Extraction of extracellular polymeric substances (EPS) from indigenous bacteria of rare earth tailings and application to removal of thorium ions (Th⁴⁺)

Thorium, as a radionuclide, mainly exists in thorium ore, uranium ore and rare earth minerals. In nature, thorium occurs mainly in the form of tetravalent compounds (Th⁴⁺). The Bayan Obo rare earth mine in Baotou has the largest thorium content in China, accounting for 77.3% of the total thorium reserves in China (Xu et al. 2005; Chen et al. 2022). Nowadays, Baotou Bayan Obo rare earth mine has been widely used in energy conservation, environmental protection, green industry, and other low-carbon economic fields. With the popularity of concepts including clean energy, and carbon neutrality, the demand for high-property rare-earth permanent magnetic materials is growing rapidly. The rare-earth permeability is likely to continue to increase.

While rare-earth is fully and efficiently utilized, thorium is not utilized due to its low industrial value, and is often discharged in the form of thorium wastewater, loss, and flying, which not only causes serious pollution to the soil environment around the mining area, but also greatly threatens the safety of groundwater (Xu et al. 2005; Chen et al. 2022). Thorium is not biodegradable and it tends to accumulate in the human body, causing serious damage (Cadena 1983; Yang et al. 2017). Currently, common treatment methods for thorium containing wastewater include adsorption, ion exchange, membrane technologies, solvent extraction, and chemical precipitation (Li et al. 2018). Traditional chemical and physical treatment methods have disadvantages such as high cost, complicated operation, and unstable effects. However, adsorption is a simple technique that does not require any special set up and the originality resides in the material itself (Hamane et al. 2015). Biosorbents are safe and less polluting to the environment. This is a new heavy metal treatment method that has been proven to have the most potential for development by previous research.

Extracellular polymeric substances (EPS), as a new kind of biological adsorbent, have been widely studied because of their unique advantages in heavy metal adsorption. EPS are high-molecular-weight mixtures that mainly consist of polysaccharides, proteins, humus, fat, nucleic acid, and inorganic substances (Li et al. 2018). It is produced, excreted, secreted, and absorbed by cells (mainly bacteria). EPS binds closely with cells and only be extracted by special physical or chemical methods.

In recent years, EPS have been widely applied in various fields such as wastewater treatment, cosmetic industries food, and pharmaceuticals due to their physicochemical and biological properties (Xiao & Zheng 2016). Some studies have shown that EPS extracted from different sources can effectively remove heavy metals (More et al. 2014; Nouha et al. 2016; Zhao et al. 2016). However, the raw materials used to extract EPS in the past are usually only for sludge and bacteria (Wang et al. 2014; Gupta & Batul 2016; Wei et al. 2016), while the extraction of EPS from indigenous bacteria of rare earth tailings and further treatment of thorium containing wastewater is relatively little investigated. In this study, indigenous bacteria of rare earth tailings were used to extract EPS and it was applied to the treatment of Th⁴⁺, and the optimal extraction method, content, and interaction mechanism of EPS with Th⁴⁺ were explored.

Anthrone, EDTA, and Coomassie Blue (G-250) were from Sinopharm Chemical Reagent Co., Ltd., HCl from Nanjing Chemical Reagent Co., Ltd., H₂C₂O₄ from Fuchen (Tianjin) Chemical Reagent Co., Ltd., C₂₂H₁₈As₂N₄O₁₄S₂ and NaOH from Tianjin Chemical Reagent Factory I. H₂SO₄ was from the Beijing Chemical Plant. Bovine serum albumin (BSA) from Tianguangyuan (Xian) Biotechnology Co., Ltd., Glucose from Xilong Scientific Co., Ltd., and Th(NO₃)₄·4H₂O was supplied by the Beijing Research Institute of Chemical Metallurgy of China National Nuclear Industry Co.. The reagents used in the experiments were analytically pure. All the solutions in the experiment were made from deionized water.

Tailings collected from the rare earth tailings dam in Baotou city, Inner Mongolia autonomous region, China were used in this study. The tailings are washed with water, dried, crushed, and sieved. Of these, tailings with an average diameter of less than 2 mm were used for the following studies.

Rare earth tailings samples were used as the source of strains, and the target strains were screened by the diluted plate method. The 1 g of tailings sample was transferred to a conical flask containing 99 mL of sterile saline water and shaken to make a suspension, which was allowed to stand for 1 min. The suspension was diluted in a 10-fold gradient, inoculated on LB solid medium and cultivated for 1–2 d at 32 °C in a constant temperature incubator (Han et al. 2020). The most numerous individual colonies on the medium with regular edges and smooth upper surfaces and larger diameters (about 3 mm) were selected and streaked repeatedly on the same solid medium for separation. The target strains were obtained when the colony morphology was consistent on the medium. A bacterial strain, named K-1, was isolated and screened from the tailings sample, and the strain K-1 was sent to Shanghai Meiji Biomedical Technology Co., Ltd for 16S rDNA sequence analysis (Han et al. 2020). The ITS sequence was compared with BLAST sequence similarity in the NCBI online database, and it was found that strain K-1 and Lysinibacillus sp. strain LJ75 were located in the same branch with 100% similarity, so strain K-1 was identified as Lysinibacillus fusiformis.

All the EPS samples were measured immediately to avoid the degradation of EPS during storage. After the extraction, the protein and polysaccharide were quantified, and the concentration of EPS was used to express the sum of the two contents. The concentration of EPS was used to perform unit conversions (Kang et al. 2016).

The functional groups of EPS were also analyzed by Fourier Transform Infrared spectrometer (FTIR) (BRUKER TensorII, Germany) with a resolution of 4 cm⁻¹ and scan a range of 400–4000 cm⁻¹. EPS was freeze dried by a lyophilizer (Alpha 1-2 LDplus, Christ, Germany) before FTIR determination. The EPS before and after adsorption was added to a 1 cm quartz cuvette and placed in the 3D-EEM (HITACHIF4600, Hitachi Limited, Tokyo, Japan) to determine its composition changes. The scanning interval was 5 nm, while the scanning speed was 12,000 nm/min. The infrared and fluorescence spectrum were analyzed by Origin 2018.

The protein contents were determined using the Coomassie Blue method, with bovine serum albumin (BSA) as the standard. The polysaccharide contents were measured using the anthrone-sulfuric acid method, with glucose as the standard.

For EPS extracted by ultrasonic and heating, the same characteristic bands can be observed on every EPS FTIR spectra presented in Figure 2. Bands 3200–1592 cm⁻¹ can be attributed to protein and polysaccharide functional groups. Carboxylic groups, under acid or basic salt form, are responsible for some less intense bands and they suggest, when combined with the other observed bands, the presence of uronic acids and of humic substances (Comte et al. 2006). The band at 2347 cm⁻¹ of EPS FTIR spectrum extracted by the heating method may be caused by sample contamination in the testing process.

The FTIR spectra of EPS extracted by chemical methods (Figure 3) show particular bands which do not appear for EPS extracted by the physical methods. For the FTIR spectrum obtained for EPS extracted by the treatment with EDTA, the bands 1332 and 1240 cm⁻¹ are seen to be associated with C–N stretching. Moreover, bands 1592 cm⁻¹ are attributed to carboxylate functions. This demonstrates contamination of EPS by the chemical reagent. This is consistent with previous experimental results. The FTIR spectrum obtained for EPS extracted by the NaOH method shows a band at 2345 cm⁻¹. These bands could be interpreted as specific bands for the result of a NaOH and EPS reaction (Comte et al. 2006).

EPS of the indigenous bacteria of rare earth tailings were extracted following the procedures in section 2.4. The quantities of proteins and polysaccharides were measured to represent the EPS yields, as they are the main component of EPS. The experimental results are shown in Table 2. The results show that the content of polysaccharides in EPS extracted from the indigenous bacteria of rare earth tailings was higher than that of protein. This is similar to the research results of Zhang et al. (2008). The results indicated that the yields of EPS were highly dependent on the extraction method. The content of total EPS and protein extracted by the ultrasonic method is the highest among the four extracted methods indicating that ultrasonic method is the best method to extract this sample EPS. Thus, all EPS extracted by the ultrasonic method was applied in the subsequent experiments.

As shown in Table 2, the contents of polysaccharides of EPS extracted by ultrasonic method are the highest. This is because the ultrasonic method uses ultrasonic shear force, cavitation, and so on to make EPS off the cell surface into the water. The action intensity is mild, which can effectively separate the effective components of EPS at the same time, but can avoid damaging the effective components of EPS. The content of polysaccharides extracted by heating method was the highest among the four methods. Increasing the temperature will lose the structure of the sample and could decrease van der Waals forces and hydrogen bonding between EPS and cells (Li & Yang 2007; Guo et al. 2010; Zhou et al. 2016), and extract the firmly bound EPS while the proteins and polysaccharides in EPS can be hydrolyzed in the extraction process. In Table 2, the biochemical composition content was lower in the EPS extracted by treatment with NaOH than in other methods. The addition of NaOH solution caused the groups, such as carboxylic groups, to be ionized, resulting in a strong repulsion and then separation between the EPS and the cells, which lysed the cells and increased the possibility of cell rupture (Huang et al. 2021). The EDTA method had the lowest polysaccharide content, and the protein content was only 0.07 mg/L higher than the NaOH method. Liu & Fang (2002) suggested that there was formation of complexes between EDTA and the EPS. These EDTA-EPS complexes were not removed by membrane separation and thus the EDTA remained in the EPS. Chemical extraction EPS will be contaminated by chemical reagents which may affect the determination of EPS components.

In the subsequent experiments, EPS was extracted by ultrasonic method. The EPS concentration was 68.95 mg/L, which was used as the basis for the EPS dosage in the adsorption test.

Notably, when the EPS is 30 mL, the Th⁴⁺ removal efficiency curve has an inflection point. Before this inflection point, the Th⁴⁺ removal efficiency increased with the increase in EPS dosage. After this inflection point, the increase in the EPS dosage did not obviously increase the Th⁴⁺ removal efficiency. Thus, 30 mL as best dosing quantity was applied in the subsequent experiments.

Langmuir isotherm model is mainly used to study the maximum monolayer adsorption Freundlich isotherm model is usually better to describe the adsorption in aqueous solution (Xiong et al. 2009).

The Th⁴⁺ adsorption isotherms of EPS were described by classical Langmuir and Freundlich models.

The better adsorption capacity of Th⁴⁺ might be due to the presence of various functional groups at the surface of EPS which can attach to Th⁴⁺ via physicochemical interactions. This also confirms the conclusion obtained by FTIR spectral analysis in section 3.2.1.

The parameters ΔG, ΔS and ΔH are listed in Table 4.

It can be seen from Table 3 that the Th⁴⁺ adsorption increases slightly from 28.81 to 31.22 mg/g with increasing temperature from 15 to 30 °C, indicating an endothermic process. Also, the positive value of ΔH confirms the heat-absorbing nature of EPS adsorption and the negative ΔG at different temperatures indicates spontaneous adsorption. Furthermore, the decrease of ΔG with an increasing temperature suggests that the adsorption is more favorable at higher temperatures. The positive value of ΔS indicates that the confusion degree of the system increases, reflecting the affinity of EPS for Th⁴⁺.

From the perspective of energy conservation, all experiments were carried out at 25 °C.

Pseudo-first-order, and pseudo-second-order kinetic equations were used to conduct the adsorption kinetics of EPS in the sorption process to analyze the adsorption mechanism.

• 1.

  The ultrasonic extraction method has the best effect on extracting EPS from indigenous bacteria of rare earth tailings, and the physical extraction method is generally more efficient than the chemical extraction method.

• 2.

  The key factor of EPS adsorbed Th⁴⁺ was functional groups, including amide I and amide II of proteins, C–H from aliphatic, as well as O–H and C = O from carboxylic acid.

• 3.

  The hydrophobic interaction of tryptophan and tyrosine in EPS plays an important role in the adsorption process.

• 4.

  The pH, initial Th⁴⁺ concentration, and EPS dosage affected the equilibrium uptake of Th⁴⁺ to EPS, and the optimum pH, initial Th⁴⁺ concentration, and EPS dosage were determined as 3.5, 20 mg/L, and 30 mL, respectively.

• 5.

  An increase in the adsorption of Th⁴⁺ deals with increasing temperature up and then followed by an exothermic uptake process at higher temperatures.

• 6.

  The Langmuir and Freundlich adsorption models were applied to describe the experimental data. Equilibrium data fitted very well to both the Langmuir and Freundlich isotherm models.

• 7.

  The kinetic data adsorption of EPS to Th⁴⁺ was found to follow the pseudo-second-order model. Chemical adsorption is the main adsorption mechanism.

The authors wish to thank the Natural Science Foundation of Inner Mongolia Autonomous Region of China (No. 2020MS02015, No. 2020MS0547), the National Natural Science Foundation of China (No. 42167029), the Open Program of Tianjin Key Laboratory of Green Chemical Engineering Proceed Engineering of Tianjin University (GCEPE20190108) for their support of this study.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.