A high Mn(II)-tolerance strain, Bacillus thuringiensis HM7, isolated from manganese ore and its biosorption characteristics

Samples and main materials

Soil samples (the content of Mn(II) was about 15225.17 mg/kg, pH was about 4.5–4.7) were collected from Xiangtan Mn ore, Hunan, China (112°45′E∼122°55′E, 27°53′N∼28°03′N), which has serious heavy metal pollution (Ouyang et al., 2016). Samples were taken from 0–10 cm soil from Mn(II) ore wasteland, 100 mesh sieved, mixed evenly and placed in a sterile centrifugal tube, and stored at −80 °C in the laboratory.

Basic beef-Protein medium: Peptone 1%, beef extract 0.3%, and NaCl 0.5%, pH 5.6–6.0 (plus agar 1.5% for solid medium); Selection medium: MnSO₄⋅H₂O was added to the basal medium and adjusted to the desired concentration. Required pH was adjusted by 1 mol/L NaOH and 1 mol/L HCl.

Selection of Mn(II)-tolerant bacterial strains

Ten gram of soil sample was dissolved in 90 ml of distilled water and coated on 500 mg/L Mn(II) solid selection medium (basic beef-Protein medium), at 30 °C for 3 days cultured in a biochemical incubator (SPX-250B, China). In the aseptic console (SW-CJ-2F, China), the selected colonies were subsequent culturing/ sub-transferring incubated with increasing concentrations of Mn(II) (500, 1,000, 1,500, 2,000, 3,000, and 4,000 mg/L) at 30 °C for 3 days in solid basic beef-Protein medium. Finally, the high Mn(II)-tolerant strain was picked up and repeatedly streaked on solid basal medium to obtain pure culture bacteria, stored at −4 °C in the Beef-Protein slant medium.

Identification of the Mn(II)-tolerant strain

Bacterial characterisation

By screening, a Mn(II)-tolerant strain was isolated and characterised morphologically, and biochemically. Gram staining, catalase test, hydrogen sulphide test, methyl red test, Voges Proskauer test, oxidase test, glucose test, and gelatine liquefaction test were detected by the standard methods in Bergey’s Manual of Determinative Bacteriology (Holt, 1994).

16S rDNA-based identification

To determine the classification of the strain, 16S rDNA analysis was performed on HM7. The strain was subjected to PCR amplification using bacterial universal primers 1492R (5′TAC GGY TAC CTT GTT ACG ACT T 3′) and 27F (5′AGA GTT TGA TCM TGG CTC AG 3′). Purification and sequencing (Illumina Hiseq 2500, California, USA) were carried out by Shanghai Majorbio Technology Company, China (http://www.majorbio.com). The obtained nucleotide sequence data was deposited in the NCBI GeneBank sequence database, the online program BLAST was used to figure out the relevant sequences with known taxonomic information in the Genebank (http://www.ncbi.nlm.nih.gov/BLAST) to identify the strain. The phylogenetic tree was built by software Mega 7.0 (Kumar, Stecher & Tamura, 2016) which based on the neighbour-joining (Saitou & Nei, 1987).

Biosorption tests

Experimental cultured conditions as follows, the concentration of Mn(II) was 1,000 mg/L, the cultured time was 72 h, the temperature was 30 °C, the inoculation biomass was one mL (with an approximate optical density of 1.0 at 600 nm, approximately 1.0 × 10¹³ cells/L), the pH was 5.6–6.0. All tests were done in 250 ml conical Erlenmeyer flasks with 100 ml selection medium on a rotary shaker at 120 r/min (ZHWY-211C, China).

In order to detect the effect of different factors on the adsorption of Mn²⁺ by the strain, one of the corresponding parameters were changed according to the above conditions. (i) Effect of the different initial Mn(II) concentrations: the cultured solutions were prepared containing Mn²⁺ at concentrations of 0, 200, 400, 600, 800, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, and 10,000 mg/L with MnSO₄⋅H₂O. (ii) Effect of the cultured time: the contact time was investigated of 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 144 h. (iii) Effect of the temperature: the biosorption tests were studied at 5, 10, 15, 20, 25, 30, 35, and 40 °C. (iv) Effect of the inoculation biomass: the cultured solutions inoculated 0.1, 0.5, 0.5, 1.0, 2.0, and 5.0 mL of biomass separately. (v) Effect of the pH: the pH of the culture solution was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 by using 1 mol/L HCl and 1 mol/L NaOH.

For the purpose of detecting the strain growth and Mn(II) removal rate in the tests, the samples were carried out at a specific time. The growth of HM7 was determined by ultraviolet spectrophotometer (UV-2450, China) at 600 nm (OD₆₀₀). The specific method was preheating the ultraviolet spectrophotometer for 20 min before the experiment, and adjusting the wavelength to 600 nm. After the sample reaction was completed, 3-5mL of the bacteria suspension were taken into the quartz cuvette and placed it in the UV spectrophotometer to read the OD₆₀₀ value of the strain immediately. Than the bacteria suspension was centrifuged at 4,000 rpm for 15 min (TD5A, China), and the residual Mn(II) concentration of the supernatant was analyzed by flame atomic absorption spectrophotometer (AA7000, Japan). The pH was tested by Rex laboratory pH meter (PHS-3C, China). All experimental tests were repeated three times, data processing and variance analysis were determined by SPSS 20.0 (Li & Chen, 2010). ${\displaystyle \mathrm{Q}=\frac{\left({\mathrm{C}}_0-{\mathrm{C}}_{\mathrm{e}}\right)}{{\mathrm{C}}_0}\times 100\text{\%}}$

where Q is removal rate of Mn(II), C₀ is the initial concentration of Mn(II) (mg/L), and C_e is the residual concentration of Mn(II) (mg/L).

Scanning electron microscopy (SEM) analysis

SEM analysis (JSM-6380LV, Japan) was used for the purpose of observing the changes in surface morphology of cells before and after adsorption of Mn²⁺ (under the conditions of initial Mn(II) concentration 1,000 mg/L, temperature 30 °C and pH 5.6–5.8 for 72 h). Mn(II)-loaded and Mn(II)-free strain samples were examined after cell fixation with glutaraldehyde, dehydrated with ethanol and freeze-drying with a vacuum.

Fourier transform infrared (FTIR) analysis

In order to figure out the main chemical functional groups of Mn²⁺ adsorbed by the cells, the strains before and after the adsorption of Mn²⁺ were analyzed by FTIR (NICOLET 5700, USA). After culturing the strain under the conditions of initial Mn(II) concentration 1,000 mg/L, temperature 30 °C and pH 5.6–5.8 for 72 h in a liquid medium, the cells were collected by centrifugation and washed three times with deionized water, after vacuum freeze-drying, a small number of lyophilized cells were mixed with KBr, pressed at 10 t/cm² for 1 min, and determined by FTIR spectrometer (Wave number range of 400 cm⁻¹–4,000 cm⁻¹).

Property of plant growth promoting and antibiotic resistance of the strain

To figure out the promoting potential of the strain, property of plant growth promoting and antibiotic resistance were checked. The test of Indole acetic acid (IAA) of the strain was detected by the method from Sheng et al. (2008a). Mineral phosphate solubilization activity of the strain was determined in the Pikovskayas medium (Biswas et al., 2018). The siderophores production of the strain was detected by the CAS plate method (Schwyn & Neilands, 1987). The antibiotic resistance of the strain was tested in the Muller-Hinton agar medium by disk diffusion method (Bharagava & Mishra, 2017).

Selection of the optimal Mn(II)-resistant strain

In this study, a highly effective Mn(II)-tolerant strain HM7 was isolated from the soil of Mn ore by gradient acclimation culture. HM7 could tolerate 4,000 mg/L of Mn(II) concentration in screening tests and the removal rate of at low concentration (400 mg/L) reached up to 95%. The strain was used for further identification and analysis of Mn(II) removal properties.

Characterization and molecular identification of HM7

According to morphological, biochemical and physiological tests, HM7 was identified as a rod-shaped bacterium. Hydrogen sulphide test was negative, while Gram-staining, catalase, methyl red, Voges Proskauer, oxidase, glucose, and gelatine tests were positive (Table 2).

HM7 was submitted to 16S rDNA gene sequence analysis for further confirm. The sequence of 16S rDNA of HM7 was subjected to GenBank (accession number MG787231). Searched for the similar strain with known DNA sequences on the National Center for Biotechnology Information (NCBI) by using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The results indicated that HM7 had a close genetic relatedness of Bacillus and 99% homology to Bacillus thuringiensis (B. thuringiensis) (ACNF01000156). The phylogenetic tree (neighbour-joining) was constructed by MEGA7 (Kumar, Stecher & Tamura, 2016) (Fig. 1). The higher identical value confirmed HM7 to be B. thuringiensis and was named B. thuringiensis HM7 in the study.

Characteristics of biosorption under different culture conditions

Effect of initial Mn(II) concentration on biosorption

The effect of initial Mn(II) concentration on biosorption by B. thuringiensis HM7 was carried out over a range of 0–10,000 mg/L. The results showed that the growth of HM7 within a certain Mn(II) concentration range (0–800 mg /L) were larger than without Mn(II). The strain growth reached equilibrium, and the maximum OD₆₀₀ was 1.90 at 600 mg/L of Mn(II). As an essential element of biological growth, Mn(II) could promote the growth of bacteria at low Mn(II) concentration (0–800 mg/L). However, when the concentration was more than 2,000 mg/L, OD₆₀₀ had a significant downward trend and growth inhibition appeared, HM7 could hardly grow at 4,000 mg /L of Mn(II). This may be due to with increased initial Mn(II) concentration decreased the microbial activities, and finally heavy metal toxicity inhibited the growth of the bacteria (Fig. 2). Microorganisms mainly provide energy for life activities through respiration. Under the stress of high concentrations of heavy metals, oxygen consumption in respiration was significantly reduced, microbial respiration was weakened, and microbial activity was significantly inhibited (Crane, Dighton & Barkay, 2010). In addition, high concentrations of Mn stimulated the production of a large number of reactive oxygen species (ROS), and ROS could oxidize cell membrane lipids, functional proteins, DNA and other biological macromolecules, resulting in cell damage (Gong et al., 2012).

The Mn(II) removal rate first increased and then decreased with the increased of Mn(II) concentration, the maximum removal rate was 95.04% at 400 mg/L. These results indicated that when the strain provided excessive active adsorption sites, at lower concentrations, all metal ions in the solution would interact with the binding site, so the percentage of biosorption was likely to be higher than at high ion concentrations. However, an increase in the concentration of Mn(II) might lead to competition at the adsorption sites and a decrease in microbial activity, so the biosorption rate declined (Ling et al., 2011; Yu et al., 2013).

Meanwhile, the experimental results indicated that the growth of B. thuringiensis HM7 (OD₆₀₀) correlated with pH (r = 0.951, P < 0.05). Before the experimental reaction, the pH in the solution was 5.6–5.8, after reacting with different concentrations of Mn²⁺, the pH in the solution varied from 5.8 to 8.1. Therefore, we speculated that HM7 might release some alkaline substances during its growth or reaction with Mn(II). In a certain concentration range of Mn(II), the pH and OD₆₀₀increased with the increase of concentration (0–600 mg/L), while, at concentrations of 600–10,000 mg/L, OD₆₀₀ and pH decreased gradually, and the solution pH from weakly alkaline to weakly acidic. This could be described that the oxidation reaction of MnO₂ ionic oxidation involved oxygen atoms in hydroxyl ions, and a large amount of H⁺ in O-H was exchanged with Mn²⁺ as the concentration of Mn(II) increased, resulting in low pH of the system (Wang et al., 2015).

Effect of contact time on biosorption

The growth of B. thuringiensis HM7 and the removal rate of heavy metal increased rapidly in the beginning, but after 72 h the rate of biosorption and the growth of HM7 slowed down and reached an equilibrium with highest Mn(II) removal rate of 56.35% at 108 h (Fig. 3A). The growth of HM7 was positively correlated with the removal rate of Mn(II) (r = 0.97, P < 0.05). Due to the high affinity of the free Mn(II) binding sites on the biosorbent, the biosorption rate of Mn(II) was highest at the start time, but slowed and reached equilibrium after about 108 h. It might be described that when the increased biomass provided sufficient active adsorption sites on the cell surface to interact with Mn(II), and the removal rate increased. But when the biomass increased continuously, excess biomass would aggregate part of cells, and less surface area exposured, which resulted in insufficient adsorption sites for the biosorption and no excess Mn(II) adsorbed, indicating that equilibrium concentrations might be reached. In addition, the growth of HM7 was correlated with pH (S Fig. 1) (r = 0.95, P < 0.05), showing that some alkaline substances might be released when bacteria adsorbed Mn(II).

Effect of temperature on biosorption

With increasing temperature, the removal rate and biomass were raised first and then decreased (Fig. 3B). At low temperatures (5−10 °C), B. thuringiensis HM7 could not grow and Mn(II) removal rates were extremely low, the removal rate and the growth of HM7 increased with increasing temperatures from 5 to 30 °C, the maximum biomass was 1.66 (OD₆₀₀) and removal rate reached 57.68% at 30 °C. When the temperature was higher than 35 °C, the adsorption capacity and growth of the strain both decreased, this may be due to the temperature was too high, and affect the activity of the extracellular polymer, thereby reducing the biosorption of metal. It was observed that extreme temperatures reduced HM7 growth and Mn(II) oxidation, this was because excessive temperatures could lead to RNA pyrolysis, protein denaturation, and microbes to stop growing or dying. On the other hand, a low temperature would reduce the metabolism of microorganisms, the fluidity of the membrane was weak and the function of the transport system was impeded, so that nutrients could not enter the cell quickly, resulting in a low growth rate (Hao et al., 2016). Besides, the pH in solution correlated with the growth of HM7 and the strain might release alkaline substances (Fig. S1). With the increased of temperature, the growth of the strain increased and the pH of the corresponding solution also increased continuously (r = 0.97, P < 0.05), and this may be related to microbial adsorption of heavy metals.

Effect of inoculation dose on biosorption

The final biomass, removal rate, and pH were stable after 72 h of culturing with different initial inoculation dose which under the same nutritional conditions (Fig. 3C and Fig. S1). As the inoculum increased, the growth of B. thuringiensis HM7 and the removal rate of Mn(II) did not change significantly. When the inoculation dose was increased from 0.1 mL to 0.5 mL, the maximum Mn(II) removal rate was 60.50%, which slightly higher than the others. This was because biomass provided enough active adsorption sites, Mn(II) ions can easily bind with these extra sites. It suggested that in the adsorption system depended on the metal concentration, as long as the adsorption sites on the cell surface did not reach saturation, the removal rate of heavy metals increased. When the number of cells reached a stable level, the effect on the removal rate was not obvious with continuously inoculation. In the case of certain nutrient, the effect of inoculation amount on strain growth was not significant. Therefore, in order to ensure high removal rate and cost savings, excessive inoculum should not be used (Fan et al., 2008). Besides, the pH of the solution with different inoculation amount did not differ significantly.

Effect of pH on biosorption

In present tests, pH could affect the ability of B. thuringiensis HM7 adsorption (Fig. 3D). In an acidic condition (pH < 5), there was almost no removal rate and growth of the strain, this was due to the competitive adsorption at low pH, a large amounts of protons like H⁺ and H₃O⁺ increased the difficulty in binding sites of Mn²⁺ to cell walls, resulting in relatively low adsorption rate. When the pH was 5.0, the highest removal rate was reached (60.88%), and the biomass achieved a maximum OD₆₀₀ of 1.66. This could be attributed to more negatively charged cells become available, promoting a greater metal uptake, and the anionic state of the functional group increased the attraction of ions to Mn²⁺. However, when the pH exceeded the appropriate point (pH > 7), as it increased, biomass and removal rate would soon decrease. The reasons might be that at higher pH values, more ligands with negative charges like imidazole, carboxyl, and phosphate groups were exposed which would attract the positive charge metal ions to the cell surface. It could affect the carriers to help transport and the activity of the cell enzyme, thus the adsorption and growth of strain was restricted (Pardo et al., 2003; Wang et al., 2017). In particular, it could be seen that pH had a significant effect on the growth of the strain, the strain grew well in the weak acid environment (pH5-7). HM7 reached the maximum growth amount with the initial pH of 5.0, and the pH of the solution after the reaction raised to 7.85 from the initial state. In the case of poor growth of the HM7, the pH of the solution before and after the reaction did not change significantly (Fig. S1). This indicated that in the Mn(II) biosorption, the chemical reaction of HM7 changed the pH of the solution or secreted alkaline substances (Liao et al., 2015; Wang et al., 2013).

SEM analysis

SEM analysis of B. thuringiensis HM7 to determine cell surface structure changes under 1,000 mg/L Mn(II) stress. The SEM micrographs showed that under normal growth conditions, HM7 had a morphological shape with a smooth surface and an average cell diameter of about 0.5 µm (Fig. 4A). The small size of the bacterial structure provided a large contact interface for interaction with metals during biosorption (Zouboulis, Loukidou & Matis, 2004). After adsorption of Mn(II), the cell surface morphology considerably changed, the length and size of the cell decreased, and the cells were irregular and cracked with the appearance of wrinkles on the surface, and there were many flocs on the surface of the strain (Fig. 4B). This might be due to the precipitation or adsorption of Mn(II) oxidation on the surface of bacterial cells and the morphological changes caused by the secretion of extracellular polymeric substances during metal biosorption (Chen et al., 2000).

FTIR analysis

Biosorption depends largely on the physicochemical conditions of the solution and the functional groups of the bacterial cell active site. Therefore, to better understand the types of functional groups involved in the biosorption process, FTIR analysis was performed on HM7 (Iqbal, Saeed & Zafar, 2009). Intensity and peak position changed before visibly and after adsorption of Mn(II), and indicated the interaction of the relevant functional groups with Mn(II) ions in the biosorption process (Fig. 5). The Peak at 3,480 cm⁻¹ was shifted to 3,000 cm⁻¹ after adsorption corresponded to hydroxyl (OH⁻) stretching vibrations (Iqbal, Saeed & Zafar, 2009). The significant variation of peak of 2,360 cm⁻¹ showed that sulfhydryl groups participated in the interaction with Mn(II) (Dash, Mangwani & Das, 2013). In addition, the spectrum indicated some prominent absorption peaks at 1,610, 1,500, 1,410 and 1,400 cm⁻¹ showed the presence of carboxyl and amide groups on the surface of bacterial cells (Bharagava & Mishra, 2017). Adsorption peak at 1,400 cm⁻¹ was protein amide I band. The peak ranges of sugars were appeared at 1,000–1,200 cm⁻¹ (Deng et al., 2013). The Mn(II) exposed strain biomass also showed two prominent peaks at 830 and 860 cm⁻¹, which represented CH=CH of trans-di-substituted alkenes and -CH out of plane deformation interact with Mn(II) (Bharagava & Mishra, 2017). The results showed that the main component of HM7 surface and strain secretion was a polysaccharide compound containing a large amount of pyrrole ring and protein hydroxyl group. Comparison of FTIR spectra of HM7 before and after Mn(II) adsorption showed that the functional groups such as carboxyl, hydroxyl, sulfhydryl groups, amino, amide I and amide II bands might participate in the complexation of Mn(II).

Characteristics of the B. thuringiensis HM7 in plant growth promoting and antibiotic resistance ability

It is reported that bacteria increased the biomass yield of plants and reduce the metal toxicity on metal contaminated soil by producing beneficial substances like mineral production of IAA, phosphate solubilization and siderophores (Ma et al., 2016; Zhang et al., 2015). In this study, B. thuringiensis HM7 might produce IAA at the level of 2.55 mg/L (Table 3), the released of IAA usually caused nutrient absorption and plant biomass to increase, thereby indirectly increased the accumulation of metal in their host (Zhang et al., 2011). In addition, HM7 was able to solubilize mineral phosphate and reached 8.76 mg/L, which would improve the bioavailability of rhizosphere phosphate (Nautiyal et al., 2000). Moreover, HM7 produced siderophores which could promote plant growth and alleviate metal toxicity of the supplement for iron nutrition in plant roots. The production of siderophores formed a strong bond with Mn(III) during Mn(II) oxygen reaction. Thus, Mn(III) was stabilized by the siderophores-Mn(III) chelate in the reaction (Barboza, Guerra-Sá & Leão, 2016; Barzanti et al., 2007).

Resistant for Amikacin, Norfloxacin, Gentamycin and Ciprofloxacin. Antibiotics and heavy metals in the environment are potential health hazards due to these properties are usually associated with transmissible plasmids, thus leading to a simultaneous selection of resistance factors for metal ions and antibiotics to suit a variety of harsh environments (Bharagava & Mishra, 2017). Therefore, we believe that the object strain could be used as the inoculant for Mn(II) contaminated water, which may have survival advantages in the rhizosphere, as co-resistance to Mn(II) and antibiotics, and have potential in the ability to promote plant growth and alleviate metal toxicity.