Journal of Earth Science  2019, Vol. 30 Issue (2): 397-406   PDF    
Vertical Variation of Nitrogen Fixers and Ammonia Oxidizers along a Sediment Profile in the Dajiuhu Peatland, Central China
Xu Ying , Wang Hongmei , Xiang Xing , Wang Ruicheng , Tian Wen     
State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan 430074, China
ABSTRACT: To investigate the vertical variation of microbial functional groups particular nitrogen fixers and ammonia oxidizers, sediment samples from a 155 cm deep peat profile were collected from the acidic Dajiuhu Peatland and subsequently subjected to clone library construction and quantification. Results showed that nifH gene abundance varied between 105-108 copies per gram dry sediment and reduced gradually with depth. The abundances of ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) abundance were generally comparable in each sample. More AOA was observed with a depth ≤55 cm, whereas AOB was more abundant with a depth >55 cm. Phylogenetically nifH could be divided into 94 OTUs which mainly affiliated with α-Proteobacteria. AOA were affiliated with Nitrosotalea from Group 1.1a associated (nearly 90%) and Nitrososphaera from Group I.1b. All AOB belonged to Nitrosospira. Notably, DJH11 with the depth of 50-55 cm was observed to have the highest abundance and the highest diversity of nitrogen fixers and ammonia oxidizers among all the samples. Pearson's correlation analysis showed a positive relationship between water content and pH with the nifH gene abundance. Our results offer the first insight about the microbial community composition and diversity involved in nitrogen cycles in the Dajiuhu Peatland.
KEY WORDS: nifH    amoA    acidic sediment profile    abundance    community structure    

Peatlands are widely distributed around the world except Antarctica (Wang et al., 2013), which reserve one-third soil carbon of the world and thus served as important carbon sinks in the global carbon cycle (Luo et al., 2016). Moreover, peatlands also contain 9%-16% of soil nitrogen (N) (Limpens et al., 2006) and play a fundamental role in the global nitrogen cycling. Accumulation of nitrogen in peatlands is a consequence of several factors, including low rates of N mineralization, low concentrations of dissolved inorganic N in pore waters, negligible gaseous N fluxes and low N losses by hydrologic export (Bobbink et al., 2010; Limpens et al., 2006). The nitrogen in bog ecosystems highly depends on atmospheric precipitation and plant nitrogen fixation (Pankratov et al., 2008). More recently, methanotrophs has been demonstrated to contribute greatly to plant N supply (1/3 of the total N in Sphagnum-dominant peatland) via symbiosis bacteria (Kip et al., 2010), which indicated the important role of microorganisms in nitrogen cycling and the coupling of carbon and nitrogen cycles in peatland ecosystems.

Nitrogen-fixing microbes can fix N2 from the atmosphere into NH4+ which can be subsequently utilized by other microorganisms and plants such as the dominant moss species, Sphagnum, in most peatlands (Kang et al., 2013). Nitrification process converts NH4+ to NO3- via two steps, in which ammonia oxidation process (NH4+→NO2-) is the first and rate-limiting step. Both bacteria (mainly β-Proteobacteria) and archaea (Thaumarchaeota) are involved in the first step of nitrification (Oved et al., 2001). More recently, comammox bacteria which mainly affiliated to Nitrospira have been isolated and confirmed to be capable of oxidizing ammonia to nitrite and then to nitrate all by themselves (Kits et al., 2017; van Kessel et al., 2015). However, their contribution to nitrification in natural environments is still poorly understood. Since nitrification greatly enhance the mobility and reduce the retention time of nitrogen in sediments (Bernot and Dodds, 2005), it is crucial to understand these fundamental processes in the peatland ecosystem in order to better sustain the ecosystem in the perspective of peatland management.

The nifH gene, encoding one of the metalloproteins of the nitrogenase complex, is one of the oldest functional genes and highly conserved, which also shows high congruence in phylogeny with that of 16S rRNA gene (Barron et al., 2009; Yeager et al., 2004). Therefore, nifH is a widely used molecular marker in the investigation of nitrogen-fixing microbial communities. It has been demonstrated that nifH-bearing microbial communities are highly diverse and vary from one place to another. In the ocean, single-celled Trichodesmium (Cyanobacteria) is the key nitrogen-fixer (Moisander et al., 2010), whereas more microbes including Proteobacteria, Firmicutes, Chlorobi, Bacteroidetes, Verrucomicrobia and Euryarchaeota are responsible for N2 fixation in sea sediments collected from different depths (Dang et al., 2013). Cyanobacteria, Proteobacteria and Firmicutes with nifH are detected in alkali soils (Kang et al., 2013). In oligotrophic high-altitude ice-free terrestrial Antarctic habitats, Cyanobacteria, particularly members of the Nostocales, dominate the N2 fixation communities within surface samples (Tahon et al., 2016). Recently, symbiotic methanotrophs rather than Cyanobacteria have been demonstrated to be substantially contributed to N2-fixation in Sphagnum mosses at pristine peatlands (Vile et al., 2014). Nevertheless knowledge about the microbial communities of nitrogen fixers and their correlation with environmental conditions in the peatland ecosystem is still poorly understood to date.

Ammonia oxidation is driven by both archaea and bacteria via ammonia monooxygenase encoded by amoA gene (Rotthauwe et al., 1997). Ammonia oxidizing archaea (AOA) are all affiliated with the archaeal phylum Thaumarchaeota which mainly includes Nitrosopumilus, Nitrososphaera (Tourna et al., 2011; Hatzenpichler et al., 2008), Nitrosocaldus (de la Torre et al., 2008) and Nitrosotalea (Lehtovirta-Morley et al., 2011). Three major clades are further defined as Group I.1a (marine), Group 1.1a-associated and Group 1.1b (soil) according to the phylogeny of amoA gene (Stahl and Torre, 2012). In contrast, ammonia oxidizing bacteria (AOB) are dominated by Nitrosospira and Nitrosomonas of β-Proteobacteria (Kowalchuk and Stephen, 2001) and Nitrosococcus of γ-Proteobacteria. Recently, Nitrospira has been confirmed to be capable of oxidizing ammonium to nitrate by themselves from both the genomic level and pure culture investigation (Kits et al., 2017; Cláudio, 2016; Daims et al., 2015).

To date, ammonia oxidizers in acidic peatlands are still far less investigated. Acidic peatlands are ammonia-limited and oligotrophic for ammonia oxidizers, which offer a unique ecosystem to study the microbially mediated nitrogen cycles. Therefore, to understand the distribution, diversity of microbial communities involved in nitrogen cycling and their correlation with environmental factors in the Dajiuhu Peatland, samples along a sediment core were collected and subjected to quantification and clone library construction of nifH and amoA genes. Multivariate statistical analyses were conducted between microbial communities and environmental factors. The results will offer new knowledge about the diversity of N2 fixers and ammonia oxidizers, and help us to better understand the biogeochemical processes in acidic, oligotrophic peatland ecosystems.

1 MATERIALS AND METHODS 1.1 Sample Collection and Physicochemical Measurements

Samples were taken from an acidic peat sediment profile in the Dajiuhu Peatland with an elevation of 1 750 m which located in the southwest edge in Shennongjia forest region, Hubei, China (31°29′27″ N, 109°59′44″ E) with an annual temperature of 7.2 ℃ and annual rainfall of 1 560 mm (Wang et al., 2017; Qin et al., 2010).

Five sediment samples along a 1.55 m deep peat profile were collected at the depth of 0-5 (DJH1), 20-25 (DJH5), 50-55 (DJH11), 100-105 (DJH21) and 150-155 cm (DJH31), respectively. Sampling tools were sterilized before use and samples were stored in sterile centrifuge tubes and subsequently stored on dry ice, and transported to the State Key Laboratory of Biogeology and Environmental Geology in China University of Geosciences, Wuhan. Samples were kept at -80 ℃ for future use.

Physicochemical parameters including pH, water content, total organic carbon (TOC), total nitrogen (TN) and contents of different nitrogen species were measured. Dry samples were mixed with ultrapure water with a ratio of 1 : 5 (W/V) followed by fully stirring with a glass rod for 10 min and quietly sitting for half an hour. Conductivity and pH of the supernatant were measured with a portable multi-parameter analyzer (STFW-7, Shanghai, China) and the mean of three measurements were reported. The TOC and TN in sediment samples were analyzed with an Elemental Analyzer (Elementar Vario EL Ⅲ) with a precision of ±0.4% (Yang et al., 2012). Water content refers to the difference between wet weight and dry weight (frozen dried with a freeze drier, ALPHA 1-2LD, Germany). NO2-, NO3- and NH4+ were measured via a Dionex ICS600 ion chromatograph (Thermo Fisher, USA) with a ratio of 1 : 10 (dry samples/ultrapure water) in the State Key Laboratory of Biogeology and Environmental Geology, China University of Geosciences, Wuhan.

1.2 DNA Extraction, Amplification of Functional Genes and Clone Library Construction

Sediment samples were freeze dried and homogenized with a free-drier (ALPHA 1-2LD, Germany). Genomic DNA from 3 subsamples was extracted with FastDNA®Spin kit for soil (MP, USA) according to manufacture instruction with minor modification. Guanidine Thiocyanate (GT) solution was added prior to eluting DNA from the glass matrix in order to further remove humic acid. Extracted DNA from subsamples was pooled together in order to enhance the representativeness of the samples and stored at -20 ℃ for future use after the concentration measured with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

Primer sets of Arch-amoAF/Arch-amoAR (Francis et al., 2005), amoA_1F/amoA_2R (Rotthauwe et al., 1997) and nifH-F/nifH-R (Gaby and Buckley, 2012) were used to construct clone libraries of archaeal amoA, bacterial amoA and nifH gene, respectively. Reaction protocols included high-temperature pre-denaturation of template DNA, cyclic amplification and terminal filling-in of product. The high-temperature pre-denaturation and terminal filling-in were under condition of 95 ℃ for 5 min and 72 ℃ for 10 min, respectively. Thirty-five cycles were used for amplifying amoA gene and 30 cycles for nifH gene. To be noted, anneal temperatures were: 53 ℃ for archaeal amoA, 55 ℃ for bacterial amoA and 56 ℃ for nifH gene, respectively. A total of 20 µl reaction mixture contained 10 µL of Ex Taq premixture (Takara, Japan), 1 µL of each primer, 6.5 µL of sterile water, 0.5 µL 0.1% BSA and 1 µL DNA template for the PCR reaction in replicates. We successfully amplified AOA in samples of DJH1, DJH5 and DJH11, AOB in DJH1, DJH5, DJH11 and DJH21 and nifH gene in all samples.

PCR products were visualized with 1% gel electrophoresis and were excised and purified with the QIAquick gel extraction kit (QIAGEN). The purified PCR products were linked into the pMD19-T vector (Takara, Japan), transformed into competent E. coli DH5α cells and then cultured on LB agar plates for 12 h. PCR amplifications with the primer set of M13-47 and Rv-M were used to confirm the insertion of appropriate-sized DNA fragments. Finally, the tested clones were sequenced by commercial companies (Wuhan Tsingke Biotechnology Limited Company, China and Wuhan Tianyi Huiyuan Biotechnology Limited Company, China). All the PCR reactions were run on a Master Cycler 5331 Gradient PCR (Eppendorf, Hamberg, Germany).

1.3 Quantification of Functional Genes

Real-time fluorescent quantitative PCR (CFX96 Bio-Rad) was employed to measure the abundance of functional genes (amoA and nifH). Primer sets of Arch-amoAF/Arch-amoAR, amoA_1F/amoA_2R and nifH-F/nifH-R were used for the quantification of amoA gene of AOA, AOB and nifH gene, respectively. The reaction cycling conditions were in accordance with the PCR procedures described above. Each reaction was performed in a 20 µl mixture including 10 µL of SYBR-Green PCR Master Mix (Takara, Japan), 1 µL of each primer (20 µM), 1 µL of DNA template, 0.5 µL 0.1% BSA and 6.5 µL of sterile water. Plasmids containing the correct inserts were returned from Wuhan Tsingke Biotechnology Limited Company. Standard samples were obtained with series of dilution of plasmid ranged from 109 to 102 copy/μL with EASY dilution (Takara, Japan). In the process, standards and samples were run in triplicates. R2 values of standard curves were above 0.99 with efficiencies of 99.30%-90.80%.

1.4 Sequencing and Phylogenetic Analysis

The sequencing was commercially performed by companies (Wuhan Tianyi Huiyuan Biotechnology Limited Company and Wuhan Tsingke Biotechnology Limited Company) using the forward primer Rv-M. Sequences with twin peaks, high GC and weak signals were excluded for further analysis and vector check was conducted via the software at the website of or MEGA 6.0 (Tamura et al., 2013).

High-quality sequences were then clustered into Operational Taxonomic Unit (OTU) via the mothur pipeline ( after singletons were excluded. AOA amoA were grouped at the threshold of 94% identity and others with the cutoff of 95% identity (Meng et al., 2017). The affiliation of representative sequences was performed with blast within NCBI database. The phylogenetic analyses were carried out with Maximum Likehood (ML) with MEGA6.0 software. The most abundant OTUs (≥2 clones) of nifH were used for the construction of the phylogenetic tree (Zhou et al., 2016).

1.5 Statistical Analysis

Mothur was used for clone libraries rarefaction analysis and to calculate diversity indices, such as Chao, Simpson and Shannon-Wiener. Coverage is the ratio between the observed OTUs and the total OTUs which is used to evaluate the sequencing depth. The associations between the environmental variables and AOA/AOB amoA gene and nifH gene community abundances were conducted with Pearson's correlation analysis in R software (RCore 2013).

1.6 Data Availability

The sequence datasets generated from the current study are available from the NCBI GenBank repository, under BioProject MG132115-MG132125 for AOA, MG132126-MG132134 for AOB and MG132135-MG132171 for nifH gene.

2 RESULTS 2.1 Physicochemical Characteristics of Peat Sediment Samples

Peat samples along the profile were overall acidic with the pH ranging from 4.55 to 6.10 (Table 1). The pH of the surface sediment was highest with a value of 6.10 among all the samples. The C/N ratio and ammonium nitrogen (NH4+) content varied from 18.17 to 21.91 and below the detected limit to 21.57 mg/kg, respectively and increased with the increase of sampling depth. In contrast, NO2- content, NO3- content and water content varied from 4.11 to 1.26, 9.42 to 3.90 mg/kg and 87% to 66%, respectively and decreased with the increase of the depth. An obvious transition of physicochemical properties was observed in samples with a depth below and above 55 cm. To be specific, in samples of DJH1, DJH5 and DJH11 (depth≤55 cm), TN, TOC, water content and organic matter were relatively high with a range of 1.71%-2.07%, 34.03%-38.98%, 76%-87% and 0.74%-0.83%, respectively. In samples with a depth > 55 cm, TN (1.16%-1.32%), TOC (25.11%-28.91%), water content (66%-67%) and organic matter (OM 0.48%-0.56%) were lower than those in the upper samples.

Table 1 Physicochemical characteristics of peat sediment along the profile in the Dajiuhu Peatland
2.2 Abundance of nifH and amoA Genes

The abundance of nifH gene varied from 105-108 copies per gram dry weight, and the amoA gene copies were about 102-104 copies per gram dry sediment (Fig. 1). Overall the copy numbers of nifH gene decreased with the depth except the sample of DJH11 with a depth of 50-55 cm. DJH11 showed a high copy number of 7.63×107 for nifH, and the highest copies of 4.67×104 for AOA amoA and 2.36×104 for AOB amoA. To be noted, AOB abundance was two times higher than that of AOA below 55 cm. In contrast, AOA outnumbered AOB above 55 cm (samples of DJH1 and DJH11) except DJH5.

Figure 1. Quantification of nifH and amoA (AOA and AOB) genes in the sediment samples along the peat profile in the Dajiuhu Peatland. Samples DJH1, DJH5, DJH11, DJH21 and DJH31 were collected at the depth of 0-5, 20-25, 50-55, 100-105, and 150-155 cm, respectively. Replicate of sediment samples was individually subject to qPCR assays and error bars represent standard deviation of mean (n=3).

Pearson's correlation analysis indicated that the abundances of archaeal and bacterial amoA genes showed no significant correlations with the measured physicochemical parameters (P > 0.05). In contract, nifH gene abundance showed a significant positive correlation with pH (P=0.04, R2=0.90) and water content (P=0.02, R2=0.93).

2.3 Diversity of nifH and amoA Genes

A total of 253 bacterial nifH sequences and 184 archaeal and 131 bacterial amoA sequences were retrieved, which were further grouped into 94, 12 and 9 OTUs, respectively (Table 2). High coverage and plateaued rarefaction curves suggest that amoA gene clone libraries constructed can well represent the microbial nitrification communities in all samples. However the rarefaction curves for nifH genes were very steep which showed a high possibility to see new OTUs with more clones sequenced.

Table 2 Overview of clone libraries constructed for functional genes

The Shannon-Wiener indexes of nifH gene, amoA gene of AOA and AOB varied from 2.43 to 3.67, 0.56 to 1.40, and 0.00 to 1.83, respectively. Overall, higher diversity was observed in the samples with a depth ≤55 cm, which consisted with the results of other indices (Table 2). The highest numbers of OTUs for nifH gene were observed in DJH11 and lowest in DJH31, which was consistent with those of diversity indices. The highest AOA OTU number was also observed in DJH11 whereas no clones of AOA were retrieved from samples of DJH21 and DJH31. AOB was detected throughout the whole profile except DJH31. Twenty-two percent of the total AOB clones and OTUs were found in DJH21. Besides, 38.9% of the total AOB clones and 77.8% of the total AOB OTUs were also observed in DJH11.

2.4 Phylogenetically of nifH and amoA Genes

Phylogenetically nifH clones (Fig. 2) affiliated with α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria and Verrucomicrobia. One OTU was found in each phylum of β-Proteobacteria, γ-Proteobacteria and Verrucomicrobia which were represented by 5, 2 and 2 clones, respectively. δ-Proteobacteria was divided into Group 1 and Group 2 according to the classification proposed by Niederberger et al. (2012) with 2 and 3 OTUs, respectively. The rest 30 OTUs with 115 representative sequences affiliated to α-Proteobacteria which was further divided into two groups (Fig. 2). nifH communities were dominated by α-Proteobacteria throughout the whole profile (Fig. 3).

Figure 2. Phylogenetic tree of the nifH gene sequences retrieved from sediments of the Dajiuhu Peatland by the ML method with a bootstrap of 1 000. All reference sequences were obtained from GenBank. Sequences in bold were from this study. Numbers after the OTU indicated the clone numbers in this OTU. Scale bar represents 0.05 nucleic acid substitutions per nucleotide position. Bootstrap values ≥50% was shown close to the nodes of the tree.
Figure 3. Bubble chart of the nifH clone libraries constructed from the sediment samples along the peat profile in the Dajiuhu Peatland. The area of each dot is proportional to the percentage of each group.

AOA was mainly clustered into Group 1.1a associated (Lehtovirta-Morley et al., 2011) and Group 1.1b (Tourna et al., 2011). However, Group1.1a was not observed in our samples (Fig. 4). Instead, 164 sequences (89.13% of the total AOA clones) fell into Group 1.1a associated, and the rest clones fell into Group 1.1b which included 8 OTUs. Group 1.1a associated sequences were observed in DJH1, DJH5 and DJH11, which accounted for 78%, 97% and 94% of the total AOA clones, respectively (Table 2).

Figure 4. Phylogenetic tree of the archaeal amoA sequences retrieved from sediments of the Dajiuhu Peatland by the ML method with a bootstrap of 1 000. All reference sequences were obtained from GenBank. Sequences in bold were from this study. Numbers after the OTU indicated the clone numbers in this OTU. Scale bar represents 0.05 nucleic acid substitutions per nucleotide position. Bootstrap values ≥50% was shown close to the nodes of the tree.

In contrast to the diverse community structure of AOA, all the AOB clones belonged to Nitrosospira of β-Proteobacteria (Fig. 5). Three clones retrieved from DJH11 affiliated to Cluster b and others had high identity with Cluster a, which was further divided into Cluster a-1, Cluster a-2 and Cluster a-3.

Figure 5. Phylogenetic tree of the bacterial amoA sequences retrieved from sediments of the Dajiuhu Peatland by the ML method with a bootstrap of 1 000. All reference sequences were obtained from GenBank. Sequences in bold were from this study. Numbers after the OTU indicated the clone numbers in this OTU. Scale bar represents 0.05 nucleic acid substitutions per nucleotide position. Bootstrap values ≥50% was shown close to the nodes of the tree.
3 DISCUSSIONS 3.1 Highly Diverse nifH Communities

Even though the coverage of our nifH clone libraries was low, we still observed a high diversity of diazotrophic bacteria in peat sediments of the Dajiuhu Peatland, with 94 OTUs identified and α-Proteobacteria dominated in all samples. Our results are consistent with the observation in other acidic Sphagnum peat bogs (Warren et al., 2017; Leppänen et al., 2014; Bragina et al., 2012; Zadorina et al., 2009). Previous studies also indicated a dominance of methanotrophs in diazotroph in Sphagnum-dominated peatlands (Larmola et al., 2014; Vile et al., 2014). However, in our study we did not observe any methanotrophs in our nifH gene clone libraries constructed from peat sediments, which may result from the low coverage of our clone libraries and the different primer pairs used. On one hand, more clones (> 200-300) were needed in order to fully evaluate the diversity of natural nitrogen-fixing communities (Zadorina et al., 2009). On the other hand, various nifH primer pairs were used to investigate the diversity of N2 fixers in natural environments and each pair set has its own specificity and limitations (Gaby and Buckley, 2012). Primer pairs targeting nifH gene include nifH-F/nifH-R (this study), polF/polR (Liebner and Svenning, 2013), F1/R6 (Vile et al., 2014), IGK/DVV (Bellenger et al., 2014), FGPH19/polR+polF/AQER (Leppänen et al., 2014) and 19F/nifH3+nifH1/nifH2 (Bellenger et al., 2011). Among these nifH primer pairs, IGK/DVV is proposed to have the highest coverage for methanotrophs in peatlands (Warren et al., 2017; Coelho et al., 2008). However, the dominant diazotrophs are still in debate in oligotrophic, acidic peatland. Early studies indicated a dominance of Cyanobacteria (Basilier et al., 1978) or heterotrophic bacteria (Schwintzer, 1983) based on microscopic observation, while more recent molecular analyses argue for the importance of α-Proteobacteria as major diazotrophs in Sphagnum peat bogs (Warren et al., 2017; Bragina et al., 2012; Zadorina et al., 2009). In fact, Cyanobacteria is found to be symbiotic with Sphagnum in peatlands (Kostka et al., 2016; Bragina et al., 2013) and may experience co-evolution with their host species (Papaefthimiou et al., 2008). Therefore the dominant N2 fixers in Sphagnum and peat sediment may vary greatly due to the difference of samples investigated.

Besides the high diversity, we also observed high abundance of nifH in the Dajiuhu Peatland with a range of 105 to 108 per gram dry sediments, which was very close to the result from an ombrotrophic peat bog (Warren et al., 2017) and much higher than those reported from forest soils (Berthrong et al., 2014) and agricultural soils (Hayden et al., 2010). Our results showed a positive correlation between water content and diazotrophic bacteria abundance within moisture ranging of 66%-87%. This observation is consistent with the result which indicated a significant relationship between soil water content and the survival of the diazotrophs (Oliveira et al., 2004). Moreover, water content is also demonstrated to be able to increase the organic matter content and minerals concentrations, which subsequently result in the increase of the microbial quantity, especially free-living nitrogen fixing bacteria (Drenovsky et al., 2004). Additionally, N2 fixation is an energy-consuming process to obtain nitrogen from the atmosphere at the high expense of ATPs, and usually occurs in nitrogen limited environmental conditions. As for the Dajiuhu Peatland, dissolved inorganic nitrogen (NH4++NO2-+NO3-, ~27.65 mg/kg) is fairly low compared with that in sandy soils (61.1-124.9 mg/kg) (Silva et al., 2011), which leads to the high abundance of diazotrophs in order to sustain the ecosystem. Nevertheless, more experiments are needed for further verification of nitrogen fixation rate.

Our observation of the high diversity and high abundance of nifH gene in the Dajiuhu Peatland is of great significance to understand nitrogen cycle in peatland ecosystem. Despite of the nitrogen deficiency in the Dajiuhu Peatland, the highly diverse nitrogen fixers with high abundance may play a fundamental role in nitrogen supply via nitrogen fixation and thus sustain the ecosystem. Our results about community composition of diazotrophs in the Dajiuhu Peatland also help people to understand nitrogen-fixing function of different microbial groups.

3.2 Abundance and Communities of AOA and AOB in the Dajiuhu Peatland

Overall, the abundance of AOA and AOB (102 to 104 per gram dry sediment) in the Dajiuhu Peatland was much lower than those reported from other environments, such as acidic paddy soils (105 to 108 per gram dry sediment) (Chen et al., 2011) and alkaline wetland sediments (105 to 108 per gram dry sediment) (Ye et al., 2011). It is indicated that pH is usually one of the important factors (Hayden et al., 2010) affecting the abundance of ammonia oxidizers. For example, archaeal abundance shows a positive relationship with pH in soils (pH 3.7-5.8) (He et al., 2007) while bacterial abundance increases with the increase of pH from 4.9 to 7.5 at the transcriptional level in soils (Nicol et al., 2008). However, we did not see any statistically significant correlation between amoA abundance and pH in the Dajiuhu Peatland sediment samples. This may attribute to the relative small variation of pH (4.55-4.90, 6.10) or limited number of samples (5 samples) in our study.

Strikingly, the dominant groups in ammonia oxidizers communities are consistent with those observed in acidic environments. For example, our observation with the dominance of Group 1.1a-associated in AOA communities was in agreement with the results under acidic pH regimes (Hatzenpichler, 2012; Lu et al., 2012). And Group 1.1a-associated sequences have been proven to be the dominant group in sediments with NH4+-N < 93 mg/kg sediment (Sun et al., 2013). AOB exclusively affiliated with Nitrosospira, which matched well with most reports of Nitrosospira as the most commonly detected genus in acidic natural environments (Chen et al., 2011; Kowalchuk and Stephen, 2001). Nitrosococcus is mostly found in neutral aerobic soil (Mary et al., 1999) and greatly sensitive to ambient temperature. Nitrosococcus and Nitrosomonas are typical mesophiles which can survive between 25 and 40 ℃ (Zhang et al., 2006). These results strongly indicate that our data is highly reliable despite of the low abundance of ammonia oxidizers in our samples. As reported, AOA communities are highly diverse, but not AOB. Under low pH, NH4+-N becomes the dominant species and free ammonia decreases sharply. The limitation of available NH3 subsequently limits the bloom of AOB, who depends much on NH3 concentration for nitrification (Frijlink et al., 1992).

Collectively, our results offer brand new knowledge about nitrification in the Dajiuhu Peatland, which would be helpful to fully understand the nitrogen cycle in acidic peatland ecosystems. Further study is needed to evaluate the rate of nitrification.

3.3 Vertical Distribution of AOA and AOB

AOA was more abundant than AOB in the upper samples with a depth ≤55 cm (DJH11), whereas AOB was more abundant than AOA in samples with a depth > 55 cm. Notably, the sample DJH11 with the depth of 50-55 cm showed the highest abundance and the most complex community structure of nifH gene and amoA gene among all samples. This interesting phenomenon may relate with the fluctuation of water table level at the Dajiuhu Peatland which results in a variation of O2 content at this depth. Mounting evidence has shown that amoA-containing autotrophic AOA and heterotrophic AOB prefer to live under aerobic conditions (Liu et al., 2011) which was consistent with our results of relative higher abundance of AOA and AOB in aerobic conditions in the upper 55 cm. In O2-limited condition with the depth > 55 cm, AOA prefers to the conditions with relative higher redox state compared to AOB (Sun et al., 2013; Jiang et al., 2009). Meanwhile, AOB can grow well with higher-ammonia concentration than AOA (Liu et al., 2011). The relatively reducing state and higher NH4+in samples with a depth > 55 cm may account for the observation of higher abundance of AOB, leading to a remarkable vertical variation of ammonia oxidizers along the sediment profile.


In conclusion, highly diverse nifH communities with high abundance were observed along the peat sediment profile in the Dajiuhu Peatland which was dominated by α-Proteobacteria, indicating a great potential of microbial contribution to nitrogen supply via N2-fixation in peatland ecosystem. In contrast, ammonia oxidizers were found in low diversity and low abundance, which may suggest a low rate of nitrification. Vertically the sample of DJH11 harbored the most complicated nifH communities and highest abundance of ammonia oxidizers. Our results give the first picture about functional genes of nitrogen cycle in the Dajiuhu Peatland, both in abundance and biological diversity, which greatly enhance our understanding about the nitrogen cycling in peatland ecosystem. However more studies are needed in order to evaluate the contribution of different microbial groups in N2 fixation and nitrification.


This work was supported by the National Natural Science Foundation of China (No. 41572325). The final publication is available at Springer via

Barron, A. R., Wurzburger, N., Bellenger, J. P., et al., 2009. Molybdenum Limitation of Asymbiotic Nitrogen Fixation in Tropical Forest Soils. Nature Geoscience, 2(1): 42-45. DOI:10.1038/ngeo366
Basilier, K., Granhall, U., Stenström, T. A., et al., 1978. Nitrogen Fixation in Wet Minerotrophic Moss Communities of a Subarctic Mire. Oikos, 31(2): 236-246. DOI:10.2307/3543568
Bellenger, J. P., Wichard, T., Xu, Y., et al., 2011. Essential Metals for Nitrogen Fixation in a Free-Living N2-Fixing Bacterium: Chelation, Homeostasis and High Use Efficiency. Environmental Microbiology, 13(6): 1395-1411. DOI:10.1111/j.1462-2920.2011.02440.x
Bellenger, J. P., Xu, Y., Zhang, X., et al., 2014. Possible Contribution of Alternative Nitrogenases to Nitrogen Fixation by Asymbiotic N2-Fixing Bacteria in Soils. Soil Biology and Biochemistry, 69: 413-420. DOI:10.1016/j.soilbio.2013.11.015
Bernot, M. J., Dodds, W. K., 2005. Nitrogen Retention, Removal, and Saturation in Lotic Ecosystems. Ecosystems, 8(4): 442-453. DOI:10.1007/s10021-003-0143-y
Berthrong, S. T., Yeager, C. M., Gallegos-Graves, L., et al., 2014. Nitrogen Fertilization Has a Stronger Effect on Soil Nitrogen-Fixing Bacterial Communities than Elevated Atmospheric CO2. Applied and Environmental Microbiology, 80(10): 3103-3112. DOI:10.1128/AEM.04034-13
Bobbink, R., Hicks, K., Galloway, J., et al., 2010. Global Assessment of Nitrogen Deposition Effects on Terrestrial Plant Diversity: A Synthesis. Ecological Applications, 20(1): 30-59. DOI:10.1890/08-1140.1
Bragina, A., Berg, C., Müller, H., et al., 2013. Insights into Functional Bacterial Diversity and Its Effects on Alpine Bog Ecosystem Functioning. Scientific Reports, 3(1): 1995. DOI:10.1038/srep01995
Bragina, A., Maier, S., Berg, C., et al., 2012. Similar Diversity of Alphaproteobacteria and Nitrogenase Gene Amplicons on Two Related Sphagnum Mosses. Frontiers in Microbiology, 2: 275.
Chen, X., Zhang, L. M., Shen, J. P., et al., 2011. Abundance and Community Structure of Ammonia-Oxidizing Archaea and Bacteria in an Acid Paddy Soil. Biology and Fertility of Soils, 47(3): 323-331. DOI:10.1007/s00374-011-0542-8
Cláudio, A. N., 2016. Microbial Ecology: Do It Yourself Nitrification. Nature Reviews Microbiology, 14(2): 61-61. DOI:10.1038/nrmicro.2015.20
Coelho, M. R. R., de Vos, M., Carneiro, N. P., et al., 2008. Diversity of nifH Gene Pools in the Rhizosphere of Two Cultivars of Sorghum (Sorghum Bicolor) Treated with Contrasting Levels of Nitrogen Fertilizer. FEMS Microbiology Letters, 279(1): 15-22. DOI:10.1111/fml.2008.279.issue-1
Daims, H., Lebedeva, E. V., Pjevac, P., et al., 2015. Complete Nitrification by Nitrospira Bacteria. Nature, 528(7583): 504-509. DOI:10.1038/nature16461
Dang, H. Y., Yang, J. Y., Li, J., et al., 2013. Environment-Dependent Distribution of the Sediment nifH-Harboring Microbiota in the Northern South China Sea. Applied and Environmental Microbiology, 79(1): 121-132. DOI:10.1128/AEM.01889-12
de la Torre, J. R., Walker, C. B., Ingalls, A. E., et al., 2008. Cultivation of a Thermophilic Ammonia Oxidizing Archaeon Synthesizing Crenarchaeol. Environmental Microbiology, 10(3): 810-818. DOI:10.1111/emi.2008.10.issue-3
Drenovsky, R. E., Vo, D., Graham, K. J., et al., 2004. Soil Water Content and Organic Carbon Availability are Major Determinants of Soil Microbial Community Composition. Microbial Ecology, 48(3): 424-430. DOI:10.1007/s00248-003-1063-2
Francis, C. A., Roberts, K. J., Beman, J. M., et al., 2005. Ubiquity and Diversity of Ammonia-Oxidizing Archaea in Water Columns and Sediments of the Ocean. Proceedings of the National Academy of Sciences, 102(41): 14683-14688. DOI:10.1073/pnas.0506625102
Frijlink, M. J., Abee, T., Laanbroek, H. J., et al., 1992. The Bioenergetics of Ammonia and Hydroxylamine Oxidation in Nitrosomonas Europaea at Acid and Alkaline pH. Archives of Microbiology, 157(2): 194-199. DOI:10.1007/BF00245290
Gaby, J. C., Buckley, D. H., 2012. A Comprehensive Evaluation of PCR Primers to Amplify the nifH Gene of Nitrogenase. PLoS ONE, 7(7): e42149. DOI:10.1371/journal.pone.0042149
Hatzenpichler, R., 2012. Diversity, Physiology, and Niche Differentiation of Ammonia-Oxidizing Archaea. Applied and Environmental Microbiology, 78(21): 7501-7510. DOI:10.1128/AEM.01960-12
Hatzenpichler, R., Lebedeva, E. V., Spieck, E., et al., 2008. A Moderately Thermophilic Ammonia-Oxidizing Crenarchaeote from a Hot Spring. Proceedings of the National Academy of Sciences, 105(6): 2134-2139. DOI:10.1073/pnas.0708857105
Hayden, H. L., Drake, J., Imhof, M., et al., 2010. The Abundance of Nitrogen Cycle Genes AmoA and nifH Depends on Land-Uses and Soil Types in South-Eastern Australia. Soil Biology and Biochemistry, 42(10): 1774-1783. DOI:10.1016/j.soilbio.2010.06.015
He, J. Z., Shen, J. P., Zhang, L. M., et al., 2007. Quantitative Analyses of the Abundance and Composition of Ammonia-Oxidizing Bacteria and Ammonia-Oxidizing Archaea of a Chinese Upland Red Soil under Long-Term Fertilization Practices. Environmental Microbiology, 9(9): 2364-2374. DOI:10.1111/emi.2007.9.issue-9
Jiang, H. C., Dong, H. L., Yu, B. S., et al., 2009. Diversity and Abundance of Ammonia-Oxidizing Archaea and Bacteria in Qinghai Lake, Northwestern China. Geomicrobiology Journal, 26(3): 199-211. DOI:10.1080/01490450902744004
Kang, W., Tai, X., Li, S., et al., 2013. Research on the Number of Nitrogen-Fixing Microorganism and Community Structure of Nitrogen-Fixing (nifH) Genes in the Alkali Soils of Alpine Steppe in the Qilian Mountains. Journal of Glaciology and Geocryology, 35(1): 208-216.
Kip, N., van Winden, J. F., Pan, Y., et al., 2010. Global Prevalence of Methane Oxidation by Symbiotic Bacteria in Peat-Moss Ecosystems. Nature Geoscience, 3(9): 617-621. DOI:10.1038/ngeo939
Kits, K. D., Sedlacek, C. J., Lebedeva, E. V., et al., 2017. Kinetic Analysis of a Complete Nitrifier Reveals an Oligotrophic Lifestyle. Nature, 549(7671): 269-272. DOI:10.1038/nature23679
Kostka, J. E., Weston, D. J., Glass, J. B., et al., 2016. The Sphagnum Microbiome: New Insights from an Ancient Plant Lineage. New Phytologist, 211(1): 57-64. DOI:10.1111/nph.13993
Kowalchuk, G. A., Stephen, J. R., et al., 2001. Ammonia-Oxidizing Bacteria: A Model for Molecular Microbial Ecology. Annual Review of Microbiology, 55(1): 485-529.
Larmola, T., Leppanen, S. M., Tuittila, E. S., et al., 2014. Methanotrophy Induces Nitrogen Fixation during Peatland Development. Proceedings of the National Academy of Sciences, 111(2): 734-739. DOI:10.1073/pnas.1314284111
Lehtovirta-Morley, L. E., Stoecker, K., Vilcinskas, A., et al., 2011. Cultivation of an Obligate Acidophilic Ammonia Oxidizer from a Nitrifying Acid Soil. Proceedings of the National Academy of Sciences, 108(38): 15892-15897. DOI:10.1073/pnas.1107196108
Leppänen, S. M., Rissanen, A. J., Tiirola, M., 2014. Nitrogen Fixation in Sphagnum Mosses is Affected by Moss Species and Water Table Level. Plant and Soil, 389(1/2): 185-196.
Liebner, S., Svenning, M. M., 2013. Environmental Transcription of mmoX by Methane-Oxidizing Proteobacteria in a Subarctic Palsa Peatland. Applied and Environmental Microbiology, 79(2): 701-706. DOI:10.1128/AEM.02292-12
Limpens, J., Heijmans, M. M., Berendse, F., 2006. The Nitrogen Cycle in Boreal Peatlands. Boreal Peatland Ecosystems, 188: 195-230. DOI:10.1007/978-3-540-31913-9
Liu, Z. H., Huang, S. B., Sun, G. P., et al., 2011. Diversity and Abundance of Ammonia-Oxidizing Archaea in the Dongjiang River, China. Microbiological Research, 166(5): 337-345. DOI:10.1016/j.micres.2010.08.002
Lu, L., Han, W. Y., Zhang, J. B., et al., 2012. Nitrification of Archaeal Ammonia Oxidizers in Acid Soils is Supported by Hydrolysis of Urea. The ISME Journal, 6(10): 1978-1984. DOI:10.1038/ismej.2012.45
Luo, L., Wang, Z., Mao, D., et al., 2016. Connotation and Differentiation of Terminology on Main Kinds of Wetlands in English. Chinese Journal of Ecology, 35(3): 834-842.
Mary, A. B., Stephen, J. R., Kowalchuk, G. A., et al., 1999. Comparative Diversity of Ammonia Oxidizer 16S rRNA Gene Sequences in Native, Tilled, and Successional Soils. Appl. Environ. Microbiol., 65(7): 2994-3000.
Meng, H., Katayama, Y., Gu, J. D., 2017. More Wide Occurrence and Dominance of Ammonia-Oxidizing Archaea than Bacteria at Three Angkor Sandstone Temples of Bayon, Phnom Krom and Wat Athvea in Cambodia. International Biodeterioration & Biodegradation, 117: 78-88.
Moisander, P. H., Beinart, R. A., Hewson, I., et al., 2010. Unicellular Cyanobacterial Distributions Broaden the Oceanic N2 Fixation Domain. Science, 327(5972): 1512-1514. DOI:10.1126/science.1185468
Nicol, G. W., Leininger, S., Schleper, C., et al., 2008. The Influence of Soil PH on the Diversity, Abundance and Transcriptional Activity of Ammonia Oxidizing Archaea and Bacteria. Environmental Microbiology, 10(11): 2966-2978. DOI:10.1111/emi.2008.10.issue-11
Niederberger, T. D., Sohm, J. A., Tirindelli, J., et al., 2012. Diverse and Highly Active Diazotrophic Assemblages Inhabit Ephemerally Wetted Soils of the Antarctic Dry Valleys. FEMS Microbiology Ecology, 82(2): 376-390. DOI:10.1111/fem.2012.82.issue-2
Oliveira, A. L. M., Canuto, E. L., Silva, E. E., et al., 2004. Survival of Endophytic Diazotrophic Bacteria in Soil under Different Moisture Levels. Brazilian Journal of Microbiology, 35(4): 295-299. DOI:10.1590/S1517-83822004000300005
Oved, T., Shaviv, A., Goldrath, T., et al., 2001. Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil. Applied and Environmental Microbiology, 67(8): 3426-3433. DOI:10.1128/AEM.67.8.3426-3433.2001
Pankratov, T. A., Serkebaeva, Y. M., Kulichevskaya, I. S., et al., 2008. Substrate-Induced Growth and Isolation of Acidobacteria from Acidic Sphagnum Peat. The ISME Journal, 2(5): 551-560. DOI:10.1038/ismej.2008.7
Papaefthimiou, D., van Hove, C., Lejeune, A., et al., 2008. Diversity and Host Specificity of Azollacyanobionts. Journal of Phycology, 44(1): 60-70. DOI:10.1111/jpy.2008.44.issue-1
Qin, Y. M., Wang, J. X., Xie, S. C., et al., 2010. Morphological Variation and Habitat Selection of Testate Amoebae in Dajiuhu Peatland, Central China. Journal of Earth Science, 21(S1): 253-256. DOI:10.1007/s12583-010-0228-4
RCore, T., 2013. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Online:
Rotthauwe, J. H., Witzel, K. P., Liesack, W., 1997. The Ammonia Monooxygenase Structural Gene amoA as a Functional Marker: Molecular Fine-Scale Analysis of Natural Ammonia-Oxidizing Populations. Applied and Environmental Microbiology, 63(12): 4704-4712.
Schwintzer, C. R., 1983. Nonsymbiotic and Symbiotic Nitrogen Fixation in a Weakly Minerotrophic Peatland. American Journal of Botany, 70(7): 1071. DOI:10.1002/j.1537-2197.1983.tb07908.x
Silva, M. C. P. E., Semenov, A. V., van Elsas, J. D., et al., 2011. Seasonal Variations in the Diversity and Abundance of Diazotrophic Communities Across Soils. FEMS Microbiology Ecology, 77(1): 57-68. DOI:10.1111/fem.2011.77.issue-1
Stahl, D. A., de la Torre, J. R., 2012. Physiology and Diversity of Ammonia-Oxidizing Archaea. Annual Review of Microbiology, 66(1): 83-101.
Sun, W., Xia, C. Y., Xu, M. Y., et al., 2013. Distribution and Abundance of Archaeal and Bacterial Ammonia Oxidizers in the Sediments of the Dongjiang River, a Drinking Water Supply for Hong Kong. Microbes and Environments, 28(4): 457-465. DOI:10.1264/jsme2.ME13066
Tahon, G., Tytgat, B., Stragier, P., et al., 2016. Analysis of CbbL, nifH, and PufLM in Soils from the Sør Rondane Mountains, Antarctica, Reveals a Large Diversity of Autotrophic and Phototrophic Bacteria. Microbial Ecology, 71(1): 131-149. DOI:10.1007/s00248-015-0704-6
Tamura, K., Stecher, G., Peterson, D., et al., 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution, 30(12): 2725-2729. DOI:10.1093/molbev/mst197
Tourna, M., Stieglmeier, M., Spang, A., et al., 2011. Nitrososphaera Viennensis, an Ammonia Oxidizing Archaeon from Soil. Proceedings of the National Academy of Sciences, 108(20): 8420-8425. DOI:10.1073/pnas.1013488108
van Kessel, M. A. H. J., Speth, D. R., Albertsen, M., et al., 2015. Complete Nitrification by a Single Microorganism. Nature, 528(7583): 555-559. DOI:10.1038/nature16459
Vile, M. A., Kelman, Wieder R., Živković, T., et al., 2014. N2-Fixation by Methanotrophs Sustains Carbon and Nitrogen Accumulation in Pristine Peatlands. Biogeochemistry, 121(2): 317-328. DOI:10.1007/s10533-014-0019-6
Wang, M., Liu, Z., Ma, X., et al., 2013. Distribution Law of Peat in the World. Wetland Science, 11(3): 339-346.
Wang, R. C., Wang, H. M., Xiang, X., et al., 2017. Temporal and Spatial Variations of Microbial Carbon Utilization in Water Bodies from the Dajiuhu Peatland, Central China. Journal of Earth Science, 29(4): 969-976.
Warren, M. J., Lin, X. J., Gaby, J. C., et al., 2017. Molybdenum-Based Diazotrophy in a Sphagnum Peatland in Northern Minnesota. Applied and Environmental Microbiology, 83(17): 01174-17.
Yang, H., Ding, W. H., Wang, J. X., et al., 2012. Soil PH Impact on Microbial Tetraether Lipids and Terrestrial Input Index (BIT) in China. Science China Earth Sciences, 55(2): 236-245. DOI:10.1007/s11430-011-4295-x
Ye, L., Zhu, G., Wang, Y., et al., 2011. Abundance and Biodiversity of Ammonia-Oxidizing Archaea and Bacteria in Littoral Wetland of Baiyangdian Lake, North China. Acta Ecologica Sinica, 31(8): 2209-2215.
Yeager, C. M., Kornosky, J. L., Housman, D. C., et al., 2004. Diazotrophic Community Structure and Function in Two Successional Stages of Biological Soil Crusts from the Colorado Plateau and Chihuahuan Desert. Applied and Environmental Microbiology, 70(2): 973-983. DOI:10.1128/AEM.70.2.973-983.2004
Zadorina, E. V., Slobodova, N. V., Boulygina, E. S., et al., 2009. Analysis of the Diversity of Diazotrophic Bacteria in Peat Soil by Cloning of the nifH Gene. Microbiology, 78(2): 218-226. DOI:10.1134/S0026261709020131
Zhang, H., Li, P., Hu, X., et al., 2006. Screening and Cultivation Conditions of Two Nitrosobacteria Strains. Environmental Protection of Chemical Industry, 26(5): 366-369.
Zhou, H. X., Dang, H. Y., Klotz, M. G., 2016. Environmental Conditions Outweigh Geographical Contiguity in Determining the Similarity of nifH-Harboring Microbial Communities in Sediments of Two Disconnected Marginal Seas. Frontiers in Microbiology, 7(236): 1111.