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

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.

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.

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

Screening of NH₄ ⁺-N-degrading strains

The screening experiment showed that 45 strains with the NH₄ ⁺-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.

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 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.

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

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).

Based on the morphological, biochemical and physiological properties and phylogenetic analysis of 16S rDNA sequences, strain 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 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).

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

Burkholderia is a genus rich in 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.

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 growth of strain Gan-35 in the high 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.