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Microb Ecol
Microb Ecol
Microbial Ecology
0095-3628
1432-184X
Springer US New York

39231820
2428
10.1007/s00248-024-02428-6
Research
Effects of Detoxifying Substances on Uranium Removal by Bacteria Isolated from Mine Soils: Performance, Mechanisms, and Bacterial Communities
Song Xin 1
Li Jun jun.li@cqu.edu.cn

2
Xiong Zhiyu 1
Sha Haichao 3
Wang Guohua 1
Liu Qin 1
Zeng Taotao biowater@126.com

1
1 https://ror.org/03mqfn238 grid.412017.1 0000 0001 0266 8918 Hunan Province Key Laboratory of Pollution Control and Resources Reuse Technology, University of South China, Hengyang, 421001 China
2 grid.190737.b 0000 0001 0154 0904 College of Environment and Ecology, Key Laboratory of the Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing, 400045 China
3 https://ror.org/00z3td547 grid.412262.1 0000 0004 1761 5538 Shaanxi Key Laboratory of Earth Surface System and Environmental Carrying Capacity, College of Urban and Environmental Sciences, Northwest University, Xi’an, 710127 China
5 9 2024
5 9 2024
2024
87 1 11131 5 2024
19 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
In this study, we investigated the effect of detoxifying substances on U(VI) removal by bacteria isolated from mine soil. The results demonstrated that the highest U(VI) removal efficiency (85.6%) was achieved at pH 6.0 and a temperature of 35 °C, with an initial U(VI) concentration of 10 mg/L. For detoxifying substances, signaling molecules acyl homoserine lactone (AHLs, 0.1 µmol/L), anthraquinone-2, 6-disulfonic acid (AQDS, 1 mmol/L), reduced glutathione (GSH, 0.1 mmol/L), selenium (Se, 1 mg/L), montmorillonite (MT, 1 g/L), and ethylenediaminetetraacetic acid (EDTA, 0.1 mmol/L) substantially enhanced the bacterial U(VI) removal by 34.9%, 37.4%, 54.5%, 35.1%, 32.8%, and 47.8% after 12 h, respectively. This was due to the alleviation of U(VI) toxicity in bacteria through detoxifying substances, as evidenced by lower malondialdehyde (MDA) content and higher superoxide dismutase (SOD) and catalase (CAT) activities for bacteria exposed to U(VI) and detoxifying substances, compared to those exposed to U(VI) alone. FTIR results showed that hydroxyl, carboxyl, phosphorus, and amide groups participated in the U(VI) removal. After exposure to U(VI), the relative abundances of Chryseobacterium and Stenotrophomonas increased by 48.5% and 12.5%, respectively, suggesting their tolerance ability to U(VI). Gene function prediction further demonstrated that the detoxifying substances AHLs alleviate U(VI) toxicity by influencing bacterial metabolism. This study suggests the potential application of detoxifying substances in the U(VI)-containing wastewater treatment through bioremediation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00248-024-02428-6.

Keywords

Uranium-containing wastewater
Detoxifying substances
Bacterial community
Ecological response
http://dx.doi.org/10.13039/501100001809 National Natural Science Foundation of China 52170164 Zeng Taotao Science and Technology Innovation Program of Hunan Province of China2022RC1184 Zeng Taotao issue-copyright-statement© Springer Science+Business Media, LLC, part of Springer Nature 2024
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pmcIntroduction

In recent years, the increasing demand for uranium for nuclear power has led to the rapid development of the uranium mining and metallurgy industry [1]. A substantial volume of uranium-laden waste streams is inevitably produced as an inherent byproduct of the uranium mining process [2]. When these waste streams enter natural water bodies without adequate treatment, they pose a significant threat to aquatic life [3]. Additionally, uranium can indirectly enter the human body through the food chain, leading to negative health effects [4].

In nature, uranium primarily exists in two oxidation states U(IV) and U(VI), each with significantly different properties regarding mobility, bioavailability, and toxicity [5]. U(IV), which mainly exists as uraninite (UO2), has limited solubility. In contrast, U(VI) is toxic and highly mobile [6]. Therefore, it is imperative to remove U(VI) from wastewater streams. Various physical and chemical methods (e.g., chemical precipitation and membrane separation) have been developed for U(VI) removal. However, these approaches are generally costly, complicated to operate, and prone to secondary contamination [7]. Simple and eco-friendly alternatives, such as bioremediation, are thus urgently needed [8]. It has been reported that bacteria, fungi, yeast, and algae exhibit a high removal capacity for U(VI) [9]. To further enhance this capacity, flavin mononucleotide and humic acid were applied to promote the generation of bacterial extracellular polymers, enhance bacterial electron transfer efficiency, and regulate bacterial activity [10]. Apart from this, chelating agents (e.g., citrate, nitrilotriacetic acid, and ethylenediamine tetraacetic acid) and clays were also reported to promote the immobilization of U(VI) through forming more stable complexes [11]. For instance, a previous study has introduced anthraquinone-2-sulfonate to remove U(VI) by Shewanella oneidensis under anaerobic conditions. The results showed that when the concentration of anthraquinone-2-sulfonate increased to 0.5–1.0 mmol/L, the reduction rate of U(VI) increased due to the formation of stable uranium oxide [12]. Similarly, other studies have demonstrated that in the presence of 0.1 mM of the reducing agent anthraquinone-2, 6-disulfonic acid (AQDS), D. radiodurans can effectively remove 82–89% of U(VI) within 21 days, over a concentration range of 5 to 100 mM [13]. However, the effects of AHL on the enhancement of U(VI) removal by microorganisms and their microbial communities have been rarely studied. Accordingly, the mechanism by which it affects U(VI) removal has also been overlooked. As the added chemicals can be thought as detoxifying substances, it is important to clarify the effects of detoxifying substances on uranium removal by microorganisms. This can be benefit for enhancement of U(VI)-containing wastewater treatment or bioremediation efficiency.

Therefore, this study aimed to explore (1) the effect of detoxifying substances on the removal efficiency of U(VI) by the mixed bacteria isolated from mine soil, (2) the microbial mechanism of the effect of detoxifying substances on the removal of U(VI) by the mixed bacteria, and (3) the bacteria community characterization upon uranium stress and typical detoxifying substance protection.

Materials and Methods

Mine Bacteria Isolation

The bacteria used for U(VI) removal in this study were all isolated from soil collected from mining areas in Southern China, which contained excessive levels of Cd, Pb, Zn, As, and Cu. The bacterial isolation procedure was followed as described in our previous study [14]. In brief, the collected soil was passed through a 2.54-mm sieve mesh. Subsequently, 10 g of the sieved soil was suspended in 90 mL distilled water and stirred to form a homogenous mixture. The supernatant was then inoculated into a liquid growth medium (5 g/L beef extract, 10 g/L tryptone, and 10 g/L sodium chloride, at pH 7.2) and cultivated for 24 h at 30 °C. The isolated bacterial suspension was inoculated into Luria–Bertani (LB) medium and incubated with continuous agitation at a speed of 150 rpm with a temperature of 30 °C until reaching the stationary growth phase (48 h). Afterward, the bacterial culture was centrifuged at 7012 g for 10 min at 4 °C to obtain a bacterial suspension, followed by washing three times with sterile water for further U(VI) removal trials.

Effects of Environmental Factors and Detoxifying Substances on the Removal of U(VI) by Mixed Bacteria

Ten milliliters of the centrifugated bacterial suspension (mentioned in the above section) was inoculated in a 150-mL flask containing 100 mL of U(VI) solution for batch experiment under different ambient conditions. U3O8 was used as raw material to prepare a stock standard solution of uranyl ion at a concentration of 1.0 g/L, which was then diluted with distilled water to obtain various U(VI) solutions. The effects of pH (4, 5, 6, and 7), temperature (20 °C, 25 °C, 30 °C, and 35 °C), and initial U(VI) concentration (1, 5, and 10 mg/L) on the removal of U(VI) were investigated. The samples were agitated at 150 rpm and collected at every time-point 1, 2, 4, 6, 8, 12, and 24 h, respectively. The collected samples were then centrifuged at 8944 g for 10 min at 4 °C. The supernatant was extracted and filtered through a 0.22-µm membrane for U(VI) analysis, following the method described by Das et al. [15]. 2-(5-bromo-2-pyridylazo-5-diethylaminophenol) (Br-PADAP) was used as a chromogenic reagent to complex with U(VI). U concentration was measured using ultraviolet spectrophotometry at the wavelength of 578 nm. The optimum conditions for U(VI) removal were thus screened from the results.

For effects of detoxifying substances, different concentrations of AHLs (0.1, 0.5, 1 µmol/L), Se (1, 5, 10 mg/L), AQDS (0.1, 0.5, 1 mmol/L), GSH (0.1, 0.5, 1 mmol/L), MT (1, 5, 10 g/L), and EDTA (0.1, 0.5, 1 mmol/L) were added into the 150-mL flasks contained 100 mL of U(VI) solution, respectively. The group with the U(VI) application alone served as the control. The mixtures were agitated and collected after 6 and 12 h for U(VI) analysis after phase separation. The reaction conditions were selected based on the results of prior experiments of this study. All experiments were performed in triplicate.

Bacterial MDA Content and Oxidase Activity Analysis

Bacterial suspension was added into the medium containing the selected optimal concentrations of detoxifying substances and U(VI) from the prior experiments within this study, and incubated for 12 h with continuous agitation at 150 rpm. The group without any application severed as the blank and noted as M0. The group with 10 mg/L U(VI) application was noted as M1. Similarly, the group spiked with both U(VI) and optimal AHL concentrations was noted as M2. The bacterial cultures were added into centrifuge tubes. After centrifugation, the supernatant was discarded with extraction solution with proportion of 5 million cells paste by 1 mL extraction solution. The bacteria were broken in an ultrasonic crusher (power 200 w, ultrasonication for 3 s at 10 s intervals for 30 repetitions). The broken bacteria were centrifuged for 10 min at 4 °C with 7012 g. The supernatant was collected for the SOD, CAT, and MDA contents analysis following the instructions of the kit (Solarbio Science & Technology Co., Ltd, Beijing, China). Three parallel groups of samples were set up for all the experiments.

Bacterial Morphology and Functional Group Analysis

Bacteria suspension collected from the experimental group spiked with both U(VI) and optimal AHL concentrations (noted as M2), blank (M0), and control (M1) group was selected for micromorphological and elemental analyses using scanning electron microscopy and energy dispersive X-ray analysis (SEM–EDS, Zeiss Ultra Plus-360, Germany). The sample preparation for SEM–EDS followed the procedure described in Khan et al. [16]. Fourier transform-infrared spectroscopy (FTIR, Thermoscientific Nicolet 6700, USA) was used to characterize the functional groups on the surface of biomass, and the scanning wave number ranged from 4000 to 400 cm−1. Samples were ground with potassium bromide (KBr) and pressed into a thin pellet for analysis [17]. X-ray photoelectron spectroscopy (XPS) was conducted with an ESCALAB 250i-XL electron spectrometer using 300 W Al Kα radiation. The analyzer mode was CAE: pass energy 30.0 eV with a 0.1 eV energy step size (Thermo Fisher Scientific). The XPS peak fitting and the valence state analysis of composited elements were determined by XPS Peak 4.1 software (Thermo Fisher Scientific) [18].

Bacterial Community Characterization

M0, M1, and M2 samples were homogenized individually, and their genomic DNA was extracted using the E.Z.N.A. Soil DNA Kit (OMEGA, BioTek, Winooski, VT, US) following the manufacturer’s instructions. The V4 hypervariable region of the 16S rRNA gene was amplified using the 515FmodF and 806RmodR primers [19]. High-throughput sequencing was performed using an Illumina MiSeq platform by Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China).

Following the application of quality control to remove short fragments and low-quality sequences that did not meet the requirements, the acquired sequences were classified into operational taxonomic units (OTUs) at a 97% similarity threshold using Usearch software v. 7.0 (http://drive5.com/uparse/) [20]. Taxonomic assignment was performed using RDP Classifier software v. 2.6 to establish the bacterial community composition and abundance at the genus level [21]. The prediction of bacterial function was carried out through the PICRUSt program based on the Clusters of Orthologous Groups of proteins (COG, http://www.ncbi.nlm.nih.gov/COG/) and Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) databases [22]. The obtained sequences were submitted to the Sequence Read Archive database in the National Coalition Building Institute, and the access numbers of SRR27012731-27012733 were acquired.

Statistical Analysis

Descriptive statistics were performed using Microsoft Excel 2019. Results are expressed as mean ± SD. FTIR and XPS characterization of bacteria was analyzed using OMNIC and Avantage software, respectively. Figures were plotted using Origin 2018. One-way analysis of variance (ANOVA) was conducted to assess significant differences, followed by Duncan’s multiple comparison tests. P values less than 0.05 were considered significant, while P values less than 0.01 were deemed highly significant, and P values less than 0.001 were regarded as extremely significant.

Results and Discussion

Influence of Environmental Factors on the Removal of U(VI) by Mixed Bacteria

The effect of pH (4–7) on U(VI) removal by microflora along with incubation time is shown in Fig. 1a. The removal rate firstly increased to the maximum value (71.32–85.60%) with the extension of incubation time, while it sharply decreased to 35.05–44.94% afterwards until 12 h. At a pH value of 4, the bacterial removal rate of U(VI) in 2 h reached a maximum value of 71.32%, while it decreased as the reaction proceeded. At pH 5, 6, and 7, the removal rate reached the maximum after 4, 6, and 8 h, respectively. Besides, the overall removal rate increased gradually with pH from 4 to 6, but the pH value of 7 led to its decrease. The lower U(VI) removal rate at lower pH levels could be attributed to the limited growth and functional expression of mixed bacteria, while with the ambient pH increasing to 6, bacterial activity is accordingly activated under the optimum conditions, resulting in a higher U(VI) removal rate. At low pH, U(VI) mainly exists in solution in the form of UO22+. H+ with smaller ionic radius is predominant in the acidic solution, which is more beneficial to occupy the active adsorption sites of bacteria. H+ on the surface of bacteria reduces the number of UO22+ binding sites, resulting in the low removal efficiency of U(VI) [23]. However, when the pH value is around 6, UO22+ ions are more easily adsorbed on the surface of the adsorbent [24], and the removal efficiency of U(VI) is therefore higher accordingly. When the pH value is greater than 6, it may cause U(VI) hydrolysis to produce negative/uncomplexed substances, such as UO2(OH)3− and UO3(OH)7−[25]. The repulsion between these ions and the bacterial surface is enhanced [26], resulting in the reduction of U(VI) removal rate in aqueous solution. The results in this study are consistent with the previous finding on U(VI) removal by Paecilomyces catenlannulatus [27]. The highest U(VI) removal efficiency was achieved at pH 6 in the study. pH 6 is therefore set as the optimum pH for all subsequent experiments.Fig. 1 Effect of varying environmental factors on the U(VI) removal: a pH, b temperature, c initial U(VI) concentration

The effect of different temperatures (20–35 °C) on bacterial removal of U(VI) is shown in Fig. 1b. Results showed that the removal of U(VI) exhibited an upward trend with rising temperatures. Specifically, at 20 °C, the removal of U(VI) reached a maximum value of 64.04% at 4 h. At 25 °C, 30 °C, and 35 °C, the maximum removal of U(VI) was achieved after 8 h, reaching 63.79%, 77.49%, and 85.60%, respectively. The higher removal of U(VI) at a higher temperature was mainly due to the accelerated movement of ion diffusion and the increase in activated ions under higher temperatures, which are beneficial to the U(VI) removal [23]. Therefore, 35 °C was selected for all subsequent trials.

Figure 1c shows the efficiency of bacterial U(VI) removal at different initial U(VI) concentrations. The bacterial removal efficiency of U(VI) increased with the initial U(VI) concentration. When the concentration of U(VI) was 1 mg/L and 5 mg/L, the maximum removal efficiencies were 56.84% and 78.55%, respectively. When the initial U(VI) concentration was 10 mg/L, the bacteria had better U(VI) removal with a removal efficiency of 85.60%. Previously, Li, Li, and Cui [27] investigated the U(VI) removal efficiency of Paecilomyces catenlannulatus under different initial concentrations of U(VI). It was found that the amount of U(VI) reduction increased significantly with the increase of initial U(VI) concentration in the range of 1–7 mg/L. It can be seen that the bacteria have strong adaptability to U(VI). When the initial concentration of U(VI) is 10 mg/L, it is favorable for the removal of U(VI) by the bacteria. Therefore, the following tests were carried out under this initial concentration condition (10 mg/L).

Influence of Detoxifying Substances on U(VI) Removal by the Bacteria

Effect of AHLs, AQDS, and GSH on U(VI) Removal

0.1 µM AHL application did not exhibit a notable effect on U(VI) removal by bacteria after 6 h (Fig. 2a) (P > 0.05). Similarly, there were no significant differences among the application of 0.1, 0.5, and 1.0 µM of AHLs (P > 0.05). The removal rates of U(VI) in the control group and the other three groups (0.1, 0.5, and 1.0 µM of AHL application) were approximately 83% after 6 h. However, after 12 h, the removal rates in the control remarkably decreased to 44.67% (P < 0.05), whereas it remained around 80% in the other three groups (P > 0.05). This result demonstrated that adding AHLs could maintain the long-term removal of U(VI) by bacteria. It may be attributed that AHLs mobilized microbial quorum sensing, alleviated the toxic effect of U(VI) on bacteria, and subsequently improved bacterial activity, resulting in the stable removal efficiency along with the incubation time. Similarly, previous studies have shown that AHLs notably enhanced the tolerance of N. europaea cells to nano-ZnO stress, and AHLs significantly attenuated np-induced inhibition of N. europaea cell proliferation, membrane integrity, specific AMO activity, and amoA expression [28].Fig. 2 Effect of different concentrations of exogenous substances on the U(VI) removal by mixed bacteria at 6 h and 12 h: a AHLs, b AQDS, c GSH, d Se, e MT, f EDTA. M1 is the control group supplemented with 10 mg/L U(VI) without exogenous substance. Different letters next to each parameter represent significant differences based on Duncan’s multiple range test (n = 3, P < 0.05)

The removal rate of U(VI) by bacteria with the application of 0.1 mM and 0.5 mM AQDS is not much different from that of the control group M1 at 6 h (Fig. 2c). The U(VI) removal efficiency at M1 after 12 h was significantly lower compared to that at 6 h (P < 0.05). However, adding AQDS (0.1 and 1 mM) remarkably increased the U(VI) removal at 12 h (Fig. 2c) (P < 0.05). This result suggests that AQDS plays a positive role in maintaining the activity of bacteria. This is consistent with previous studies. For instance, the addition of AQDS significantly alleviated the toxicity of Cu2+ and SeO32− for Shewanella oneidensis MR-1 cells, activated the extracellular metabolic detoxification process, resulting in a metabolic detoxification rate one order of magnitude higher than that of the control group [29].

The removal rate of U(VI) by bacteria with three concentrations of GSH application after 6 h was similar to that of the control group, while after 12 h, the removal rate of the three experimental groups was increased and reached 96.16% when the GSH concentration was 1 mM (Fig. 2b). Glutathione is widely distributed in bacteria, plants, and animals, which participates in some important cell activities, including protecting cells from toxic metals [30, 31]. GSH serves as a chelating agent capable of directly interacting with metal ions within cells. Therefore, metal owing a robust affinity for S could bind with the sulfhydryl groups in GSH molecules to achieve detoxification. Likewise, in 100 mg/L Cd solution, the content of glutathione in Fusarium tropicalis increased by 135% [32].

Influence of Se, MT, and EDTA on U(VI) Removal

The effect of adding different concentrations of Se on bacterial U(VI) removal is shown in Fig. 2d. The removal efficiency of U(VI) by bacteria decreased with increasing Se concentration. The U(VI) removal efficiencies were 83.54%, 58.81%, and 59.59% after 6 h at initial Se concentrations of 1 mg/L, 5 mg/L, and 10 mg/L, respectively, which were significantly lower than those of the control group (87.28%) (P < 0.05). However, at 12 h, the removal efficiency of the control group decreased to 44.67%, which was obviously lower than that of the group with Se application (P < 0.05). This is partially consistent with other studies. For instance, 67 kinds of microorganisms were used to study the effect of Se on U(VI) adsorption. The results showed that Se inhibited the growth of most microorganisms [33].

MT application reduced the U(VI) removal efficiency with a 6-h reaction (Fig. 2e). More specifically, the addition of 5 and 10 g/L MT notably decreased U(VI) removal efficiency by 13.74–24.24% and 14.1–25.54% at 6 and 12 h, respectively, compared to the control group (P < 0.05). The results indicated that 5 and 10 g/L MT did not show the detoxifying ability. This could be ascribed to the relatively high concentration of MT (5 and 10 g/L), which is toxic to bacteria, resulting in the reduction of bacterial activity [34]. Besides, the U(VI) removal efficiency in the group with 1 g/L of MT application (77.28%) is significantly higher than that of the control group (44.67%) at 12 h (P < 0.05). Bacteria can be attached to clay minerals as bacterial-mineral compounds through ligand complexation or electrostatic interaction. The combination of bacteria and montmorillonite will change its aggregation state, thus affecting its retention of uranium. It is also reported in other studies that the interaction between Streptococcus putrescens and montmorillonite has a great influence on its immobilization of U(VI) [35].

As shown in Fig. 2f, the bacterial removal efficiency of U(VI) decreased with increasing EDTA concentration. The removal rate of the experimental group was always higher than that of the control group at an EDTA concentration of 0.1 mM, with the highest removal rate of 91.74% and 91.61% after 6 and 12 h. When the concentration of EDTA is 0.5 and 1 mM, the removal rate is significantly lower than that of the control group at 6 h, while obviously higher than that of the control group at 12 h, which is 81.36% and 53.10%, respectively (P < 0.05). This could be due to the toxicity of EDTA, affecting the life activities of bacteria at the beginning of 6 h. However, after 12 h, bacteria progressively acquire resistance, leading to a gradual enhancement in the efficiency of U(VI) removal. EDTA and U(VI) can generally form strong complex organic acids, which can affect the adsorption, precipitation, and redox behavior of U(VI) by forming stable complexes. This is partially consistent with the results in other studies. For instance, the effect of Shewanella putrefaciens on U(VI) reduction has been investigated in the presence of EDTA. The results showed that the presence of EDTA inhibits the precipitation of bioreductive UO2 by forming a stable U(IV)-organic complex, while the polymerization of U(VI)-organic complex can delay the reduction of U(VI) by Shewanella putrefaciens [36].

In summary, the optimal concentrations of the six exogenous substances were AHLs 0.1 µmol/L, AQDS 1 mmol/L, GSH 0.1 mmol/L, Se 1 mg/L, MT 1 g/L, and EDTA 0.1 mmol/L, respectively. It is not difficult to observe that the removal efficiency of U(VI) by M1 in the control group decreases rapidly after 8 h (Fig. 1), and the removal rate drops to 44.66% at 12 h (Fig. 1). However, after 12 h of reaction with the optimal concentrations of these six exogenous substance applications, the bacterial removal of U(VI) increased to different degrees compared with that of M1, and the increases were 34.93%, 37.40%, 54.48%, 35.12%, 32.84%, and 47.83%, respectively. Therefore, the subsequent toxic effect analysis was carried out with the application of the optimal concentration of the external substances within the duration of 12 h.

Mechanism of the Effect of Detoxifying Substances on Bacterial Removal of U(VI)

Effects of Detoxifying Substances on MDA, SOD, and CAT Activities of the Bacteria Under U(VI) Stress

As a representative product of lipid oxidation reaction in cells, MDA content could reflect the level of lipid peroxidation [37]. As can be seen from Fig. 3a, the MDA content with 10 mg/L (M1) of U(VI) exposure (0.0227 nmol/104 cell) is significantly higher than that without U(VI) exposure (M0) (0.0147 nmol/104 cell) (P < 0.05), indicating the production of MDA under the stress of U(VI) by bacteria. In this study, compared to the M1, the treatments with exogenous substances application all decreased the MDA content, except for the increase of 8.37% observed in the group with Se application (P < 0.05). This could be due to the toxicity of high Se concentration for microorganism [38]. The finding suggests that the application of detoxifying substances can facilitate the alleviation of bacterial cell damage caused by U(VI).Fig. 3 MDA content (a), SOD (b), and CAT (c) activities of the mixed bacteria after exposure with U(VI) and exogenous substances for 12-h. M0 is the blank without U(VI) and exogenous substances exposure, and M1 is the control group supplemented with only 10 mg/L U(VI). Different letters next to each parameter represent significant differences based on Duncan’s multiple range test (n = 3, P < 0.05)

Antioxidant enzymes are considered the first line of defense against oxidative stress, which can eliminate reactive oxygen radicals produced within cells to maintain metabolic balance [39]. This protective mechanism thereby secures cell membrane structure and enhances the ability of cells to tolerate adverse stress [40]. In this study, the activities of SOD and CAT were inhibited under the exposure of 10 mg/L U(VI) (M1), which were 0.64 and 0.33 times than that of the blank group (M0), respectively (Fig. 3 b and c). For the treatments with AHLs, AQDS, GSH, Se, MT, and EDTA application, the SOD and CAT activity was 1.23 and 1.46, 1.11 and 1.11, 1.08 and 1.08, 0.58 and 0.41, 1.03 and 1.05, and 1.01 and 1.03 times higher than that of the control group (M1), respectively. These results indicated that AHLs, AQDS, GSH, MT, and EDTA reduced the stress of U(VI) on bacteria and protected the metabolic activity of bacteria. Some other results showed that when heavy metal pollution stresses organisms, SOD activity will generally decrease, and a large number of active oxygen free radicals will be produced, which will harm organisms [41]. This experiment also demonstrated the inhibition of SOD and CAT activities within bacteria under conditions of U(VI) stress.

Effect of Detoxifying Substances on the Morphology and Element Constituent of Mixed Bacteria

The surface morphological characteristics and elemental composition of the bacteria in M0 (Fig. 4 a and b), M1 (Fig. 4 c and d), and M2 (Fig. 4 e and f) were measured by SEM–EDS. The SEM images revealed that the surface of bacteria in M0 was smooth, rod-shaped, and well-structured (Fig. 4a). While after reacting with U(VI), the surface of bacteria became rough with granular particles loaded (Fig. 4c). Besides, EDS analysis confirmed the existence of U(VI) on the cell surface (Fig. 4 d and f), which accounted for 0.41% and 0.70% in the bacteria collected from M1 and M2, respectively. It was observed that the phosphorus percentage on the bacterial surface increased and the oxygen percentage decreased after reaction, which contributed to the ability of many microorganisms to adapt to environmental changes through surface modification [42]. For example, the change of phospholipid structure in the cell membrane leads to the increase of P percentage, while amide groups are consumed during adsorption, resulting in the decrease of N and O percentages [14].Fig. 4 SEM–EDS analysis of the blank group M0, control group M1 of 10 mg/L U(VI), and M2 of 10 mg/L U(VI) + 0.1 µM AHLs after 12-h reaction

Effect of Detoxifying Substances on the Functional Groups of Mixed Bacteria

The FTIR spectra of bacteria before (M0) and after exposure (M1 and M2) to U(VI) are shown in Fig. 5. The main IR spectra of bacterial cells of M0 included the vibration peaks of hydroxyl at 3290 cm−1, carboxyl at 1395 cm−1, and P-O at 1237 cm−1, and the vibration peaks of protein amide I band and protein amide II band at 1654 cm−1and 1534 cm−1, respectively. After exposure, the peak position changed corresponding to the above main groups and bonds in the spectrum. For M1, the peak positions of -OH, -COO- and C=O all shifted obviously, and a new peak attributed to the asymmetric stretching vibration of uranyl ions appeared near 912 cm−1. After adding AHLs (M2), the peak intensities of P-O and UO22+ at 1237 cm−1 and 912 cm−1 are stronger. These results showed that there are strong coordination interactions between uranyl ions and hydroxyl, carboxyl, phosphorus, and amide groups on the surface of microorganisms [43].Fig. 5 a FTIR spectra of the blank group M0, control group M1 of 10 mg/L U(VI), and M2 of 10 mg/L U(VI) + 0.1 µM AHLs after 12 h reaction. b-e XPS spectra of the bacteria exposed to U with and without AHL application, b full spectrum, c U4f, d O1s, e C1s

The XPS spectra of bacteria exposed to U with and without AHL application are shown in Fig. 5 b–e. Figure 5b is the full scan of XPS spectrum after U adsorption. A distinct U4f peak appeared on the surface of the bacteria after adsorption, confirming the adsorption of uranium ions by the composite bacteria. Furthermore, there are both peaks of U(VI) (392.6 eV, 381.6 eV) and U(IV) (390.4 eV, 391.6 eV, 379.7 eV, and 379.9 eV) after U(VI) adsorption (Fig. 5c), indicating the reduction of U(VI) to U(IV) during the adsorption process. For O1s, the peak of bacteria in the AHL application is lower than that of the group without AHL, suggesting that AHL promotes the complexation of oxygen-containing functional groups on the bacterial surface with U ions (Fig. 5d). Figure 5e presents the spectra of C1s. Three peaks at 286.5 eV, 288.0 eV, and 284.8 eV were corresponding to C-O, C = O, and C–C. The potential of the C = O peak shifted from 532.4 to 532.0 eV, revealing the complexation between U(VI) and carbonyl groups within the composite bacteria after adding AHL.

Effects of Detoxifying Substances on the Bacterial Community

Diversity of Bacterial Community

A total of 200,918 valid readings were obtained from the three samples (M0, M1, and M2) by using the Illumina Miseq platform. Venn diagrams of the three samples at the generic level are shown in Fig. S1. There are 17 species of bacteria shared in the three groups, and each group has one unique species. Table 1 shows the changes in species diversity and richness under different reaction conditions. Ace and Chao indexes are both used to evaluate bacterial richness, while Shannon and Simpson indexes are used to evaluate bacterial diversity [44]. The larger the ACE index and Chao index, the higher the species richness. It was found that there was no significant difference in the ACE index and Chao index among the three groups of samples, indicating that there was no significant change in community richness before and after sample exposure. In general, the higher the Shannon index and the lower the Simpson index, the richer the species diversity of bacteria. Compared with M0 and M2, M1 had the smallest Shannon index and the largest Simpson index, indicating that the diversity of bacteria in M1 was the lowest. This indicates that bacteria diversity decreases under U(VI) stress. Table 1 Bacterial abundance and diversity index of the blank group M0, control group M1 of 10 mg/L U(VI), and M2 of 10 mg/L U(VI) + 0.1 µM AHLs after 12-h reaction

Samples	Coverage	Ace	Chao	Shannon	Simpson	
M0	1.000	18.393	18	1.356	0.333	
M1	1.000	18.537	18	1.030	0.434	
M2	1.000	18.336	18	1.082	0.406	

Bacterial Community Composition

The analysis of the samples (M0, M1, and M2) at the phylum level is shown in Table S1. The bacterial communities with a relative abundance of less than 1% are represented by “others.” Obviously, Proteobacteria and Bacteroidota bacteria were the main bacteria in the samples before and after the reaction. The relative abundances of Proteobacteria in M0, M1, and M2 were 88.53%, 39.94%, and 44.04%, respectively, while the relative abundance of Bacteroidota was 11.40% in M0, 59.92% in M1, and 55.88% in M2. The proportion of Bacteroidota after U(VI) exposure is remarkably higher, indicating that Bacteroidota plays an important role during the U(VI) exposure process. Similarly, other studies investigated the effects of U(VI) (32.4 bq/k) on bacterial diversity in the Gaoweili River and also demonstrated that Proteobacteria (47.5%) and Bacteroidota (22.4%) are the main species [45]. The Proteobacteria and Bacteroidota have also been found as the predominant microbial in U(VI) tailings waters [46].

In addition, the abundance of each community at the genus level before and after U(VI) exposure is shown in Fig. 6. For M0, the main genera were unclassified_f_enterobacteriaceae (49.89%), Alcaligenes (25.16%), Chryseobacterium (11.36%), Bordetella (8.48%), and Stenotrophomonas (3.27%). For M1, the main species of bacteria are Chryseobacterium (59.84%), unclassified_f_enterobacteriaceae (22.64%), Stenotrophomonas (15.8%), Serratia (0.78%), and Alcaligenes (0.51%). For simultaneous exposure to U(VI) and AHLs (M2), Chryseobacterium (55.85%), unclassified_f_enterobacteriaceae (27%), Stenotrophomonas (14.57%), Alcaligenes (1.61%), and Serratia (0.65%) were the domain.Fig. 6 Bacterial composition and abundance at the genus level of the blank group M0, control group M1 of 10 mg/L U(VI), and M2 of 10 mg/L U(VI) + 0.1 µM AHLs after 12-h reaction

After exposure to U(VI), the relative abundance of unclassified_f _enterobacteriaceae, Alcaligenes, and Bordetella decreased by 27.25%, 24.65%, and 8.47% respectively, compared to M0, indicating that these bacteria were adversely affected by U(VI). In contrast, the relative abundances of Chryseobacterium and Stenotrophomonas increased by 48.48% and 12.53% after U(VI) exposure, respectively. This finding demonstrated that Chryseobacterium and Stenotrophomonas have superior tolerable ability for U(VI). This is partially in line with the findings reported in other studies. For instance, Zeng et al. [47] found that Chryseobacterium significantly increased from below 0.1 to 4%, 5%, and 12% after U(VI) exposure under different pH conditions. Yuan et al. [48] demonstrated that the most abundant genus in low-grade high-fluorine uranium ore samples is Stenotrophomonas, accounting for 97.68%. Hu et al. [49] reported that Stenotrophomonas sp. shows excellent U(VI) adsorption capacity with a maximum adsorption capacity of 392.9 mg/g.

Bacterial Community Function Genes

PICRUSt is used to predict the gene function of flora in the M0, M1, and M2 samples, and the change in the gene function of flora is revealed based on the COG database. The results are shown in Fig. 7a. From M0 to M1 and M2, the regulatory abilities of amino acid transport and metabolism, inorganic ion transport and metabolism, and energy production and conversion firstly decreased and then increased. The result indicates that the transmembrane transport ability of bacteria decreased when exposed to U(VI), while it could be alleviated to some extent through AHL application. However, the abundance of cell wall/membrane/envelope biogenesis, translation, ribosomal structure and biogenesis, replication, recombination, and repair genes first increased and then decreased, indicating that bacteria may promote cell proliferation during U(VI) removal. Before and after the reaction, the function unknown gene accounted for the highest proportion, which indicates that there are a large number of unknown functional genes in bacterial flora that need to be determined. According to the annotation information in the KEGG database, there are six types of primary metabolic pathways (Fig. 7b). It can be seen that the genes related to metabolism are dominant at each stage, which abundance was increased after U(VI) exposure, indicating that the exposure of U(VI) and AHLs has a certain impact on bacterial metabolism.Fig. 7 Gene function prediction analysis of the blank group M0, control group M1 of 10 mg/L U(VI), and M2 of 10 mg/L U(VI) + 0.1 µM AHLs after 12-h reaction based on COG (a) and KEGG (b) databases

Conclusion

Detoxifying substances play a positive role in the removal of U(VI) from mixed bacteria. The maximum removal efficiency of U(VI) by the mixed bacteria isolated from metal mine soil reached 85.60% under the optimal conditions (pH 6, 35 °C, 10 mg/L U(VI)). The detoxifying substances (AHLs, AQDS, GSH, Se, MT, and EDTA) application had a promotional effect on the bacterial U(VI) removal efficiency. U(VI) is toxic to the mixed bacteria, as reflected by the increase of MDA content and the decrease of the activities of antioxidant enzymes SOD and CAT, while the detoxifying substances can affiliate to alleviate the oxidative stress of the bacteria caused by the toxicity of U(VI). The analysis showed that hydroxyl, carboxyl, phosphorus, and amide groups might be involved in the removal process of U(VI). After 10 mg/L U(VI) exposure, the dominant genera were Chryseobacterium (59.84%), unclassified_f_Enterobacteriaceae (22.64%), Stenotrophomonas (15.80%), Serratia (0.78%), and Alcaligenes (0.51%). The proportion of dominant genera changed significantly with the addition of AHLs. COG and KEGG function prediction revealed significant changes in the abundance of some metabolic genes.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary file1 (DOCX 52 KB)

Author Contribution

X.S.: Investigation, Data curation, Software, Writing – original draft. J. L.: Data curation, Writing – review & editing, Supervision. ZY. X.: Investigation, Data curation. HC. S: Investigation. GH. W and Q. L.: Resources, Writing –review & editing. TT. Z.: Conceptualization, Writing – review & editing, Funding acquisition, Supervision.

Funding

This research was supported by the National Natural Science Foundation of China (52170164) and the Science and Technology Innovation Program of Hunan Province of China (2022RC1184).

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethical Approval and Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.
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