). Soil pH was determined at a soil/water ratio of 1:2.5. All analyses were performed in triplicate on each sample. The in situ measurement of oxygen concentration was achieved by OXY Meter S/N 4164 with stainless electrode sensor (Unisense, Denmark) (Gundersen et al., 1998). Statistical analyses were performed using program spss for Windows.

DNA extraction and PCR amplification

DNA in soil and sediment samples were extracted from 0.25g samples using the Powersoil DNA isolation kits (Mobio). DNA from enriched anammox biomass was extracted according to the method described previously (Schmid et al., 2008). For the specific PCR amplification of the anammox hzsB gene, newly designed primer pair of hzsB_396F and hzsB_742R was applied based on our new findings in anammox molecular mechanism (Kartal et al., 2011; Harhangi et al., 2012). The pmoA gene of n-damo bacteria was amplified using a nested approach (first-step primer pair A189_b-cmo682, followed by primer pair cmo182-cmo568) according to Luesken et al. (2011c). The 16S rRNA gene of n-damo was amplified using a nested approach (first-step primer pair 202F-1545R, followed by primer pair qP1F-qP2R) according to Juretschko et al. (1998) and Ettwig et al. (2009). The sequences of primers and thermal profiles were shown in Table 1. PCRs were performed with the PerfeCTa SYBR Green FastMix (Quanta).

Cloning, sequencing, and phylogenetic analysis

PCR amplified fragments were cloned using the pGEM-T Easy cloning kit (Promega) according to the manufacturer's instructions. Plasmid DNA was isolated with the GeneJET Plasmid Miniprep kit (Fermentas, Lithuania). Plasmids were digested with EcoRI enzyme, and the digestion products were examined for an insert with expected size by agarose (1%) gel electrophoresis. Selected clones were sequenced using primer of M13f targeting vector sequences adjacent to the multiple cloning sites. Phylogenetic analysis was performed using mega 5.0 software (Tamura et al., 2011) by neighbor-joining (NJ) with the Jukes-Cantor correction. Diversity indices, including Chaol, Shannon, and Simpson, were generated by DOTUR for each clone library (Schloss & Handelsman, 2005).

Quantitative PCR analysis

Quantitative PCR was performed on a Bio-Rad iQ5 real-time PCR instrument (Bio-Rad) with a SYBR Green qPCR kit (Quanta). The quantitative PCR of anammox hzsB gene was performed with the same primer pair used for the PCR cloning. The quantitative PCR of n-damo 16S rRNA gene was performed with specific primers qP1F-qP1R described previously (Ettwig et al., 2009). Total bacterial numbers were quantified with the primer pair 616F-Eub338-IR specific for the 16S rRNA gene (Amann et al., 1990; Juretschko et al., 1998). Standard curves were obtained with serial dilutions of plasmid DNA containing the target genes.

Nucleic acid sequence accession numbers

The sequences reported in this study have been deposited in the GenBank database under accession numbers JN704402–JN704415 (n-damo pmoA), JN704416–JN704466 (n-damo 16S rRNA ), and JN704467–JN704568 (anammox hzsB).

Results and discussion

Biogeochemistry of the soil core

Owing to the long-term fertilizations, the concentrations of nitrogen compounds (, and total nitrogen) and total organic matter (TOM) in soil were very high (Supporting Information, Fig. S1). Most of the highest values were observed in the upper 10-cm layers except for which was peaked at 10–20cm (up to 158.8mgkg⁻¹ dry soil). For , the common electron acceptor for anammox and n-damo bacteria, the highest concentration (53.8mgkg⁻¹ dry soil) was present at 0–10cm. After a rapid decrease at 10–30cm (11.6±0.3mgkg⁻¹ dry soil), a slight increase in was observed at 30–50cm of 12.5±0.3mgkg⁻¹ dry soil, providing a potentially suitable condition for the growth of anammox and n-damo bacteria.

Anammox-specific primer design and verification

In addition to the previous work exploiting the hzsA gene (Harhangi et al., 2012), we focused on the hzsB gene in this study. A data set with hydrazine synthase β-subunit DNA and protein sequences from the known anammox bacteria of Candidatus genera ‘Jettenia’, ‘Brocadia’, ‘Scalindua’, ‘Kuenenia’, and Planctomycete KSU-1 available from metagenome sequencing projects and GenBank were aligned. Conserved regions of the aligned sequences were identified and used as the targets for designing degenerate primers (Fig. S2). Six forward and five reverse degenerate primers were designed based on the alignment. The sequences and positions on the gene were shown in Table S1 and Fig. S3. Different combinations of the designed primers were tested and evaluated with template DNA extracted from anammox enrichment cultures. High intensities of specific band (c. 365bp) were observed (Figs S4–S7) using the primer pair of hzsB_396F and hzsB_742R (at annealing temperature 59°C and with 2–2.5mM MgCl₂) by single-step amplification instead of nested PCR which was previously required for soil samples (Humbert et al., 2010; Hu et al., 2011; Zhu et al., 2011b). The PCR products were cloned and sequenced, and a phylogenetic tree of the retrieved hzsB sequences from anammox enrichment cultures was constructed (Fig. S8a). The phylogeny of hzsB was consistent with that of the 16S rRNA gene (Fig. S8b) (Schmid et al., 2008) and the hzsA gene (Harhangi et al., 2012).

For the molecular detection of anammox bacteria in soil, the 16S rRNA gene was the most common used biomarker (Humbert et al., 2010; Hu et al., 2011; Zhu et al., 2011b). However, the amplified PCR products must be verified with subsequent cloning and sequence analyses because nonanammox bacterial 16S rRNA genes were also amplified with the current PCR protocols (Song & Tobias, 2011). In this study, the specificity was promising when using functional hzsB gene as the biomarker that the retrieved sequences were all closely related to the known anammox bacteria. Four catalytic proteins (nitrite and nitrate reductases, hydrazine synthase, and hydrazine dehydrogenase) were possibly used as the biomarkers for the molecular detection of anammox bacteria in present study (Strous et al., 2006; Kartal et al., 2011). The hydrazine synthase was the most unique one (no multiple copies present) (Harhangi et al., 2012) compare with the other functional genes that were present in both anammox and nitrifying or denitrifying bacteria (Song & Tobias, 2011). The application of hzsB gene would avoid the ambiguous differentiation between the anammox and nitrifiers or denitrifiers sequences.

Community structures of anammox bacteria

The community structures of anammox from four representative depths (0–10, 20–30, 40–50, and 60–70cm) of the soil core were analyzed by amplifying their hzsB gene. Ninety-two anammox hzsB clone sequences were retrieved and shown to be closely related to the ‘Kuenenia stuttgartiensis’ hzsB gene (AB365070) present in GenBank (identities up to 82–85% for nucleotide and 90–93% for protein sequence). Phylogenetic analysis showed that the clone sequences were related to the anammox bacterial genera Candidatus ‘Brocadia’ and ‘Jettenia’ (Fig. 1). Most of the sequences (79/92) were closely related to Candidatus genus ‘Brocadia’ which comprises the ‘Candidatus Brocadia fulgida’ of group 1 (44/79) and ‘Candidatus Brocadia anammoxidans’ of group 2 (35/79). It confirmed the previous conclusion that representatives of the Candidatus genus ‘Brocadia’ were the most frequently detected anammox genus in soils (Humbert et al., 2010). Group 3 with 13 sequences was clustering in between the Candidatus genera ‘Brocadia’ and ‘Jettenia’. It was in agreement with a recent study revealing unknown anammox species distantly related to Candidatus ‘Brocadia’ and Candidatus ‘Jettenia’ in soil (Hu et al., 2011). However, this result must be interpreted with caution because of the absence of Candidatus genera ‘Anammoxoglobus propionicus’ as a reference in the phylogenetic analysis. It is noted that all the 16 sequences retrieved from the surface soil (0–10cm) were identical (difference up to 98–100% nucleotide identity) and most closely related to the ‘Candidatus Brocadia anammoxidans’ group. In contrast, sequences from other depths were very divergent. These results confirmed that the community composition of anammox bacteria in soil changed with depth (Zhu et al., 2011b).

The biodiversity and coverage analysis of the clone library targeting the hzsB gene were conducted and compared with the 16S rRNA gene at the same location of our previous study (Zhu et al., 2011b). The rarefaction curve built from the 16S rRNA gene reached the saturation easily, whereas that of the hzsB gene suggested that higher diversity could be observed if more clones were sequenced (Fig. S9). It is further confirmed by the coverage estimators of Chao1, which showed a high value of the hzsB clone library than that of the 16S rRNA gene (16.9 vs. 5). The Shannon (2.2 vs. 1.35) and Simpson (0.14 vs. 0.27) indices also implied a higher diversity of anammox bacteria by amplifying the hzsB gene. Compared with primers targeting the hzsA subunits, similarly high specificities were observed that no false positives were detected in 92 (hzsB) and 46 (hzsA) clones.

Abundance of anammox bacteria

The primer pair of hzsB_396F and hzsB_742R was applied for the quantification of anammox bacterial abundance in the soil core. The copy number measured in the surface sample (0–10cm) was 7.0±0.3×10⁵copiesg⁻¹ dry soil and decreased slightly to 2.0±0.9×10⁵copiesg⁻¹ dry soil at 20–30cm depth as shown in Fig. 2a. Below this depth, hzsB gene copy numbers increased and peaked at 40–50cm depth of 2.7±1.3×10⁶copiesg⁻¹ dry soil, which accounts for about 2.3% of total bacterial cells (Fig. 2c) assuming that the anammox bacteria contained one copy of the hzsCBA gene cluster (Strous et al., 2006; Kartal et al., 2011) and 3.8 copies of the 16S rRNA gene for all bacteria (Fogel et al., 1999). For the samples below 60cm, the copy numbers decreased below the detection limit of the qPCR assay.

The variety in anammox bacterial abundance in the soil core was more or less similar to the result based on 16S rRNA gene from the same site (Zhu et al., 2011b). Little significant correlation was observed between the abundance of anammox bacteria and environmental factors (Table 2). Similar to the anammox in stratified water columns and sediments where active anammox was restricted to specific layers (Dalsgaard et al., 2003, 2005), anammox bacteria seemed to prefer selective niches at particular depths in soil (Humbert et al., 2010). Owing to the high interfering background in soil samples, only the primers targeting the 16S rRNA gene were capable for the in situ quantification of soil sample until now (Hamersley et al., 2007; Hu et al., 2011; Zhu et al., 2011b). As the specificity and sensitivity of 16S rRNA gene detection are highly dependent on the abundance of anammox bacteria in environmental samples (Song & Tobias, 2011), the hzsB gene would be a more precise biomarker for the quantification of anammox in soil.

Community structures of n-damo bacteria

To analyze the community structure of n-damo bacteria on a functional level, primers targeting the pmoA gene were used in samples from representative depths (0–10, 20–30, 40–50, and 60–70cm). The n-damo-specific pmoA primer A189_b was combined with the widely applied cmo682 primer (Holmes et al., 1995; Luesken et al., 2011c). Following by a nested PCR approach (cmo182-cmo568) (Luesken et al., 2011c), sequences clustering with the pmoA sequence present in the genome of M.oxyfera were retrieved in the samples except the surface soil (0–10cm), which did not result in a positive PCR product at the second amplification. The absence of pmoA sequence in surface soil suggested a preferred habitat in deep soil for n-damo bacteria. The 14 sequences retrieved from the other three depths together with the published pmoA, pxmA and amoA nucleic acid sequences were phylogenetically analyzed (Fig. 3). Most of the sequences in this study showed high identity to each other and were closely related (difference up to 90–92% nucleotide and up to 94–95% protein identity) to the pmoA gene of M.oxyfera (FP565575 or CBE69519). The sequences obtained from the paddy soil formed a unique clade in the tree along with other pmoA sequences from ditch, aquifer environments, and WWTPs reported previously (Luesken et al., 2011a, c). The low diversity of pmoA sequences obtained from the paddy soil was consistent with previous studies (Deutzmann & Schink, 2011; Luesken et al., 2011c; Kojima et al., 2012). The fact that the sequences obtained were not highly divergent from each other was probably caused by the functional conservation of pmoA gene reflected by the unique oxygenic pathway of n-damo bacteria (Luesken et al., 2011c). In addition, the primers used in this study were designed based on the limited references available. It cannot be ruled out that they were too narrow to cover all the pmoA gene of the n-damo bacteria (Deutzmann & Schink, 2011). Therefore, further improvement in specific primers was needed to analyze the diversity of the n-damo at a functional level (Kojima et al., 2012).

Abundance of n-damo bacteria

Because there was no suitable primer pair targeting the pmoA gene for qPCR so far, the abundance of n-damo bacteria was estimated by quantifying their 16S rRNA gene. The copy numbers ranged from 1.0±0.1× 10⁵ (0–10cm) to 7.5±0.4×10⁴copiesg⁻¹ dry soil (30–40cm; Fig. 2b). Below 40cm depth, the abundance decreased gradually from 4.9±0.1×10⁴ (40–50cm) to 6.5±0.4×10³ (60–70cm) copiesg⁻¹ dry soil. Below 70cm depth, the abundance decreased beyond the limit of detection. As the primers used were designed based on enrichment samples and have not been previously applied on environmental samples. Therefore, the clones of 16S rRNA gene were also sequenced for a comparison with the known n-damo bacteria (Fig. S10). The phylogenetic analysis showed that sequences from 40 to 50 and 60 to 70cm depths clustered within group a, which comprises sequences closely related to the enrichment n-damo bacteria (DQ369742) (Ettwig et al., 2009), whereas sequences from 0 to 10 and 20 to 30cm depths were distantly related to the known n-damo bacteria. This means the quantification based on the 16S rRNA gene probably overestimated the abundance in the upper soils because of the less specificity of the primer set. Taken together, n-damo bacteria were most abundant at 30–70cm depth below the plough pan layer, which was in agreement with the previous study that the n-damo bacteria was only present in profundal sediment (Deutzmann & Schink, 2011). Correlation analysis showed that chemical profiles like pH and TOM correlated well with the abundance of n-damo as shown in Table 2. But in consideration of the flaws in specificity of the primers used, it was hard to find connections between the abundance of n-damo and chemical profiles. There was not a clear interpretation for the vertical distribution of n-damo bacteria in natural ecosystem so far. However, recent enrichment study of n-damo has identified that the addition of oxygen resulted in an instant decrease in methane and nitrite conversion rates (Luesken et al., 2012). Therefore, the absence of n-damo bacteria in surface soil might be caused by the possible penetration of oxygen into the surface soil that negatively affects these anaerobes.

On the whole, the results in this study showed that the anammox and n-damo bacteria co-occurred in the paddy soil. The hzsB gene was identified as a novel biomarker for the molecular detection of anammox bacteria. The quantitative PCR and clone library analyses performed in this study indicated both of anammox and n-damo bacteria were abundant in deep layers (30–60cm). Further studies are required to explore the function and relation of anammox and n-damo bacteria in paddy soil.

Acknowledgements

This research is financially supported by the National Natural Science Foundation of China (21077119), Knowledge Innovation Program of the Chinese Academy of Sciences (KZCX2-EW-410-01), and special fund of State Key Joint Laboratory of Environment Simulation and Pollution Control (12L03ESPC). Moreover, the author G.Z. gratefully acknowledges the support of Beijing Nova Program (2011095) and K. C. Wong Education Foundation, Hong Kong. The anammox research of M.S.M.J. is supported by ERC Advanced Grant 232937.

References

Supporting Information

Fig. S1. Vertical profiles of , , pH, total nitrogen (TN), total organic matter (TOM), disolved oxygen (DO) and Mn (II–IV) in the paddy soil.

Fig. S2. Sequence alignment of hzs gene β subunit and primers design.

Fig. S3. Primers designed in this study and positions indicated refer to the ‘Ca. Kuenenia stuttgartiensis‘ hzsB gene (kuste2860).

Fig. S4. PCR test result of primer combinations on enriched Kuenenia gDNA (annealing temperature 55 °C).

Fig. S5. PCR test result of primer combinations on enriched Brocadia gDNA (annealing temperature 55 °C).

Fig. S6. PCR test result of selected primer combinations on different enriched gDNA (annealing temperature 55 °C).

Fig. S7. PCR test result of selected primer combinations on enriched Brocadia gDNA in a gradient PCR with the annealing temperature ranging from 53.5 to 58.4 °C.

Fig. S8. (a) Phylogenetic analysis of hzsB gene sequences from anammox enrichment cultures with designed primer set hzsB_396F and hzsB_742R. The evolutionary history was inferred using the neighbor-joining method. The bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in > 50% bootstrap replicates were collapsed. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Jukes-Cantor method and are shown as numbers of base substitutions per site. (b) For comparison, a 16S rRNA gene-based phylogenetic tree was shown [adapted from reference (Schmid et al., 2008)]

Fig. S9. Rarefaction and diversity analysis of anammox (hzsB and 16S rRNA genes) bacteria.

Fig. S10. Phylogenetic tree of the deduced n-damo and NC10 phylum bacterial 16S rRNA gene sequences (shown in bold) from paddy soil.

Table. S1. Sequences of designed hydrazine synthase primers targeting the hzsB subunit of anammox bacteria.

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