Isolation and characterization of Burkholderia fungorum Gan-35 with the outstanding ammonia nitrogen-degrading ability from the tailings of rare-earth-element mines in southern Jiangxi, China.

The exploitation of rare-earth-element (REE) mines has resulted in severe ammonia nitrogen pollution and induced hazards to environments and human health. Screening microorganisms with the ammonia nitrogen-degrading ability provides a basis for bioremediation of ammonia nitrogen-polluted environments. In this study, a bacterium with the outstanding ammonia nitrogen-degrading capability was isolated from the tailings of REE mines in southern Jiangxi Province, China. This strain was identified as Burkholderia fungorum Gan-35 according to phenotypic and phylogenetic analyses. The optimal conditions for ammonia-nitrogen degradation by strain Gan-35 were determined as follows: pH value, 7.5; inoculum dose, 10%; incubation time, 44 h; temperature, 30 °C; and C/N ratio, 15:1. Strain Gan-35 degraded 68.6% of ammonia nitrogen under the optimized conditions. Nepeta cataria grew obviously better in the ammonia nitrogen-polluted soil with strain Gan-35 than that without inoculation, and the decrease in ammonia-nitrogen contents of the former was also more obvious than the latter. Besides, strain Gan-35 exhibited the tolerance to high salinities. In summary, strain Gan-35 harbors the ability of both ammonia-nitrogen degradation at high concentrations and promoting plant growth. This work has reported a Burkholderia strain with the ammonia nitrogen-degrading capability for the first time and is also the first study on the isolation of a bacterium with the ammonia nitrogen-degrading ability from the tailings of REE mines. The results are useful for developing an effective method for microbial remediation of the ammonia nitrogen-polluted tailings of REE mines.

Background

Rare earth elements (REEs) have wide applications and are considered as the industrial gold due to their unique optical, magnetic, and catalytic properties (Cornell 1993). Currently, China supplies over 90% of the n class="Chemical">REEs-related products to the global market, and two-thirds of the products are produced in southern Jiangxi Province (Information Office of the State Council of China 2010). Among the REE mines, the ion-absorbing middle-heavy REE deposit occupies an important position in the world market (Information Office of the State Council of China 2010). REEs in this deposit primarily occur in the weathered layer of granites and are generally adsorbed in soils/sediments in the form of ions (Bao and Zhao 2008). So far, the advanced in situ leaching method has been extensively adopted to separate and extract ion-adsorbed REEs in southern Jiangxi Province, China (Wen et al. 2013). This is an effective method for the REE exploitation. However, it depends on the injection of chemicals, such as ammonium sulfate or ammonium bicarbonate, into the soils/sediments to extract REEs. The tailings and waste water resulting from the exploitation contains high concentrations of ammonia nitrogen (NH4 +-N), which have caused severe negative impacts on local ecosystems and human health (Åström 2001). For instance, the NH4 +-N pollution in the tailings of REE mines has resulted in soil degradation, forest destruction, and threat to life (Gao and Zhou 2011). The carcinogenic effect may be induced when NH4 +-N in the polluted drinking water was transformed into nitrite nitrogen (NO2 −-N). Therefore, it is urgent and necessary to remediate the NH4 +-N-polluted tailings in REE mines for realizing a sustainable development.

Recently, diverse methods have been proposed for environmental remediation. Besides high cost, the physical and chemical methods can not thoroughly eliminate pollutants and may result in secondary pollution. Thus, they are usually used for emergent environmental restoration (Xue et al. 2015). Bioremediation has become one of the most reliable strategies for completely eliminating pollutants without secondary pollution. Recent researches on the tailings of REE mines has focused on the REE risk in soils and vegetables to n class="Species">human health (Hao et al. 2016). To our knowledge, however, studies on the bioremediation of the tailings of REE mines are rare, which is hindering the realization of pollution reduction and anticipated ecological balance in these areas. Microbial remediation is one of bioremediation methods and has been regarded as a cost-effective and eco-friendly strategy for eliminating pollutants (Al-Mailem et al. 2014; Dellagnezze et al. 2014; Hassanshahian et al. 2012). Screening microorganisms with the NH4 +-N-degrading ability in the tailings of REE mines is undoubtedly important for performing microbial remediation in these areas. However, up to date, no report on the microorganisms with the NH4 +-N-degrading ability is present in these areas.

The aim of this study is to isolate and characterize a bacterium with the outstanding NH4 +-n class="Chemical">N-degrading capability from the tailings of REE mines in southern Jiangxi Province, China. The results may contribute to developing an effective method for the microbial remediation of NH4 +-N-polluted environments, in particular the tailings of REE mines.

Methods

Sampling

Sixteen tailing samples were obtained from three REE mines with the severe NH4 +-n class="Chemical">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.

Fig. 1

Remote sensing images of three rare-earth-element mines in southern Jiangxi, China. The geomorphologic features and 16 sampling sites (blue circles) are shown in the figure

Culture media

The enrichment medium (pH 7.2–7.4) was composed of (g/L): glucose, 5; n class="Chemical">(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 class="Chemical">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 n class="Chemical">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 n class="Chemical">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.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 class="Chemical">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 class="Chemical">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 class="Chemical">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 class="Chemical">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 class="Chemical">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 n class="Species">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 n class="Chemical">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.

Results

Contents of NH4+-N, NO3−-N and NO2−-N in the tailings of REE mines

The contents of NH4 +-n class="Chemical">N in the tailings range from 483.2 to 899.4 mg/kg (Table 1), indicating that there is severe NH4 +-N pollution in the tailings of REE mines. However, the concentrations of NO3 −-N and NO2 −-N are relatively low.

Table 1

Contents of NH4 +-N, NO3 −-N and NO2 −-N in the tailings of rare-earth-element mines

Sample no.	NH4 +-N (mg/kg)	NO3 −-N (mg/kg)	NO2 −-N (mg/kg)
1	837.8	151.0	7.9
2	713.6	69.8	33.3
3	821.2	84.8	16.8
4	766.5	135.2	25.5
5	663.6	152.3	8.3
6	728.9	174.6	150.1
7	483.2	70.6	37.9
8	648.1	241.0	51.6
9	677.7	293.4	18.0
10	899.4	37.1	23.7
11	888.4	118.1	15.9
12	563.2	135.0	66.4
13	587.8	56.2	45.6
14	890.2	70.3	24.5
15	653.6	87.8	39.5
16	770.9	121.1	229.2

Contents of NH4 +-n class="Chemical">N, NO3 −-N and NO2 −-N in the tailings of rare-earth-element mines

Screening of NH4+-N-degrading strains

The screening experiment showed that 45 strains with the NH4 +-n class="Chemical">N-degrading ability were obtained from the tailings of REE mines. The degradation rates against NH4 +-N (1 g/L) ranged from 21.6 to 65.6% (Fig. 2) after incubation for two days. Strain Gan-35 exhibited the highest degradation rate and was selected for further investigation. Additionally, the concentrations of NO3 −-N and NO2 −-N in the screening medium during NH4 +-N degradation were measured, and the results showed that their contents were very low (Additional file 1: Tables S1, S2). Only 0.95% and 0.06% of NH4 +-N were transformed into NO3 −-N and NO2 −-N, respectively, suggesting that strain Gan-35 may not cause secondary pollution during NH4 +-N degradation.

Fig. 2

Strains with the NH4 +-N-degrading ability. The degradation rates were detected after incubation for 2 days. The detections were performed in triplicate, and the results were presented as mean ± standard deviation

Strains with the NH4 +-n class="Chemical">N-degrading ability. The degradation rates were detected after incubation for 2 days. The detections were performed in triplicate, and the results were presented as mean ± standard deviation

Identification of strain Gan-35

The colony of strain Gan-35 was shown to be smooth-faced, white-colored, and circular with a tidy margin (Fig. 3a). This strain is a Gram-negative bacterium (Fig. 3b). The results of scanning electron microscopy (×15,000) and transmission electronic microscopy (×5000) showed that strain n class="Chemical">Gan-35 was a rod-shaped cell with a size of (0.5–0.8) μm × (1.0–2.1) μm and contained flagella on the cell surface (Fig. 3c, d). The biochemical and physiological characteristics of strain Gan-35 were shown in Table 2. Briefly, strain Gan-35 is an aerobic and motile bacterium with the activities of catalase and oxidase. This strain is positive for nitrate reduction, starch hydrolysis, gelatin hydrolysis, and Voges–Proskauer reaction. It is negative for indole production, citrate utilization, spore formation, and production of hydrogen sulfide. In summary, the characteristics of strain Gan-35 are consistent with the descriptions of Bergey’s Manual of Determinative for Bacteriology (Holt et al. 1994) regarding Burkholderia.

Fig. 3

Morphological characteristics of strain Gan-35. a Colonies of strain Gan-35 on the LB agar plate; b a photograph of Gram staining (20 × 100); c a photograph of scanning electron microscopy (×15,000); and d a photograph of transmission electronic microscopy (×5000)

Table 2

Morphological, physiological and biochemical characteristics of strain Gan-35

Characteristics	Strain Gan-35
Morphology	Rod shaped
Colony color	White
Gram staining	−
Motility	+
Aerobism	+
Spore formation	−
Oxidase activity	+
Catalase activity	+
Glucose fermentation	−
Nitrate reduction	+
Starch hydrolysis	+
Gelatin hydrolysis	+
Methyl red test	−
Citrate utilization	−
Indole production	−
Voges–Proskauer reaction	+
Production of hydrogen sulfide	−

−, negative; +, positive

Morphological characteristics of strain Gan-35. a Colonies of strain n class="Chemical">Gan-35 on the LB agar plate; b a photograph of Gram staining (20 × 100); c a photograph of scanning electron microscopy (×15,000); and d a photograph of transmission electronic microscopy (×5000)

DNA sequencing showed that the obtained 16S rDNA sequence (1425 bp) of strain Gan-35 was highly homologous (99% identity) to that of Burkholderia strains. A phylogenetic tree (Fig. 4) was constructed according to the similar 16S rDNA sequences using the neighbor-joining method (Li 2015). The result showed that strain Gan-35 was most closely related to Burkholderia fungorum strain NBRC 102489 (accession number: NR_114118.1).

Fig. 4

Phylogenetic analysis using 16S rDNA sequences. The accession numbers of the 16S rDNA sequences are presented in the parentheses. Strain Gan-35 is indicated by a pentagram

Phylogenetic analysis using 16S rDNA sequences. The accession numbers of the 16S rDn class="Chemical">NA sequences are presented in the parentheses. Strain Gan-35 is indicated by a pentagram

Based on the morphological, biochemical and physiological properties and phylogenetic analysis of 16S rDNA sequences, strain n class="Chemical">Gan-35 was identified as Burkholderia fungorum. Strain Gan-35 was deposited in the China Center for Type Culture Collection with the preservation number of “CCTCC AB 2017087”.

Optimization of the conditions for NH4+-N degradation

The effects of incubation time, carbon source, temperature, pH, C/n class="Chemical">N ratio, inoculum dose, and rotary speed on the degradation of NH4 +-N by strain Gan-35 were investigated in this study. It can be seen from Fig. 5a that the degradation rates of NH4 +-N increase rapidly after the time point of 12 h, and after 32 h the increase is less obvious. Under the conditions of 16–30 °C, the degradation rates increase with the enhancement of temperatures. Strain Gan-35 can degrade 59.8% of NH4 +-N at 30 °C, and the degradation rates decrease when the temperatures are higher (Fig. 5b). Strain Gan-35 exhibits the highest degradation rates (60.1%) against NH4 +-N at an initial pH value of 7.0 (Fig. 5c). The degradation rates decrease when the pH values are lower or higher, suggesting that a neutral environment is more suitable for the NH4 +-N degradation by strain Gan-35. As shown in Fig. 5d, the highest degradation rate (59.3%) was obtained when the C/N ratio was set at 10:1. It also suggests that a greater C/N ratio does not necessarily increase the degradation rates. The degradation rates increase significantly when the inoculum doses are at the interval of 2–10% (Fig. 5e). The changes of degradation rates are not obvious if the inoculum doses are greater than 10%, which may be due to that the nutrients are relatively deficient when the inoculum doses are greater. A higher degradation rate was observed when the rotary speed of an orbital shaker was set at 150 rpm (Fig. 5f), suggesting that moderate dissolved oxygen is needed for the efficient degradation of NH4 +-N. Besides, strain Gan-35 exhibits the highest degradation rate of NH4 +-N (59.4%) using glucose (followed by sodium citrate and sodium acetate) as the sole carbon source (Fig. 5g).

Fig. 5

Optimization of the conditions for NH4 +-N degradation. The optimized conditions included incubation time (a), temperature (b), pH (c), C/N ratio (d), inoculum dose (e), rotary speed (f), and carbon source (g). The degradation tests were performed in triplicate, and the results were shown as mean ± standard deviation

Optimization of the conditions for NH4 +-n class="Chemical">N degradation. The optimized conditions included incubation time (a), temperature (b), pH (c), C/N ratio (d), inoculum dose (e), rotary speed (f), and carbon source (g). The degradation tests were performed in triplicate, and the results were shown as mean ± standard deviation

An orthogonal design involving five factors and four levels was performed in this study. Range analysis and variance analysis were adopted to determine the optimal conditions for NH4 +-n class="Chemical">N degradation, and the results were shown in Tables 3 and 4. The variance analysis indicates that there are differences among inoculum dose, temperature, pH, C/N ratio, and incubation time. Their effects on NH4 +-N degradation are significant (p < 0.05) and are in the following order: temperature >pH >C/N ratio >incubation time >inoculum dose. According to the range analysis, the optimal conditions for NH4 +-N degradation in an indoor laboratory was determined as follows: pH value, 7.5; inoculum dose, 10%; incubation time, 44 h; temperature, 30 °C; and C/N ratio, 15:1. A mean degradation rate of 68.6% was obtained under the above optimal conditions. The optimization of NH4 +-N degradation by strain Gan-35 contributes to designing effective methods for bioremediation of NH4 +-N-polluted environments, such as producing effective microbial inocula for bioaugmentation.

Table 3

Orthogonal design for NH4 +-N degradation

No.	Inoculum dose (%)	Temperature (°C)	pH value	C/N ratio	Incubation time (h)	Degradation rate (%)
1	6	26	6.0	5:1	44	49.7 ± 2.3
2	6	28	6.5	10:1	48	50.8 ± 1.9
3	6	30	7.0	15:1	52	66.1 ± 1.2
4	6	32	7.5	20:1	56	59.4 ± 1.5
5	8	26	6.5	15:1	56	51.2 ± 2.2
6	8	28	6.0	20:1	52	53.7 ± 2.8
7	8	30	7.5	5:1	48	66.8 ± 2.3
8	8	32	7.0	10:1	44	51.5 ± 2.0
9	10	32	7.0	20:1	48	52.4 ± 1.1
10	10	30	7.5	15:1	44	68.6 ± 1.4
11	10	28	6.0	10:1	56	65.2 ± 1.6
12	10	26	6.5	5:1	52	51.2 ± 2.5
13	12	26	7.5	10:1	52	52.1 ± 1.4
14	12	28	7.0	5:1	56	51.8 ± 2.8
15	12	30	6.5	20:1	44	65.1 ± 2.2
16	12	32	6.0	15:1	48	53.1 ± 2.4
k_1¯	56.500	51.350	55.425	54.875	59.225
k_2¯	55.800	55.975	55.325	54.900	55.775
k_3¯	59.100	66.550	55.450	59.500	55.775
k_4¯	56.275	53.800	61.475	58.400	56.900
Range	3.300	15.200	6.150	4.625	3.450

The degradation rates are presented as mean ± standard deviation

Table 4

Variance analysis for the conditions of NH4 +-N degradation

Factor	Square of deviance	df	F ratio	F critical value	Significance
Inoculum dose	26.397	3	0.170	3.290
Temperature	537.557	3	3.469	3.290	*
pH	100.752	3	0.715	3.290
C/N	68.437	3	0.442	3.290
Incubation time	31.742	3	0.205	3.290
Error	774.88	15

* Significantly different (p < 0.05)

Orthogonal design for NH4 +-n class="Chemical">N degradation

Variance analysis for the conditions of NH4 +-n class="Chemical">N degradation

Effects of strain Gan-35 on plant growth and its tolerance to the high salinity

Burkholderia is a genus rich in n class="Chemical">nitrogen-fixing and phosphate-solubilizing strains that have been isolated from various plant systems. The functions of phosphate-solubilizing bacteria in agriculture have been well documented, including enhancements in growth, yield and disease-resistance of crops (Ghosh et al. 2016). The effects of Burkholderia fungorum Gan-35 on plant growth were investigated in this study. As shown in Fig. 6, Nepeta cataria with Gan-35 inoculum grew obviously better than that without inoculation. The average plant lengths of the former are significantly greater than the latter (Fig. 7). Besides, the NH4 +-N contents in the red soils decreased obviously along with the time extension in the experimental groups with Gan-35 inoculum, and the degradation rates of NH4 +-N were between 43.37 and 51.42% (Table 5). Nevertheless, the decrease of NH4 +-N contents in the controls was not obvious, and the degradation rates were between 6.12 and 10.30%. Thus, it is suggested that the degradation of NH4 +-N performed by strain Gan-35 can relieve the growth inhibition effect caused by the high concentrations of NH4 +-N. In other words, Gan-35 inoculation in the soils with NH4 +-N pollution contributes to the growth of Nepeta cataria.

Fig. 6

The promotion effect of strain Gan-35 on the growth of Nepeta cataria. Nepeta cataria was grown in the red soils containing NH4 +-N at the concentrations of 500, 1000, 1500, and 2000 mg/kg, respectively. (−), without Gan-35; (+), with Gan-35

Fig. 7

Lengths of Nepeta cataria during growth in the red soils containing NH4 +-N. (−), without Gan-35; (+), with Gan-35. The numbers on the horizontal axis are the concentrations of NH4 +-N (mg/kg)

Table 5

NH4 +-N contents in the red soils with time extension

Time (d)	Concentration of NH4 +-N (mg/kg)
	500(+)	500(−)	1000(+)	1000(−)	1500(+)	1500(−)	2000(+)	2000(−)
0	547.92	539.69	1093.27	1121.02	1605.42	1589.48	2128.18	2131.26
2	439.76	530.27	903.55	1103.54	1481.53	1556.12	1803.37	2090.11
4	372.84	521.89	784.51	1096.26	1377.28	1539.47	1657.23	2049.15
6	340.64	510.68	700.36	1088.47	1236.01	1521.91	1492.84	2017.34
8	306.61	506.12	646.78	1071.22	1099.32	1498.98	1375.27	1995.77
10	282.79	500.09	598.32	1061.32	995.50	1482.42	1284.41	1941.36
12	266.17	496.46	540.16	1052.43	885.94	1473.22	1205.21	1911.69
Degradation rate (%)	51.42	8.01	50.59	6.12	44.82	7.31	43.37	10.30

The concentrations of NH4 +-N are presented as the average values of two determinations

d, day; (−), without Gan-35; (+), with Gan-35

The promotion effect of strain Gan-35 on the growth of n class="Species">Nepeta cataria. Nepeta cataria was grown in the red soils containing NH4 +-N at the concentrations of 500, 1000, 1500, and 2000 mg/kg, respectively. (−), without Gan-35; (+), with Gan-35

Lengths of Nepeta cataria during growth in the red sn class="Chemical">oils containing NH4 +-N. (−), without Gan-35; (+), with Gan-35. The numbers on the horizontal axis are the concentrations of NH4 +-N (mg/kg)

NH4 +-n class="Chemical">N contents in the red soils with time extension

The concentrations of NH4 +-n class="Chemical">N are presented as the average values of two determinations

d, day; (−), without Gan-35; (+), with n class="Chemical">Gan-35

The growth of strain Gan-35 in the high n class="Chemical">salt medium showed that this bacterium entered a rapid growth phase after the time points of 12 or 20 h when the concentrations of NaCl were set at 1.0, 2.0, and 3.5%, respectively (Fig. 8). The bacteria were still in the rapid growth period at the time point of 48 h in the media with 2.0 or 3.5% of NaCl, suggesting that strain Gan-35 exhibits the tolerance to the high salinity, which contributes to the bioremediation in hyperhaline NH4 +-N-polluted environments and the removal of NH4 +-N in aquatic environments in marine farms.

Fig. 8

Growth curve of strain Gan-35 during growth in the high salt medium. The concentrations of NaCl in the medium were set at 1.0, 2.0, and 3.5% (w/v), respectively. The absorbance of the culture at 523 nm was measured every 4 h

Growth curve of strain Gan-35 during growth in the high n class="Chemical">salt medium. The concentrations of NaCl in the medium were set at 1.0, 2.0, and 3.5% (w/v), respectively. The absorbance of the culture at 523 nm was measured every 4 h

Discussion

The detection of NH4 +-n class="Chemical">N contents in the tailings of REE mines suggests that the NH4 +-N pollution in these areas is severe. The studied samples were collected at the depth of 10–15 cm in the tailings. We infer that the contents of NH4 +-N may be higher at deeper positions due to the cumulative effect, and that a part of NH4 +-N in the surface tailings has been transferred into aquatic environments by rain wash. The severe NH4 +-N pollution has induced many negative effects on the surrounding ecosystems and human health (Åström 2001). Thus, reducing the NH4 +-N pollution and remediating its contaminated environment are imperative.

So far, some functions of Burkholderia strains have been demonstrated, such as biological control, promoting plant growth, bioremediation, and producing various active metabolites, including n class="Chemical">phenazine, pyroace, and monoterpenoid alkaloids (Coenye et al. 2001). Besides, Burkholderia strains had been applied as biological insecticides, biological bactericides and decomposition of toxic substances. To our knowledge, the NH4 +-N-degrading ability of Burkholderia sp. has not been discovered before. Thus, this study has reported a Burkholderia strain with the NH4 +-N-degrading capability for the first time. The obtained results provide a new insight into the promising applications of Burkholderia strains in terms of bioremediation of NH4 +-N-polluted environments. This is also the first report on the isolation and characterization of a bacterium with the NH4 +-N-degrading ability from the tailings of REE mines, laying the foundation for the bioremediation of these areas.

In situ bioremediation using indigenous microorganisms is an effective method to eliminate pollutants (Lin et al. 2012). In some cases, indigenous microorn class="Chemical">ganisms with the pollutant-degrading ability may be better adapted for bioremediation. Since strain Gan-35 is an indigenous bacterium isolated from the NH4 +-N-polluted tailings of REE mines and exhibits the ability for (i) the degradation of NH4 +-N at high concentrations, (ii) promoting plant growth, and (iii) resistance to high salinity, it is plausible that strain Gan-35 can be applied in the bioremediation of these areas. Unraveling the mechanisms for NH4 +-N degradation in strain Gan-35 and extensive field studies in the future, such as revealing the relative abundance of strain Gan-35 in the tailings of REE mines, contribute to realizing its practical applications for bioremediation.

In summary, a bacterium with the NH4 +-n class="Chemical">N-degrading capability has successfully been isolated from the tailings of REE mines. This strain is identified as Burkholderia fungorum Gan-35 on the basis of phylogenetic analysis and its phenotypic characteristics. This is the first study to report a Burkholderia strain with the NH4 +-N-degrading ability. This is also the first research on the screening of a bacterium with the NH4 +-N-degrading ability from the tailings of REE mines. The optimal conditions for NH4 +-N degradation in strain Gan-35 have been determined, which provides valuable information for designing effective methods for its applications in bioremediation. Besides, strain Gan-35 exhibits the abilities of promoting plant growth and resistance to high salinities. This work contributes to developing a cost-effective and eco-friendly method for bioremediation of the tailings of REE mines contaminated by NH4 +-N.