Sampling
Sixteen tailing samples were obtained from three REE mines with the severe NH4
+-N pollution in southern Jiangxi Province, China. The sampling sites were randomly selected near the exploitation areas of the REE mines (Fig. 1). The samples were excavated from the depth of 10–15 cm in the tailings. Then, the samples were transferred into sterile bags, sealed and kept in a nitrogen canister. After being taken back to the laboratory, the samples were stored at −20 °C until being used for analysis.
Culture media
The enrichment medium (pH 7.2–7.4) was composed of (g/L): glucose, 5; (NH4)2SO4, 5; NaCl, 2; FeSO4·7H2O, 0.4; K2HPO4, 1; and MgSO4·7H2O, 0.5. The Luria–Bertani (LB) liquid medium consisted of (g/L): yeast extract, 5; tryptone, 10; and NaCl, 10. The LB agar medium contained (g/L): yeast extract, 5; agar, 20; NaCl, 10; and tryptone, 10. The screening medium (pH 7.2–7.4) was composed of (g/L): glucose, 5; (NH4)2SO4, 5; NaCl, 1; K2HPO4, 0.5; and MgSO4·7H2O, 0.25. All the culture media were prepared using deionized water and were autoclaved for 30 min before use.
Analysis of the contents of NH4
+-N, NO3
−-N and NO2
−-N in the tailings
The concentrations of NH4
+-N in the tailings were measured by spectrophotometry using the Auto Analyser 3 System (Bran + Luebbe, Germany). Prior to analysis, 25 g of the samples were mixed with 100 mL of deionized water, respectively. The concentrations of NH4
+-N were measured using hydrazine sulphate (Kearns 1968) as a color marker. The obtained results were corrected for the amount of the samples and expressed as milligram per kilogram of the tailings. The contents of NO3
−-N were measured according to the international method (Liang et al. 2012), which was based on the absorbance of NO3
− at 220 nm. The contents of NO2
−-N were determined by measuring the absorbance of NO2
−-N solution at 540 nm according to the instructions of an international standard method (Shi and Chao 2014). This method is based on the following principle: (i) NO2
− reacts with 4-aminobenzenesul fonamide under the condition of pH 1.8, resulting in the production of diazonium salt; (ii) the diazonium salt couples with C12H14N2·2HCl to produce a red dye that can be detected at 540 nm.
Enrichment culture and screening of microorganisms with the NH4
+-N-degrading ability
The tailing samples obtained from the REE mines were mixed together (10 g per sample) for the screening experiment. Then, 50 g of the mixed sample was transferred into the enrichment medium. The mixtures were incubated at 28 °C and 120 rpm overnight. After that, 10 mL of the culture was injected into a fresh enrichment medium, followed by incubation at 28 °C and 120 rpm overnight. Then, the culture was subjected to separation using the LB agar plate to obtain single clones.
The clones were separately inoculated into the LB medium and were incubated at 28 °C and 180 rpm for 24 h. After that, the cells were collected by centrifugation (8000 rpm) and were suspended by sterilized normal saline to prepare a bacterial suspension with a density of approximately 109 cells per milliliter (OD600 ≈ 1). Then, 10 mL of the bacterial suspension was mixed with 190 mL of screening medium in a 1 L flask, followed by incubation at 28 °C and 200 rpm for 48 h. The residual NH4
+-N (from (NH4)2SO4 in the screening medium) was detected according to the method described previously (Yang et al. 2006). The screening medium without cell inoculation served as the control. The degradation rates of NH4
+-N were calculated according to Eq. (1) to evaluate the degradation capabilities of microorganisms.
$$R = (C_{0} - C_{1} )/C_{0} \times 100\%$$
(1)
where R, C
0 and C
1 represented degradation rates, the concentration of NH4
+-N in the control and the concentration of NH4
+-N in the medium with cell inoculation, respectively.
Morphological and biochemical characterization
The bacterium with the excellent NH4
+-N-degrading capability was subjected to morphological observations and biochemical characterization. Optical microscopy, transmission electronic microscopy and scanning electron microscopy were adopted to analyze its morphological features according to the conventional methods (Chao et al. 2010; Deng et al. 2014, 2016; Prior and Perkins 1974). Its biochemical and physiological characteristics were analyzed according to the methods described previously (Faller and Schleifer 1981; Holt et al. 1994; Kloos et al. 1974; Lányi 1988), including motility, aerobism, Gram staining, spore formation, catalase activity, glucose fermentation, oxidase activity, nitrate reduction, starch hydrolysis, gelatin hydrolysis, indole production, Voges–Proskauer (V–P) reaction, citrate utilization, methyl red test, and production of hydrogen sulfide.
PCR amplification of 16S rDNA and phylogenetic analysis
The bacterium with the excellent NH4
+-N-degrading ability was further identified by phylogenetic analysis. Its genomic DNA was extracted according to the method described previously (Winnepenninckx et al. 1993). The 16S rDNA was amplified using universal primers 27F (5′-AGAGATTGATCCTGGCTCTG-3′) and 1492R (5′-GGTTTCCTTGTTACGACAT-3′) (Deng et al. 2014). The primers were synthesized by Sangon Biotech (Shanghai, China). The PCR reaction mixture was composed of genomic DNA (20 ng), 27F (50 μM), 1492R (50 μM), 10 × PCR buffer, 0.5 μL of DNA polymerase (5 U/L, TaKaRa, Japan), dNTPs (10 mM), MgCl2 (25 mM), and sterile ddH2O up to a volume of 50 μL. The PCR reactions were carried out on the LongGene MGL96G (Hangzhou, China). The PCR procedure was set as follows: (i) 95 °C for 5 min; (ii) 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s; and (iii) 72 °C for 10 min. Then, the PCR product was sequenced by Sangon Biotech (Shanghai, China). The obtained 16S rDNA sequence was submitted to the GenBank database for the BLAST alignment. The MEGA 5 software (Tamura et al. 2011) was adopted to construct a phylogenetic tree using the neighbor-joining method (Li 2015).
Optimization of NH4
+-N degradation by the isolated bacterium
The effects of incubation time, carbon source, temperature, pH, C/N ratio, inoculum dose, and rotary speed on NH4
+-N degradation were evaluated to determine the optimal conditions for NH4
+-N degradation. (i) To evaluate the effect of incubation time on NH4
+-N degradation, the isolated bacterium was inoculated (10%, v/v) into the screening medium (pH 7.0) containing NH4
+-N (1 g/L), followed by incubation at 30 °C and 120 rpm. (ii) To determine the most suitable carbon source for NH4
+-N degradation, the following compounds were added into the screening medium without glucose, respectively: saccharose, lactose, sodium propionate, potassium sodium tartrate, glucose, ethanol, sodium acetate, and sodium citrate. The bacteria (10%, v/v) were incubated for 48 h at 30 °C and 120 rpm. (iii) Regarding the most suitable temperature for NH4
+-N degradation, the incubation temperature was set at 16, 20, 24, 28, 30, 32, 36, and 40 °C, respectively. (iv) To determine the most suitable pH for NH4
+-N degradation, HCl or NaOH was adopted to adjust the initial pH of the screening medium to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The bacteria were incubated at 30 °C and 120 rpm for 48 h. (v) The carbon nitrogen ratios (C/N; w/w) were set at 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, and 14:1, respectively. The bacteria were incubated at 30 °C and 120 rpm for 48 h. (vi) For the optimization of inoculum dose, the bacteria were inoculated into the screening medium (pH 7.0) containing 1 g/L of NH4
+-N, followed by incubation at 30 °C and 120 rpm for 48 h. The inoculum doses (v/v) of bacteria were set at 2, 5, 8, 10, 12, 15, and 18%, respectively. (vii) To determine the optimal rotary speed during incubation in an orbital shaker, the bacteria were inoculated (10%, v/v) into the medium (pH 7.0) containing 1 g/L of NH4
+-N and were incubated at 30 °C for 48 h. The rotary speeds were set at 100, 120, 150, 180, and 210 rpm, respectively.
The screening medium (unless otherwise specified) was used in all the optimization experiments mentioned above. The medium without cell inoculation served as the negative control. The degradation rates of NH4
+-N were calculated according to the method described above to determine the most suitable conditions for NH4
+-N degradation.
Besides, an orthogonal design containing five factors and four levels was adopted to further optimize the conditions for NH4
+-N degradation. The inoculum amount, temperature, pH, C/N ratio, and incubation time were respectively set at 6, 8, 10, 12%; 26, 28, 30, 32 °C; 6.0, 6.5, 7.0, 7.5; 5:1, 10:1, 15:1, 20:1; and 44, 48, 52, 56 h.
Effect of the isolated bacterium on plant growth
Red soils for the growth of Nepeta cataria were baked at 120 °C for 6 h to remove the original bacteria in the soils. Ten seeds of Nepeta cataria were sown in the red soils (1 kg) with different concentrations of NH4
+-N (500, 1000, 1500, and 2000 mg/kg, respectively). Then, a bacterial suspension of the isolated bacterium (1 mL, OD600 = 1) was inoculated into the red soils. The groups without bacterial inoculum served as the controls. The growth of Nepeta cataria in a humid environment was observed, and the plant lengths were measured at the time point of 12 days. Additionally, the concentrations of residual NH4
+-N in the soils were detected every two days using the method described above.
Growth of strain Gan-35 in the high salt medium
Strain Gan-35 was inoculated into the screening medium containing 1.0, 2.0, and 3.5% (w/v) of NaCl, respectively, followed by incubation at 28 °C and 120 rpm for 48 h. The absorbance of the culture at 523 nm was measured every 4 h. Then, a growth curve was drawn to evaluate the growth of strain Gan-35.
Accession number
The 16S rDNA sequence of the isolated bacterium was submitted to the GenBank database under accession number KY928114.