Effects of environmental variables on abundance of ammonia-oxidizing communities in sediments of Luotian River, China

Physiochemical analysis

The study was conducted at the Luotian River in Huanggang City, South Dabia Mountains, Hubei Province, China (30°35′N, 115°06′E) (Fig. S1). This location has a series of mountains with 70% forest coverage. Five streams originate from the Dabie Mountains and flow in the southwest direction.

The physiochemical parameters of the sediment samples were assessed in triplicate following the methods of a previous work (Bailey et al., 2013). Physiochemical parameters, including oxidation–reduction potential (ORP), total suspended solids (TSS), temperature (Temp), pH, dissolved oxygen (DO), and conductivity (Cond), were determined with a multiparameter probe (YSI Professional Plus). Moreover, magnesium (Mg), calcium (Ca), nitrate (NO${}_3^{-}$), ammonia (NH${}_4^{+}$), nitrite (NO${}_2^{-}$), total nitrogen (TN), sulfate (SO${}_4^{-2}$), free cyanide (CN), and phosphorus (PO${}_4^{-3}$-P) were also analyzed with an electronic spectrophotometer (DR2800, Hach Company, Loveland, CO, USA).

Samples collection

Three replicate samples were collected from each sampling site at the Luotian River in May, August, and October 2018. Nine stations (S1–S9), specifically, pristine upstream (S1–S4), anthropogenic activities in Luotian City (S5–S7), downstream located far from human activities (S8), and industrial zone for waste discharge (S9), were selected for this study (Fig. S1). A total of 50–100 g sediments were collected in triplicate from each site at a 0–5 cm depth, transferred to sterile impermeable plastics with liquid nitrogen and then transported to the laboratory. All samples were conserved at −80 °C before DNA extraction.

Nucleic acid extraction, PCR, cloning, and sequencing

Microorganisms from the sediments were studied by extracting DNA using a FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA). A NanoDrop (2000c) spectrophotometer (Wilmington, DE, USA) was used to quantify DNA according to the manufacturer’s instructions. Thermal cycling conditions for each primer set and primers’ names are shown in Table S1.

PCR amplification was performed with a peqSTAR 2 × double block thermocycler (PEQLAB Biotechnologies, Erlangen, Germany) with the following conditions: 50 µL mixture comprising 25 µL 2 × Master Mix (Thermo Fisher Scientific, USA), 0.2 µM of each primer, and 50–100 ng DNA. Thermal cycling was performed in an initial denaturation step for 5 min at 95 °C. The sizes of the PCR amplification products were analyzed by 1% gel electrophoresis.

The clone libraries of AOA and AOB amoA gene-amplified fragments from each sampling site were obtained on the basis of a previously reported study (Jin et al., 2011). After the extraction of DNA and PCR products of the amoA genes, the mixture of PCR products was briefly gel-purified and cloned with a pMD19-T cloning kit (TaKaRa Biotechnology, Wuhan, China). A total of >30 clone libraries were randomly tested for the positive insertion of DNA products by PCR amplification with primer M13F and M13R. Then, these libraries were sequenced using an ABI 3,730 × 1 sequencer (Wuhan, China). The DNA sequences were analyzed and edited with BioEdit software version 5.07 and MEGA version 7.

Phylogenetic analysis

The sequences were first cropped. Then, the alignments were completed by ClustalW and analyzed by BLASTn according to the nonrepetitive National Center for Biotechnology Information (NCBI) database (Morgulis et al., 2008). Operational taxonomic units (OTUs) were determined as clusters, where the sequence identities were performed (>90%) by the Ribosomal Database Project (RDP) (http://rdp.cme.msu.edu and http://www.mothur.org). Phylogenetic trees were created using MEGA 7, the neighbor-joining method of reference (Saitou & Nei, 1987) (http://www.phylogeny.fr), and FigTree v1.4.3.

AOA and AOB amoA communities were partially sequenced from the nine stations (S1–S9). Among 109 archaeal amoA clone libraries, 54 archaeal amoA genes were analyzed. As a result, 34 operational taxonomic units (OTUs) were recovered on the basis of a 1 amino acid residue cut-off (Fig. 1, Table S3). Among 83 bacterial amoA clone libraries, 61 bacterial amoA sequences were examined. Only 3 OTUs were registered on the basis of a 1 amino acid residue cut-off (Fig. S2, Table S3). BLASTn analysis identified that most sequences were close to the amoA genes of uncultured bacteria. Ammonia monooxygenase genes were inserted into the dataset for the most similar GenBank sequences according to the BLASTn results. Sequence alignment was performed. The phylogenetic trees of ammonia-oxidizing archaea and bacteria are presented in Fig. 1 and Fig. S2, respectively. A neighbor-joining tree was designed using the amoA gene sequences and the sequences related to those accessed from GenBank. All the OTU sequences of AOA and AOB were deposited in the NCBI database under accession numbers MG238515-MG238543 and MG519671-MG519707.

Quantitative PCR

Real-time PCR was conducted to measure the abundance of functional marker genes in the sediments. 16S rRNA gene copies were measured using SYBR Green I Real-Time PCR. Gene abundance was measured in triplicate by amplifying the 16S rRNA gene using specific primers and PCR conditions, as shown in Table S1 (Keshri, Mishra & Jha, 2013). Each of AOA and AOB samples was quantified by an ABI Prism sequence detection system on the basis of the SYBR Green I method. The primer sets and thermal cycling conditions for the qPCR are described in Table S1. Each reaction was performed in 25 µL of reaction mixtures containing 12.5 µL of SYBR Premix Ex Taq (TaKaRa Biotechnology, Wuhan, China), 1 µL each of forward and reverse primers, and 1 µL DNA template. The specified PCR for each target gene was verified by the generated melting curves and agarose gel electrophoresis. All assays were conducted in triplicate with 85%–95% efficiency. Cycle thresholds were made in comparison with standard curves that were constructed using 10-fold 1:10 serial dilution from 10¹ to 10⁸ gene copies per gram of dry weight. The R² values for the normal curves were ≥0.993 for all tests.

AOA and AOB Investigation by terminal-restriction fragment length polymorphism (T-RFLP)

T-RFLP was used to investigate the PCR products of AOA and AOB (several tests were removed from the AOA and AOB investigations because of inadequate PCR output). Fluorescence-labeled PCR products were obtained by applying forward primer 5′-labeled with 6-carboxyfluorescein for the AOA and AOB genes (Table S1) under the following cycling conditions: initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s, and extension at 70 °C for 60 s.

Each 50 µL of the PCR reaction contained 10 X ThermoPol reaction buffer, 1.5 mM MgCl₂, 0.1 mM dNTPs, 0.25 µL of Taq DNA polymerase (New England Biolabs Ltd., China), 0.2 µg µL⁻¹ of bovine serum albumin (Roche, Penzberg, Germany), 0.1 µM of each primer (Table S1), and 1 µL of the DNA template. The PCR fragments were analyzed by gel electrophoresis to confirm the amplification of a specific area. After purification, amplicons were selected to digest the fluorescence-labeled PCR products into two separate enzymatic digestions using HhaI and MspI for bacteria and HpyCH4V and RsaI for AOA. The amplicons were processed for 2 h in a thermocycler at 37 °C and 65 °C.

Restriction products were purified using ethanol precipitation (Sambrook & Russell, 2006) and resuspended in 20 µL of water. Then, 10 µL of aliquots was mixed with 13 µL of HiDi formamide (Applied Biosystems) and 0.3 µL of internal DNA fragment size standard GeneScan-500 LIZ (Applied Biosystems). The product was analyzed with an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems). T-RFs were defined by GeneMarker Demo software (V 1.91, Applied Biosystems). Products within 40 and 600 bp were combined in the investigation. The peak area was represented as a percentage of the entire peak area, the profile of <1.5% of the entire area was excluded from the study.

Statistical analysis

Principal component analysis (PCA) was conducted to describe whether environmental variables can be performed to discriminate among sampling sites in the Luotian River. PCA was carried out using PAST v.3.15 (Hammer, Harper & Ryan, 2001). To determine the environmental indicators that best explained the variation in ammonia-oxidizing communities, redundancy analysis (RDA) was carried out using Canoco version 4.5 for Windows (Microcomputer Power, Ithaca, New York, United States) (Ramette, 2007). Spearman correlation matrix was used to describe the relationships among environmental indicators and AOA and AOB (Cassman et al., 2019). A p <0.01 and p <0.05 were considered significant, and the matrix was created using Excel add-in XLSTAT 2017 (Fig. S3). The algorithm in BLASTn was used to define the most similar sequences. The aligned sequences were converted into amino acids by applying MEGA 7 and used for the assembly of phylogenetic tree neighbor-joining by using Poisson model distances and pairwise deletion of gaps and missing data. Bootstrapping (1,000 replicates restructured) was performed for phylogenetic restructuring.

Physical and chemical characteristics of Luotian River

The environmental parameters indicated that the sampling sites were influential, as shown by the variability of many ecological features (Table 1). The Cond, SO${}_4^{-2}$, Temp, NH${}_4^{+}$, NO${}_3^{-}$, NO${}_2^{-}$, and TN in S9 were relatively higher than those recorded in other stations. Low pH and ORP was also observed in S4 and S9, respectively. The other environmental parameters, although varied, did appear consistent between study periods or sampling sites.

Archaeal and bacterial amoA-based community structures

Most of the sequences were related to Nitrososphaera and Nitrosopumilus (three AOA clusters, I.I, I.II, and I.IV). However, the sequences connected to Nitrosotalea cluster I.III were noted in S4 (Fig. 1). Only 1 AOB cluster was related to genus Nitrosomonas (Fig. S2).

Relationships among physicochemical variables and sampling sites

The ordinations of physicochemical variables obtained by PCA were differentiated between sites (Fig. 2). The sampling sites were disseminated along PCA 1, which described 55.77% of the environmental data. PCA 1 was positively correlated (p < 0.01) with NO${}_3^{-}$, Temp, NH${}_4^{+}$, SO${}_4^{-2}$, and phosphorus, in addition to, negatively correlated (p < 0.05) to DO, ORP, and pH, which mostly indicated anthropogenic activities in the sampling sites. PCA 2 described 20.7% of the environmental data and was positively correlated (p < 0.01) to Temp and negatively correlated (p < 0.05) to ORP, pH, and DO. PCA showed the locations S9 within the Luotian River, that is, most of the sampling sites were clarified by the presence of anthropized activities due to the industrial zone for waste discharge in the river.

Relationships between the abundance of ammonia oxidization genes (AOA and AOB) and the environmental parameters were analyzed by RDA (Fig. 3). For AOA, axis1 and axis2 explained 85.75% and 13.7% of the total variation, respectively, in community structure (Fig. 3A). For AOB, axis1 and axis2 respectively explained 77.48% and 18.59% of the total variation (Fig. 3B). Our results show that ORP had important influences on AOA and AOB communities in all sediments. Furthermore, the overall composition of the AOA and AOB communities was negatively correlated (p < 0.05) with ORP (Fig. 3). However, NO${}_3^{-}$ was positively correlated (p < 0.01) with the overall composition of the AOA and AOB. The order of the dominant factors corresponding to the levels of impact on the ammonia monooxygenase communities was ORP, Temp, NO${}_3^{-}$, DO, and NH${}_4^{+}$.

Spearman correlation matrix was calculated to investigate the correlations among environmental factors (Table 1) and abundance of AOA and AOB. All correlation analyses were applied to evaluate the significant number of relations at p <0.01 and p <0.05 (p-values). The results shown in Fig. S3 indicated that AOA and AOB communities were positively correlated (p<0.05) with DO, ORP, and NO${}_3^{-}$ and negatively correlated (p < 0.01) with Temp and NH${}_4^{+}$.

Abundance of functional ammonia-oxidizing communities in sediments

The functional genes (amoA genes) of AOA and AOB from nine different stations along the Luotian River were investigated. The results showed insignificant variations in the copy number of the total microorganisms, as evaluated on the basis of 16S rRNA (Table S2). AOA amoA copy numbers ranged from 5.28 × 10⁸ gene copies (g-soil⁻¹) from S7 (May) to 2.23 × 10⁸ gene copies (g-soil⁻¹) from S3 (August). AOB amoA copy numbers ranged from 5.45 × 10⁸ gene copies (g-soil⁻¹) from S5 (May) to 3.30 × 10⁷ gene copies (g-soil⁻¹) from S4 (May). However, high numbers of gene copies in AOA and AOB (Fig. 4) were observed at S7 and S5; they were calculated to be 5.28 × 10⁸ and 5.45 × 10⁸ gene copies (g-soil⁻¹) from May, respectively. In most of the sampling sites, higher numbers of gene copies were presented by the 16S rRNA group than by the AOA group (Table S2). S2, S6, and S7 showed higher numbers in AOA than in AOB, whereas at S5 and S8, AOB was higher than AOA. However, the abundance of AOB at S4 from May showed lower numbers of gene copies of 3.3 × 10⁷ than the other sites (Fig. 4B).

T-RFLP profiles of ammonia-oxidizing communities

T-RFLP is an essential method for detecting microbes in the environment, and it can be used to observe the abundance and composition of microbial communities in a statistical manner (Lueders & Friedrich, 2003). Therefore, T-RFLP is an effective tool to determine the diversity and abundance of the AOA group in and around the sampling locations in this work. The T-RFLP investigation of the AOA group by RsaI and HpyCH4V enzymes resulted in 8 distinct terminal restriction fragments (TRFs) across all the treatments and sediment fractions. In each sampling site, TRF-116 and TRF-486 were identified in two dominant genotypes (Fig. 5). All 8 T-RFs may be related to the clones of the AOA gene clone library, as shown in Table S4. The T-RFs of 116 and 486 bp were correlated with AOA in Nitrososphaera and Nitrosopumilus. However, the T-RF of 382 bp was mostly obtained from AOA in Nitrosotalea. At S3 and S5–S9, the relative abundances of Nitrososphaera and Nitrosopumilus were shown by the T-RF of 486 bp. The abundance of AOA in Nitrosotalea sp. was shown by the T-RF of 382 bp. Similar T-RFs in various tests appeared from different AOA groups while the T-RFs of 116 and 237 bp were collected by AOA in three groups with various ratios. The diversity of AOA groups between the sampling sites was intricate.