Detection and Potential Abundances of Anammox Bacteria in the Paddy Soil

Introduction

Anaerobic ammonium oxidation (anammox) process is one of the distinct microbial processes, which is responsible for the release of nitrogen gas to the atmosphere [1]. Even though its molecular mechanism is not completely understood, it has been proposed that the reaction proceeds through hydrazine (N₂H₄), which is produced from an ammonia and nitric oxide (NO). Nitrite is reduced to NO, and then ammonium is oxidized to yield hydrazine (N₂H₄), followed by hydrazine oxidation to N₂, yielding four electrons [2-4].

Anammox bacteria have been discovered mostly in wastewater sludge since the early 1990s, and have been reported to belong to Planctomycetes [5,6]. These anammox bacteria have been divided into five genera i.e., Kuenenia [4,7], Brocadia [5,8], Anammoxoglobus [9], Jettenia [10], and Scalindua [11-13]. Scalindua has been reported mainly in the marine environment, and some have been also detected in freshwater ecosystems and wastewater treatment plants. Anammox bacteria have been detected also in permafrost and agriculture soils based on 16S rRNA gene probing [14] using different sets of PCR primers for their profiling [15].

The growth rates of these bacteria are slow and doubling times are approximately 2-3 weeks [16,17]. Many factors are believed to be responsible for their slow growth rates such as ammonium, nitrite, pH, temperature, organic matter, and iron content in natural habitats. Efforts have been made to optimize the growth of the bacteria in laboratory conditions by mimicking similar environments as in the natural habitats using enrichment cultures and bioreactors such as fed-batch reactor and sequencing batch reactor [2,18].

Anammox bacteria play a prominent role in the nitrogen cycle [19]. They participate in the biogeochemical nitrogen production in paddy soils by 4-37% [20] and 50% of nitrogen loss from marine environment [21,22]. Besides the ecological aspect, their practical applications in the elimination of ammonia from wastewater sludges, industrial effluents, and leachates suggest sustainable and environment-friendly technological aspects of the microorganism [3].

Even though increased interests on anammox bacteria, their diversity, distribution, and the ecological processes mediated by them have not been well understood over the last two decades, and the information about these is limited, especially on anammox in the terrestrial habitats. It seems that the slow growth rateand the difficult buildup of the biomass of these bacteria contributed to the limited information [23]. The main aim of this work was to find the distribution and abundance of anammox bacteria in the terrestrial niche, particularly of rice paddy soil by using molecular approaches such as clone library and next-generation sequencing (NGS), for the better understanding of these bacteria, in the terrestrial habitat rather than the oceanic and sludge environments.

Materials and Methods

Sampling and analyses of physico-chemical parameters of paddy soil

A rice paddy field site was selected for soil sampling, that is located inside Rural Development Administration, Jeonju, South Korea (latitude 35°49’42.4’’N and longitude 127°02’38.4’’E; Fig. S1). Soil samples were taken at 30 cm depth (RPS1) from the ground level and from the surface level (RPS2) of a rice paddy field by using a small shovel. Three spots were sampled into individual tubes for each soil depth. Soil slurry with water was prepared in the ratio of 1:5 by shaking for 30 min at shaker to measure the pH and electrical conductivity by using portable probes and meter (Thermo Scientific, Pittsburgh, PA, USA). Measurement of organic carbon was done by detecting the carbon loss on ignition at 360℃ [24]. Total organic nitrogen, nitrate, and ammonia were measured by using Dahnke method [25]. Chromic acid digestion and spectrophotometric procedure were used to measure the total organic carbon [26] and total iron was measured by using acid digestion method by inductively coupled plasma-atomic emission spectroscopy.

DNA extraction from soil samples and PCR

Genomic DNA was extracted from the rice paddy soils by using MoBio Powersoil DNA kit (Carlsbad, CA, USA), following the manufacturer protocol using 0.25 mg of the soil samples. The triplicate DNA extracts from each soil depth were pooled into one tube for downstream experiments. The DNA quantity was measured by fluorometry (Qubit 3.0 Fluorometer, Invitrogen, Pittsburgh, PA, USA). Genomic DNA was diluted to 1-5 ng μl-1 for further use. Nested PCR was performed in order to amplify 16S rRNA genes of anammox bacteria. Bacterial universal primer set of 27F/1391R was used for first round of PCR, followed by the second round of PCR using the primer sets of Pla46F/Amx820R [7,27], and Amx368F/Amx820R [7,28]. All the primers used in this study are summarized in Table S1 (Supplementary Information). A total of 50 μl was prepared for the PCR reactions, including 1-2 μl of genomic DNAs (1-5 ng μl-1), and 1 μl of forward and reverse primers (10 pmol). The thermal cycles were operated with an initial denaturation at 95℃ for 2 min followed by 30 cycles of denaturation at 94℃ for 30 sec, annealing at 50℃ for 30 sec, and an extension at 72℃ for 1 min 30 sec. Final extension was done at 72℃ for 5 min. The quality and quantity of purified DNA were checked, respectively by gel electrophoresis and fluorometry.

Clone library construction and confirmation of positive clones

DNA templates of specific band size after amplification from the nested PCR and purification were ligated into pTOP TA V2 (vector) of TOP cloner TA kit (Enzynomics, Daejon, South Korea) and transformed into E. Coli DH5α^TM competent cells (Enzynomics, Daejon, South Korea). Blue-white screening was performed by using ampicillin (0.1 mg ml^-1), IPTG (0.1 mM), and X-gal (40 μg ml^-1) in LB (lysogeny broth) agar plate. White colonies were selected and incubated in LB broth containing 0.1 mg ml^-1 of ampicillin for overnight. Plasmid DNA extraction was done by using Inclone mini plasmid preparation kit (Inclone biotech, Yongin, South Korea). Plasmid DNAs isolated from the white colonies were subjected to PCR amplification using the multiple cloning site (MCS) primer set of M13F (-20) and M13R (-40) and the PCR products were checked on 1% agarose gel for confirmation of the positive clones. DNAs isolated from the clone library were sequenced at Genotech (Daejeon, South Korea).

Phylogenetic analysis of the clones

The forward and reverse nucleotide sequences of the cloned DNAs of 16S rRNA gene fragments were assembled together. The obtained sequences were compared to NCBI GenBank by using BLAST search [29], in order to find the known sequences so far, particularly for anammox bacteria and clones. Phylogenetic trees for the cloned anammox sequences were constructed together with the similar sequences retrieved from the GenBank by using Mega 7 [30]. The evolutionary history was inferred using the neighbor-joining method [31,32].

Soil microbial community analysis

Two genomic DNAs isolated from the soil samples (surface and 30 cm-deep soils) were subjected to next-generation sequencing (NGS) for the V3-V4 region of 16S rRNA gene using 341F/805R primer set by Miseq system (Illumina, San Diego, CA, USA), which generated paired- end sequences of approximately 428 bp-long (250 bp x 2). The raw sequence data were analyzed using the Mothur version 1.43.0 [33], referring to the Miseq sequence standard operating procedure [34]. The sequences were contig-assembled, filtered, and screened for the undesirable sequences such as low-qualityreads, sequencing errors, chimers, archaea, etc. resulting in the total of 122,456 sequences from the initial 508,977 reads. Chimeras were screened by Vsearch [35] and the sequences were aligned with Silva database release v. 132 [36]. The sequences were clustered into 14,403 operational taxonomic units (OTUs) by 3% dissimilarity and classified to taxa by using RDP database [37].

Nucleotide accession Number

All the 16S rRNA gene sequences from clone library related to uncultured bacterial species, Planctomycetes , and anammox bacteria were deposited in GenBank with accession numbers of KY883355 to KY883367 respectively for clones PA-1, PA-2, PA-3, PA-4, AA-6, AA-7, PA-9, PA-11, PA-12, PA-13, PA-14, AA-15, and AA-17, and MF171191 and MF171192 for clones AA-5 and AA-16, respectively. The Miseq sequenced data were deposited at NCBI Sequence Read Archive (SRA) with accession number SRR7791714.

Results

There are many microorganisms in the agricultural soils, which contribute to the biogeochemical nitrogen cycle such as anammox bacteria. Although the anammox contribution to nitrogen loss by the generation of N₂ gas in the paddy soils is not as high as in the marine [20,29], investigating the anammox reaction in the rice paddy soils would provide a better understanding of the reaction in the terrestrial environments and the contribution to the nitrogen cycle. In this study, we have selected the soil samples from the rice paddy field, to study the distribution and abundances of microbial species with more focus on the anaerobic ammonium oxidation bacteria.

Physico-chemical parameters of soil

The measurement, as well as knowledge of physicochemical parameters of the soil, is very important because it will determine the quality, health of the soil, as well as the diversity of the microorganism [38]. The physico-chemical parameters of the soil (RPS1) such as soil texture, pH, electrical conductivity (EC), ammonia, nitrate, organic matter, total organic carbon, total nitrogen, and total iron is listed in (Table S2). The soil was found to be in good condition for agriculture.

Amplification of 16S rRNA gene from soil sample and clone library construction

Amplification of 16S rRNA gene from the rice paddy soil genomic DNA (RPS1) with 27F/1391R yielded a DNA band of approximately 1.4 kbp fragment (Fig. S2 A) and the subsequent amplification using Planctomycetes-specific and anammox-specific primer sets of Pla46F/Amx820R and Amx368F/Amx820R yielded approximately 700 bp - 800 bp and approximately 500 bp fragments, respectively (Fig. S2 B and C).

After a series of procedures for clone library construction such as the purification of amplicons, ligation, and transformation, among the white colonies by the blue-white screening, 15 colonies were taken and subjected to plasmid extraction. Positive clones were confirmed by PCR using the MCS primer set of M13F and M13R and the amplicons of cloned gene fragments were sequenced.

Seq uencing and phylogenetic analysis

Among the white colonies, 15 selected clones (PA-1, PA-2, PA-3, PA-4, AA-6, AA-7, PA-9, PA-11, PA-12, PA-13, PA-14, AA-15, and AA-17 with accession numbers KY883355 to KY883367; AA-5, and AA-16 with accession numbers MF171191 and MF171192) showed sequence similarities ranging from 94% to 99% with an uncultured bacterium. Some clones showed sequence similarities with Planctomycetes (PA-4 at 98%; PA-12, PA-13, and PA-14 at 97 %; PA-1 at 87 %) and 4 clones with anaerobic ammonium oxidation bacteria (PA-4 at 94%, PA-12, PA-13, and PA-14 at 95%). Rest of the clones showed sequence similarities with other microorganisms such as salt marsh clones, Proteobacteria and Firmicutes. The Phylogenetic trees for the evolutionary relationship on the basis of sequence similarities with the uncultured bacterium, Planctomycetes , anammox bacteria, and other microbial species like Firmicutes, saltmarsh clones etc. are shown in Figs. 1, 2, and 3. We have identified clues of Planctomycetes in the rice paddy fields by clone library construction, sequencing, and phylogenetic analysis.

Microbial community analysis by Miseq sequences

To examine diversity of microorganism including anammox bacteria in the particular field samples, microbial community analysis was conducted. The numbers of nucleotide sequences of the two samples (RSP1 from 30 cm- depth and RPS2 from the surface) were normalized to 53,532 to perform alpha-diversity analysis, which showed Good’s coverage values of 91.2% and 91.5% respectively, for RPS1 and RPS2 (Table S3), indicating sufficient resolution of the microbial communities. Bacterial species richness, which is a count of species, calculated by Chao1 was found to be 23,146 and 26,638, suggesting higher bacterial counts at the surface soil (RPS2) (Table S3). The bacterial diversity indices of Shannon and inverse Simpson, reflecting both the richness and species evenness showed opposite patterns of the values, while inverse Simpson index was in consistent pattern with Chao1 index, indicating higher bacterial diversity in RPS2.

Excluding the most abundant unclassified Bacteria (27.8% and 38.5%, respectively for RPS1 and RPS2) at phylum level, the distribution of bacteria showed that Chloroflexi was abundant at 21.7% and 19.0%, respectively for soils RPS1 and RPS2, followed by Acidobacteria at 12.4% and 21.6%, respectively (Fig. 4). Phylum Planctomycetes, where the anammox bacteria belong was at 1.7% and 1.2%, respectively for the 30-cm depth (RPS1) and the surface (RPS2) soils. The similar type of result was reported by [39], where no difference in abundance of anammox was detected between the surface (0-5 cm) and subsurface (20-25 cm) soils. The community composition at genus level suggested as shown by sequence abundance revealed that unclassified Bacteria were the most abundant at 27.8% and 38.5%, respectively for the RPS1 and RPS2, followed by unclassified Anaerolineaceae (16.7% and 12.8%, respectively) and unclassified Gp3 (4.9% and 7.8%) (Fig. 5). We detected the anammox bacterial 16S rRNA genes, but their abundances were found to be low which was supported by the microbial community data by Miseq sequencing. Although relatively high abundances of the phylum Planctomycetes were found with 1.7% and 1.2% for two soil samples, anammox bacterial sequences were very minor.

Discussion

Based on the above-presented results, we could suggest that there were very low abundances of anammox bacteria in the paddy soils. Although the paddy field is not a completely natural environment, it is also very different from an engineered environment of wastewater sludges from which most anammox bacteria have been enriched. Various factors such as excessive use of fertilizers, oxygen availability, pH and/or total organic carbon in the soil environment, could be responsible for low abundance of these bacteria [40]. In addition, several studies have shown that the commonly used 16S rRNA gene primers could not extend the diversity of these anammox bacteria, and analysis on functional genes could be a better option for future [16]. Rice paddy fields have been focused as a model for habitats of diverse microorganisms [41]. Still many aspects of questions, however, are remained about the indigenous microorganisms, especially on the anammox bacteria in the paddy soils, and their potential roles on the biogeochemical nitrogen cycle.

Bacteria-mediated anaerobic ammonium oxidation (anammox) resulting in conversion of ammonium and nitrite to dinitrogen gas is one of the important processes in environments such as wastewater, ocean, freshwater, and terrestrial habitat. Researchers have identified and reported anammox reaction and anammox bacteria in the above-mentioned specific environments, but still there is only limited information regarding their diversity and roles in terrestrial habitats. This study will help to contribute towards understanding and exploring the anammox bacteria in terrestrial habitat.

Note

The authors declare no conflict of interest.

ACKNOWLEDGEMENT

This research was supported by the “Basic Science Research Program” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1D1A3B01012231).

Tables & Figures

Fig. 1. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of clones PA-1, PA-2, PA-3, PA-9, and PA-11 obtained from the clone library with known anammox bacterial 16S rRNA gene sequences.

Fig. 2. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of clones PA-4, PA-8, PA-12, PA-13, and PA-14 obtained from the clone library with known anammox bacterial 16S rRNA gene sequences.

Fig. 3. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of clones AA-5, AA-6, AA-7, AA-15, AA-16, and AA-17 obtained from the clone library with known bacterial 16S rRNA gene sequences.

Fig. 4. Relative sequence abundance of 21 bacterial phyla out of total 29, based on OTUs clustered at 97% similarity (excluding phyla less than 0.01%).

Fig. 5. Relative sequence abundance of bacterial genera. (A) 19 out of total 349 genera with exclusion of genera whose sequence abundances were less than 1% in both the samples. (B) 10 genera classified as Planctomycetes .

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