2.1 Source of soil sample and determination of soil basic properties
Soil sample was collected from a lead and zinc mine in Shangyu, Zhejiang, China (29°59′N, 120°46′E) that has East-Asian monsoon climate with an annual average precipitation of 1400 mm and temperature of 16.4°C. The soil sample was packed in a sterile plastic bag and stored at 4°C before use. The physicochemical properties of soil were determined as described previously (Bao 2000). Heavy metals were extracted with aqua regia-HClO4 and analyzed with inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 8000, Perkin Elmer Co, USA).
2.2 Growth media and buffers
Liquid media and buffers were prepared by dissolving the below-mentioned components into deionized water to make up a total volume of 1000 mL. For solid media, 1.8% (w/v) agar was added to the liquid media. The media were sterilized at 121°C for 30 min.
LB medium: 10.00 g tryptone, 5.00 g yeast extract, 10.00 g NaCl.
Iron-free modified King B (MKB) medium: 5.00 g acid hydrolyzed casein, 15 mL glycerinum, 2.50 g MgSO4·7H2O, 2.50 g K2HPO4. Trace iron was removed with 8-hydroxyquinoline.
Chrome azurol sulphonate (CAS) semisolid medium (Wang et al. 2014): 60.50 mg chrome azurol, 72.90 mg hexadecyl trimethyl ammonium bromide (HDTMA), 10 mL of 1 mmol·L− 1 FeCl3·6H2O (dissolved with 10 mmol·L− 1 HCl), 50 mL of 0.1 mol·L− 1 phosphate buffer, 0.9% (w/v) agar.
Dworkin and Foster (DF) medium: 2.00 g (NH4)2SO4, 4.00 g KH2PO4, 6.00 g Na2HPO4, 0.20 g MgSO4·7H2O, 2.00 g glucose, 2.00 g gluconic acid, 2.00 g citric acid, 1.00 mg FeSO4·7H2O, 10.00 µg H3BO3, 11.19 µg MnSO4·H2O, 124.60 µg ZnSO4·7H2O, 78.22 µg CuSO4·5H2O, 10.00 µg MoO3, pH 7.2.
ADF medium: as DF medium, except that the N source is 3.0 mmol ACC instead of (NH4)2SO4.
National Botanical Research Institute’s Phosphate Growth (NBRIP) medium: 10.00 g glucose, 0.50 g (NH4)2SO4, 0.30 g NaCl, 0.30 g KCl, 0.30 g MgSO4·7H2O, 5.00 g Ca3(PO4)2.
1 mol·L− 1 phosphate buffer: 5.91 g NaH2PO4·2H2O, 24.27 g Na2HPO4·12H2O, 2.50 g NH4Cl, 0.75 g KH2PO4, 1.25 g NaCl, pH 6.8.
2.3 Isolation of bacteria
10.0 g soil sample was suspended into 80 mL of sterile water, and the suspension was shaken at 150 rpm for 30 min at 28°C. Ten-fold serial dilutions of the suspension were inoculated onto Luria-Bertani (LB) agar plates as described previously (Liu et al. 2019). Plates were incubated at 28°C for 48 h. Well-separated colonies were picked and inoculated onto LB agar plates until a pure culture was obtained.
2.3.1 Qualitative analysis of SPB
The isolates were inoculated onto iron-free modified King B (MKB) medium agar plates and cultured at 28°C for 48 h. Chrome azurol sulphonate (CAS) semisolid medium was cooled to approximately 60°C and poured onto the MKB agar. After 2–4 h, a color change from blue to orange around a colony indicated siderophore production. Forty-four isolates with distinct orange circles around the colonies were chosen for further analyses. The analyses here and further on were done in triplicate.
2.3.2 Quantitative analysis of SPB
The isolates were cultured in iron-free MKB medium at 28°C for 48 h at 150 rpm. After centrifugation at 10,000 rpm for 10 min, the supernatant was collected and 1 mL supernatant was mixed with 1 mL of CAS reagent (Arora and Verma 2017; Schywn and Nielands 1987). A negative control was made by mixing 1 mL sterile water and 1 mL of CAS reagent. After incubation for 30 min at room temperature, the absorbance value of the mixture (As) was measured at the wavelength of 630 nm. The absorbance of uninoculated iron-free MKB medium mixed with CAS reagent was determined as the reference ratio (Ar). The experiment was carried out in triplicate. The percent siderophore unit (SU) was calculated using the following formula:
% SU = (Ar − As) / Ar × 100
The larger the SU value, the stronger the ability of an isolate to produce siderophore. The isolate with highest % SU value, referred as strain SX9 from hereon, was chosen for further analyses.
2.4 Characterization of strain SX9
The morphological and chemotaxonomic characterization of strain SX9 was done as described in Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). Genomic DNA of strain SX9 was extracted using Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech Co., Ltd, Shanghai, China). The 16S rRNA gene of the strain was amplified with polymerase chain reaction (PCR) using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-ACGGCTACCTTGTTACGACTT-3') (Weisburg et al. 1991). The PCR reaction conditions were as follows: initial denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 90 s, and a final extension at 72°C for 10 min. The PCR products were checked in 1.0% agarose gel and purified using DiaSpin DNA Gel Extraction Kit (Sangon Biotech Co., Ltd, Shanghai, China). The amplified product was sequenced at Sangon Biotech Co., Ltd (Shanghai, China). The 16S rRNA gene sequence of the strain SX9 was compared to closely related sequences in the National Centre of Biotechnology Information (NCBI) GenBank database using BLAST online (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was constructed using neighbor-joining method in MEGA 6.0 with a bootstrap value of 1,000 (Koichiro et al. 2013). The sequence was submitted to GenBank with the accession number MN658668.
2.5 Siderophore type determination
Arnow’s test (Arnow 1937), Csaky’s test (Csáky et al. 1948) and Shenker’s test (Shenker et al. 1992) were used to determine catecholate type, hydroxamate type and hydroxycarboxylate type siderophores, respectively.
Arnow’s test. l mL of culture supernatant was mixed with 0.l mL of 5 mol·L− 1 HCl. Subsequently, l mL nitrite-molybdate reagent (10.00 g NaNO2 and 10.00 g Na2MoO4 dissolved in 100 mL of deionized water) was added. In the presence of catechol, the nitrite in the solution is decomposed to form a yellow NO ligand. Next, 0.5 mL of 2 mol·L− 1 NaOH was added. In the presence of catecholate siderophore, the solution becomes red and remains unchanged for at least 1 h.
Csaky’s test. 1 mL of culture supernatant was mixed with 1.0 mL of 1 N H2SO4, boiled for 6 h, 3 mL of 35% sodium acetate solution was added and mixed well, and 1 mL of the solution was diluted with deionized water in a 1:5 ratio. Next, 0.5 mL of sulfanilic acid solution (1.00 g sulfanilic acid dissolved in 100 mL of 30% acetic acid) and 0.2 mL of iodine solution (1.30 g iodine dissolved in 100 mL acetic acid) were added and the solution was incubated at room temperature for 5 min. After this, 0.2 mL of sodium thiosulphate (2.00 g sodium thiosulphate dissolved in 100 mL deionized water) and 0.1 mL of α-naphthylamine solution (3.00 g α-naphthylamine in 1,000 mL of 30% acetic acid) were added and the solution was incubated at room temperature for 20–30 min. In the presence of hydroxamate siderophore, the solution becomes pink.
Shenker’s test. 1 mL supernatant was mixed with 1 mL 250 µmol·L− 1 CuSO4 and 2 mL acetic acid buffer (pH 4), and incubated at room temperature for 30 min. The presence of hydroxycarboxylate siderophore–copper compound was determined using full wavelength scanning. A maximum absorption peak at 190–280 nm indicated that the solution contains hydroxycarboxylate siderophore.
2.6 Siderophore production by strain SX9
To study siderophore production over time, strain SX9 was grown for 24 h and 2.5 mL of the culture, adjusted to A600 of 1.0, was inoculated into 250 mL iron-free MKB, followed by incubation at 28°C for 48 h at 150 rpm. Cell density as OD600 and siderophore production as % SU were recorded at 4 h intervals.
The effects of pH and NaCl and Fe3+ concentrations on the growth and siderophore production of strain SX9 were studied using MKB medium, inoculation volume of 1% (v/v) and incubation at 28°C and 150 rpm. The effect of temperature was studied similarly, except that the cultures were incubated at temperatures from 15 to 35°C. The effect of pH was examined in medium with the initial pH adjusted to 5.0, 6.0, 6.8, 7.4, 8.0 and 9.0. Salt tolerance was assessed at 0, 0.5, 1, 2, 3 and 5% (w/v) NaCl concentrations. The effect of Fe3+ concentration was assessed at 0, 0.5, 1, 2, 3, 5, 10 and 20 µmol·L− 1 Fe3+ concentrations.
2.7 Minimal inhibitory concentrations (MICs)
To determine the MICs of twelve metal salts and eight antibiotics, strain SX9 was grown in LB medium with predetermined concentrations of metal salts and antibiotics (Table 1) at 28°C for 48 h at 150 rpm (Yu et al. 2013).
Table 1 Physiological and biochemical characteristics of strain SX9 isolated from lead and zinc mine soil in Shangyu, Zhejiang, China.
Item
|
Result
|
Item
|
Result
|
Methylene red
|
+
|
Gram-staining
|
-
|
Voges-Proskauer
|
-
|
Fluorochrome assay
|
+
|
Oxidase
|
+
|
Catalase
|
+
|
Amylohydrolysis assay
|
-
|
Nitrate reductase
|
-
|
Indol reaction
|
-
|
Urease activity
|
-
|
H2S production
|
-
|
Gelatin hydrolysis
|
-
|
Fructose utilization
|
+
|
Mannose utilization
|
+
|
Xylose utilization
|
+
|
Maltose utilization
|
+
|
Lactose utilization
|
+
|
Galactose utilization
|
+
|
2.8 Solubilization of heavy metals
50 mL of liquid MKB with 50 mg of Cu2+ as CuO, 50 mg of Zn2+ as ZnO or 50 mg of Cd2+ as CdCO3 was inoculated with 1 mL of logarithmic phase strain SX9 inoculum and incubated at 28°C and 150 rpm. After 72 h, 5 mL of the culture was centrifuged at 8,000 rpm for 10 min and filtered through a 0.22 µm needle cartridge membrane filter (Jin Teng, China). The Cu2+, Zn2+ and Cd2+ concentrations in the filtrate were determined using inductively coupled plasma optical emission spectrometry (ICP-OES).
2.9 Complexation capacity assays
To assess the ability of siderophore to chelate metal ions under alkaline conditions, a standard curve was made using demertamine mesylate, and the content of siderophores was determined using the CAS method (Ferreira et al. 2019). FeCl3·6H2O, CuSO4·5H2O, ZnSO4·7H2O and Cd(NO3)2·4H2O were added to a fixed volume of the culture filtrate (Ferreira et al. 2019). The pH of the solution was set to 9.0 ± 0.1, the solution was incubated in room temperature for 30 min, the pH of solution was adjusted to 9.0 again, and the solution was incubated for 3 h. The solution was centrifuged at 3,000 g for 10 min and filtered through a 0.45 µm pore size membrane. The concentrations of metal ions in the solution was determined using ICP-OES. The iron complexation capacity of the siderophore in solution was calculated by plotting the ratio [metal ion]soluble/[S] versus [metal ion]added/[S], where [metal ion]soluble is the concentration of metal ion in the filtrate, [metal ion]added is the total concentration of metal ion added and [S] is the concentration of siderophore determined using the CAS method (Ferreira et al. 2019).
2.10 Plant growth promoting characteristics
IAA synthesis assay. The strain SX9 was inoculated into LB liquid medium containing 200 mg·L− 1 L-tryptophan and incubated at 150 rpm for 48 h at 30°C. 5 mL of the culture was centrifuged at 8,000 rpm for 10 min. 2 mL of the supernatant was mixed with 4 mL Salkowski’s reagent, incubated for 30 min at room temperature in the dark, and the absorbance of the solution was measured at 530 nm (Bahadur et al. 2020). A standard curve was made by measuring the absorbance of IAA standard solution diluted to concentrations of 0, 3, 5, 8, 10 and 15 mg·L− 1.
ACC deaminase activity assay. The strain SX9 was inoculated into 20 mL Dworkin and Foster (DF) medium. After incubation at 150 rpm for 24 h at 30°C, the cells were collected by centrifugation at 8,000 rpm for 10 min at 4°C. The cells were washed twice with DF liquid medium without (NH4)2SO4, suspended in 10 mL of ADF medium and incubated at 150 rpm for 24 h at 30°C. Protein content was determined using colorimetry with bovine serum albumin as the standard (Penrose and Glick 2010). The ACC deaminase activity was calculated using the standard curves of α-ketobutyric acid (α-KA) and protein as the amount of α-KA generated per mg of bacterial protein per hour (Penrose and Glick 2010). The ACC deaminase activity unit is µmol α-KA·(h·mg)−1.
Phosphate solubilization. The strain was inoculated into National Botanical Research Institute’s Phosphate Growth (NBRIP) medium and incubated at 30°C for 7 days at 150 rpm (Kong and Hong 2020). After centrifugation at 6,000 rpm for 10 min, phosphorus content of 1 mL supernatant was determined using the Mo-Sb colorimetric method. A not inoculated culture was used as a negative control.
2.11 Seed germination tests
To assess the effect of strain SX9 inoculation on Lolium perenne seed germination in the presence of heavy metal contaminants, Lolium perenne seeds were soaked in 75% alcohol for 5 min, washed 4 times with sterile deionized water, 50 seeds per plate were spread on filter paper in glass petri dishes, and inoculated with 5 mL of bacterial culture, bacterial culture supernatant or uninoculated growth medium. Metal salt solutions with increasing concentrations of Cu2+ (0, 100, 200, and 400 mg·L− 1), Zn2+ (0, 200, 500, and 1,000 mg·L− 1) and Cd2+ (0, 5, 10, and 20 mg·L− 1) were added to the plates. The plates were kept at a constant temperature of 28°C. Seeds were considered germinated when the root protruded from the seed coat by at least 2 mm. The final germination rate was calculated after 7 days. Five seedlings per plate were randomly chosen to determine the shoot and root lengths (Aka and Babalola 2016).
2.12 Statistical analysis
Statistical analyses and visualization were done using Microsoft Excel (version 2016) and Origin Pro 2018C (OriginLab Corporation, Northampton, USA), respectively. Statistical significance of the differences was tested using one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) test. Differences were considered statistically significant at p < 0.05.