Lead (II) adsorption efficiency and mechanisms by a heavy metal-tolerant yeast Cystobasidium oligophagum QN-3

Lead (II) adsorption efficiency and mechanisms by a heavy metal-tolerant yeast Cystobasidium oligophagum QN-3 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lead (II) adsorption efficiency and mechanisms by a heavy metal-tolerant yeast Cystobasidium oligophagum QN-3 Wen Li, Tao Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6901159/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The lead resistance and adsorption capacity of Cystobasidium oligophagum QN-3 were studied. The strain exhibited high Pb 2+ resistance, withstanding concentrations up to 6,000 mg/L on PDA plates and 26,000 mg/L in liquid PDA medium. The Pb 2+ adsorption capacity of QN-3 was significantly affected by Pb 2+ concentration, temperature, pH, and incubation time. Scanning electron microscopy (SEM) revealed notable morphological changes after adsorption of Pb 2+ , including cell surface wrinkling and elongation. Fourier transform infrared spectroscopy (FTIR) identified key functional groups (–NH 2 , -OH, C=O, COO-, P=O, C-N and -CH) on cell surface participating in Pb 2+ adsorption. Chemical modification of functional groups combined with zeta potential measurements at varying pH and Pb 2+ concentrations confirmed that electrostatic interactions and complexation were the predominant Pb 2+ adsorption mechanisms. Pb 2+ was primarily bound to the cell wall, with minimal intracellular accumulation. The decreased Pb 2+ uptake observed in the presence of metabolic inhibitor DCC suggested an ATP-dependent transport process following initial surface biosorption. Pretreatments including ultrasonication, boiling and alkaline treatment enhanced Pb 2+ adsorption efficiency. The exceptional Pb 2+ tolerance and adsorption capacity of Cystobasidium oligophagum QN-3 highlighted its potential for bioremediation applications. Lead Biosorbent Cystobasidium oligophagum Heavy metals Bioremediation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key points Pb 2+ adsorption capacity of Cystobasidium oligophagum QN-3 was affected by environmental factors. Pb 2+ binding induced morphological alterations and was mediated with groups on the cell surface. Pb 2+ adsorption involved ATP-dependent bioaccumulation following initial surface biosorption. Introduction Heavy metal pollution remains a critical environmental issue due to its persistence, toxicity, and detrimental effects on ecosystems and human health (Zaynab et al. 2022 ). Lead (Pb), as the second most toxic metal, is extensively utilized in industries such as battery production, mining, automobiles, and electronics, leading to its widespread release into soil, air and water systems (Kshyanaprava and Das 2020). It can enter the human body through water, food, and direct contact with the materials that contain this element, it might cause anemia, abdominal pain, damage to the kidneys and nervous system, brain damage, respiratory syndromes, and cardiovascular disorders (Li et al. 2016 ; Narjala 2020). To remove heavy metals from the environment, conventional remediation techniques, including chemical precipitation, membrane filtration, flocculation, ion exchange, electrodeposition, and electrochemical techniques are often limited by high costs, secondary pollution, and inefficiency in treating low-concentration contaminants (Atkovska et al. 2018 ; Burakov et al. 2018 ; Chwastowski et al. 2022; Le et al. 2021 ; Pei et al. 2021 ). In contrast, bioremediation, which employs microorganisms to adsorb and immobilize heavy metals, has emerged as a sustainable, cost-effective, environment friendly alternative (Heidari et al. 2020). Among various microorganisms, yeast has gained significant attention due to its high adsorption capacity, safety, rapid growth, larger amounts of biomass from fermentation, and ease of cultivation (Massoud et al. 2019 ). So far, numerous studies have demonstrated the remarkable adsorption capabilities of various yeast strains for heavy metal removal from aqueous solutions, such as lead (Pb), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), zinc (Zn), gold (Au), arsenic (As)and so on (Aibeche et al. 2022 ; Bakkaloglu et al. 1998; Hadiani et al. 2018 ; Lin et al. 2005 ; Özer & Özer 2004; Roy et al. 2013 ; Zhao et al. 2018 ;). Among these strains, Saccharomyces , Candida, Rhodotorula, Wickerhamomyces and Pichia species (Aibeche et al. 2022 ; Banwo et al. 2021 ; Vidal and Wong Dávila 2022) have been reported to be used as particularly effective biosorbents for lead contaminants. These microorganisms have shown significant potential for bioremediation applications due to their efficient metal-binding properties and capacity to withstand extreme conditions (e.g., acidity, osmotic stress) (Wang and Hu 2018). Besides biosorbent attributes, the effectiveness of adsorption performance can depend on environmental conditions such as pH, temperature, adsorption time and so on. For example, Saccharomyces Cerevisiae is the most widely utilized type of yeast in water treatment. The optimum pH, contact time, and temperature for heavy metal adsorption by Saccharomyces Cerevisiae as found from literatures are 5.0 to 7.0, 60 min, and 20°C to 30°C respectively. (Jena et al. 2022). Rapid uptake of metal ions by yeast cells has stimulated a growing interest in metal-yeast interaction and applicability of this phenomenon. To our best knowledge, the adsorption properties, mechanism of Cystobasidium oligophagum toward lead have been rarely studied. The microorganisms can detoxify and remove Pb + 2 by various complex mechanisms, such as biosorption, bioaccumulation, and so on (Shan et al. 2023 ). Biosorption is a biochemical process of reversible and rapid binding of metal ions to the functional groups present on the surface of microbial biomass. It is an autonomous metabolism-independent process. Cell wall composition of yeast is rich in polysaccharides, proteins, and functional groups such as carboxyl, hydroxyl, and phosphate (Kordialik-Bogacka 2011 ). These functional groups facilitate the biosorption of metal ions through mechanisms such as ionic interaction, micro-precipitation, and covalent bonding (Torres 2020 ). Bioaccumulation is an active metabolism-dependent process in which microorganism intakes metal particles, which then accumulate within the cells. The metal adsorption performance of yeast exhibits significant interspecies and intraspecies variability, as adsorption capacity, specificity, and adsorption mechanisms are strongly influenced by properties of metal solution, types of yeast, and environmental parameters. This study aims to explore the lead tolerance and adsorption capacity of a novel yeast strain, Cystobasidium oligophagum QN-3, isolated from contaminated environments, evaluate the effects of environmental factors on adsorption performance, and elucidate the underlying mechanisms driving the process. Pb 2+ -induced morphological changes were analyzed by scanning electron microscopy (SEM), functional groups associated with Pb 2+ adsorption were characterized using Fourier transform-infrared spectroscopy (FTIR) combined with chemical modification. Zeta potential measurements were conducted under varying pH and Pb 2+ concentrations to assess surface charge dynamics. Pb 2+ accumulation in different cellular parts at different Pb 2+ concentrations was determined. Furthermore, the impacts of energy inhibitor, and various pretreatment methods on Pb 2+ binding capacity were evaluated. Materials and methods Materials Pb (NO 3 ) 2 were of analytical grade and purchased from Fuchen Chemical Reagent Co.Ltd. (Tianjin, China). Sodium dodecyl sulfate (SDS) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.5% glutaraldehyde were of analytical grade and purchased from Shanghai Gefan Biotechnology Co., Ltd. (Shanghai, China). All the other chemical agents were of analytical grade and purchased from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China). Strain and pre-culture conditions Strain Cystobasidium oligophagum QN-3 was isolated from soil sample in Xuzhou, China, and maintained at 40% glycerinum at -20 °C. The sequence of C. oligophagum QN-3 strain was deposited in NCBI Genbank under the accession number MK417801. The strain is currently deposited in Jiangsu Province Engineering Research Center for Food Biological Transformation and Safety Detection. It showed high tolerance of cadmium, and could remove Cd 2+ from aqueous solution effectively (Li and Wang 2020). QN-3 was pre-cultured through two successive transfers in potato dextrose agar (PDA) liquid medium (containing 1% potato and 2% dextrose) in 250 mL Erlenmeyer Flasks at 30 °C at 120 r/min for 72 h. Pb 2+ tolerance assays The pre-cultured yeast cells were inoculated on potato dextrose agar (PDA, containing 1% potato, 2% dextrose and 2% agar) plates with 0, 500, 1000, 1500, 2000, 4000, 6000, 8000 mg/L Pb 2+ by ten-fold serial dilutions with NaCl solution, which were also cultivated in PDA liquid medium with the addition of 0, 500, 1000, 2000, 4000, 6000, 8000, 12000, 16000, 20000, 24000, 26000 mg/L Pb 2+ . To access the amount of the yeast biomass in the cultured liquid medium, the value of OD 600 was determined by a microplate reader (Thermo Varioskan Flash, USA). Pb 2+ binding by QN-3 Pb 2+ binding assay was performed following the method of Halttunen et al. (2008) with minor modifications. Pre-cultured C. oligophagum QN-3 cells were collected through centrifugation (8000 g, 15 min, Sigma 3-30 Krefrigerated Centrifuge, Germany) and washed twice with ultrapure water before use. 0.1 g of wet biomass was added into 20 mL of Pb 2+ solutions (100 mg/L). The mixed sample was shaked at 120 r/min at a constant temperature of 30°C for 2.5 h. Following the Pb 2+ binding, yeast cells were separated (8000 g, 15 min) to obtain the supernatant. The residual Pb 2+ concentration in the supernatant was determined by flame atomic biosorption spectrophotometry (A3, AFG-13, Beijing Purkinje General Instrument Company, Beijing). The yeast cells were dried by vacuum freezing and weighted. The percentage of Pb 2+ removed by yeast biomass and amount of Pb 2+ adsorbed by 1 g of yeast biomass (dry weigh) were calculated using the following equations: where C is the initial Pb 2+ concentration, and C b is the final Pb 2+ concentration after binding. M is the wight of yeast cells and V 0 is the volume of the Pb 2+ solution. Lead calibration curve was obtained based on six levels (0, 2, 4, 6, 8, 10, and 12 mg/L) of lead standard solution. Effect of initial Pb 2+ concentration, temperature, pH, and incubation time on Pb 2+ binding capacity of QN-3 To determine the effect of Pb 2+ concentration on Pb 2+ binding, the yeast biomass was added with 30, 50, 100, 300, 500, 700, 900 and 1500 mg/L Pb 2+ solutions while the other conditions remained similar to those in the Pb 2+ binding assay. The Pb 2+ concentration of samples before and after binding were then detected by flame atomic biosorption spectrophotometry. The influence of incubation temperature on Pb 2+ binding was performed in Pb 2+ solution incubated at 26 °C, 30 °C, 34 °C, 38 °C, and 42 °C, respectively. Then the samples were analyzed. The effect of pH on binding ability was conducted in Pb 2+ solution with pH levels of 4.0, 5.0, 6.0, 7.0 and 8.0 adjusted by 1.0 M HCl or 1.0 M NaOH. Samples were collected by centrifugation and determined. The effect of incubation time was studied during 180 min. The samples were collected by centrifugation after incubation for 0, 30, 60, 90, 120, 150, and 180 min and then analyzed by flame atomic biosorption spectrophotometry. Scanning electron microscope (SEM) analysis The samples for SEM were prepared as described earlier with slight modifications (Chakravarty and Banerjee 2008). After the Pb 2+ binding assay, the yeast cells incubated in 0, 500, and 1500 mg/L of Pb 2+ solutions were collected by centrifugation at 3,000 g, 4℃ for 15 min, washed with phosphate buffer (0.1 M, pH 7.2), and then fixed with 2.5% glutaraldehyde in the same phosphate buffer for 8 h. The cells were then washed with the phosphate buffer thrice for 10 min each, and dehydrated with series of ethanol (gradient concentration of 50%,70%,80%,90%, and 100%). After that, tert-butyl alcohol was added to displace the alcohols three times for 30 min each; Then the samples were freeze-dried. Finally, lyophilized samples were vacuum gold coated and viewed under a SU8010 scanning electron microscope (Hitachi, Tokyo, Japan). FTIR analysis Infrared spectra of pristine and Pb 2+ binding yeast cells were recorded on a FTIR spectrometer (Nicolet is10, Thermo, USA) in the region 4000 to 400 cm −1 to characterize the surface functional groups. Biosorbent before and after Pb 2+ binding with 0, 500 and 1500 mg/L of Pb 2+ concentrations were collected by centrifugation for 15 min at 8000 g, 4°C. After washing three times by ultrapure water, the yeast cells were dried by vacuum freezing. The dried samples were pressed into spectroscopic quality KBr micro slide with a sample/KBr ratio about 1/100. They were scanned by FTIR spectrometer after dried under infrared light. Chemical modification The pre-cultured yeast cells were subjected to chemical treatments for modifications of functional groups as described below according to the procedure reported by Das et al. (2007) and Panda et al. (2006). Hydroxyl groups: 1 g of biomass was stirred for 3 min at room temperature (30 ℃) by acetylation with 0.1% acetic anhydride dissolved in ultra dry petroleum ether. Carboxyl groups: 1 g of biomass was stirred for 4h with 200 ml of methyl iodide. Amino groups:1 g of biomass was refluxed for 4 h with 30 ml of formic acid and 15 mL of formaldehyde. Phosphate groups: 1 g of biomass was refluxed with 20 ml of nitromethane and 25 mL of triethyl phosphite for 6 h. The modified biomass was washed twice with deionized water and used for Pb 2+ binding assay with Pb 2+ concentration of 100 mg/L as described in Section 2.4. Zeta potential measurement Surface charge properties of yeast biomass were carried out by zeta potential measurement according to the protocol of Das et al. (2007) with some modifications. Pre-cultured yeast cells were washed with potassium phosphate (10 mM ) buffer to remove the liquid medium, and then the yeast biomass were suspended in distilled water, adjusted to pH 1.0 to 7.0 and 0 mg/L to 700 mg/L of Pb 2+ solutions (pH 6.0). OD 600 of above yeast suspension was adjusted to 0.5, and zeta potential was measured by Zetasizer (Beijing Puchan General Instrument Co., LTD). Distribution of Pb 2+ in different cellular parts of yeast cells Pb 2+ binding assay was performed in different concentrations of Pb 2+ solutions (50, 100, 200 mg/L) as described in Section 2.4. Yeast Cells were collected and dried by vacuum freezing. Then Pb 2+ accumulation in different cellular parts was extracted by the following procedures as previous report (Sheng et al. 2016). (i) Cytoplasmic water-soluble Pb 2+ : 300 mg of dried cells were mixed with 5 mL of Tris-HCl (10 mM, pH 8.0), after treated by ultrasonic vibration, the supernatant was obtained by centrifuged at 8000 g for 10 min, then the Pb 2+ concentration in the supernatant was determined by flame atomic biosorption spectrophotometry; (ii) Cellular soluble protein bound Pb 2+ : the precipitate of procedure i was incubated with SDS solution (10 mM, pH 8.0) at 90 ℃ for 60 min, then centrifuged at 8000 g for 10 min, Pb 2+ concentration in the supernatant was determined; (iii) Cell-surface bound Pb 2+ : the precipitate of procedure ii was digested in 10 mL of 65% nitric acid solution, then the Pb 2+ concentration was detected; (iv) Total Pb 2+ : 300 mg of dried cells were digested in HNO 3 /HClO 4 (4:1, n/n) solution, then the total Pb 2+ accumulation were detected. Time course of Pb 2+ binding under metabolic inhibitors N,N’-dicyclohexyl carbodiimide (DCC) Pb 2+ binding course in the presence of metabolic inhibitors DCC was monitored by the procedure of Li et al. (2008) with minor modifications. The pre-cultured yeast cells were grown in PDA liquid medium. At the mid-exponential growth phase, DCC (400mM) was added to the liquid medium. After 15 min of incubation, the cells were collected, washed twice, and used for Pb 2+ binding assay in 100 mg/L of Pb 2+ solution, then the Pb 2+ concentrations before and after binding assay were determined respectively as described in Section 2.4. The DCC-untreated yeast cells were used as control sample. Physical and chemical pretreatment of yeast cells Precultured yeast cells were collected and washed as described in Section 2.4. They were pretreated in three ways as follows: Heat-treated cells were prepared by mixing 2 g of yeast cells with 20 mL of distilled water and heating at 100 °C for 60 min. After that, yeast cells were collected by centrifugation at 8000 g for 10 min. Ultrasonication-treated cells were prepared by suspending 2 g of yeast cells with 20 mL of distilled water and treated by ultrasonic vibration at 600W using a JYD-650 sonifier (Zhixin, Shanghai, China). Then the treated cells were collected by centrifugation at 8000×g for 10 min. NaOH-treated cells were prepared by mixing 2 g of yeast cells in 20 mL of 0.1M NaOH solution for 60 min. Then, yeast cells were collected by centrifugation at 8000 g for 10 min and washed three times with distilled water to natural pH. After pretreatment, the yeast cells were resuspended in Pb 2+ solution for Pb 2+ binding assay as described in Section 2.4. The untreated yeast biomass was used as control sample. Statistical analysis Each sample was subjected to three independent experimental trials, and the mean values were determined. The statistical significance of the results was assessed using One-way ANOVA with SPSS 17.0 Statistics. A p-value of less than 0.05 was deemed to indicate statistical significance. Results Pb 2+ tolerant ability of Cystobasidium oligophagum QN-3 Table 1 presents the growth of QN-3 on PDA plates with varying concentrations of Pb 2+ . As shown in the table, QN-3 exhibited robust growth (+++) at Pb 2+ concentrations up to 1500 mg/L, indicating a strong tolerance of the strain to high levels of Pb 2+ . When the Pb 2+ concentration increased to 2000 mg/L and 4000 mg/L, the growth of QN-3 slightly declined but remained relatively good (++). However, at a Pb 2+ concentration of 6000 mg/L, the growth of QN-3 significantly weakened (+), and at 8000 mg/L, QN-3 ceased to grow entirely (-). Figure 1 illustrates the growth of QN-3 under varying concentrations of Pb 2+ , ranging from 0 to 26,000 mg/L. The growth of QN-3 remained relatively stable at lower concentrations, with a slight decline observed as the Pb 2+ concentration increased. Notably, at concentrations above 1,000 mg/L, the growth of QN-3 showed a more pronounced decrease, indicating a threshold beyond which Pb 2+ significantly inhibited microbial activity. The results demonstrated that yeast cells exhibited significant tolerance to Pb 2+ , withstanding concentrations as high as 26000 mg/L which is 52,000 times greater than the allowable limit of 0.5 mg/l for Pb 2+ in industrial wastewater. Table 1 Growth of QN-3 on PDA plates with different concentrations of Pb 2+ Pb 2+ (mg/L) Growth 0 +++ 500 1000 1500 2000 4000 6000 8000 +++ +++ +++ ++ ++ + - +++ indicated growth best; ++ indicated growth better; + indicated growth; - indicated no growth. Effects of Pb 2+ concentration, temperature, pH, and incubation time on Pb 2+ adsorption ability of QN-3 As illustrated in Fig. 2 A, QN-3 exhibited a remarkable capacity for Pb²⁺ adsorption, particularly at lower concentrations. The removal efficiency approached 100% when the Pb 2+ concentration was 10 and 50 mg/L, and it remained above 60% for concentrations below 300 mg/L. However, a general trend of declining adsorption capacity was observed as the Pb 2+ concentration increased. It was also observed that with the Pb 2+ concentration increasing from 30 to 300 mg/L, the adsorption amount enhanced from 7.28 mg/g to 36.39 mg/g rapidly. When the Pb 2+ concentration exceeded 300 mg/L, the adsorption amount increased slightly, and reached a saturation value of 39.82 mg/g at 700 mg /L concentration of Pb 2+ . The uptake capacity was reduced when Pb 2+ concentration was higher than 700 mg/L. The temperature is a critical factor influencing the energy-dependent mechanisms involved in biosorbent-mediated metal removal. As shown in Fig. 2 B, the adsorption capacity exhibited an enhancement within the temperature range of 26°C to 30°C, with maximum removal rate and adsorption amount of Pb 2+ 68.53% and 25.50 mg/g at 30°C, respectively, and then decreased when the temperature was higher than 30°C. The influence of pH on Pb 2+ adsorption by QN-3 biomass is illustrated in Fig. 2 C. As the pH increased from 4.0 to 8.0, the removal efficiency and adsorption amount both initially rose and subsequently declined, with the maximum adsorption observed at pH 6.0. Metal ion binding occurs rapidly during the initial phase. As seen in Fig. 2 D, within the first 30min, Pb 2+ were rapidly adsorbed by the yeast biomass, but after 30 min, the Pb 2+ binding activity increased more slowly and reached equilibrium at about 150 min. Scanning Electron microscopy analysis Scanning electron microscopy (SEM) was utilized to examine the surface morphology of the yeast cells before and after the binding of Pb 2+ (Fig. 3 ). The Pb 2+ binding biomass exhibited significant morphological changes compared to the original biomass. In the absence of Pb 2+ , the cells appeared as single, oval-shaped structures with smooth surfaces (Fig. 3 A). However, under 500 mg/L Pb 2+ stress, some cells retained their original shape, while others exhibited rough, wrinkled, and sunken surfaces (Fig. 3 B). At a higher concentration of 1500 mg/L Pb 2+ , most cells lost their original morphology, displaying sunken and distorted structures with rough surfaces, some cells were elongated. Additionally, numerous white crystalline aggregates adhered to the cell surfaces, and high levels of cell aggregation and overlapping were evident (Fig. 3 C). FTIR analysis The FTIR spectrum is a powerful tool for identifying functional groups capable of binding to Pb 2+ . Each functional group exhibits a distinct absorption peak. Upon interaction with Pb 2+ , the absorption peaks of these functional groups may shift to either higher or lower wavenumbers (Chen et al. 2021 ). The FTIR spectrum of QN-3 biomass without or with different Pb 2+ concentrations are shown in Fig. 4 . A prominent absorption band at 3377 cm⁻¹, corresponding to -OH or -NH stretching vibrations. Under Pb 2+ stress conditions of 500 mg/L and 1500 mg/L, the peak at 3377 cm⁻¹ exhibited a rightward shift of 20 cm⁻¹ and leftward shift of 5 cm⁻¹, respectively, compared to the control group. This shift suggested the participation of -OH and -NH groups from polysaccharides, fatty acids, and proteins in the biosorption process (Pouya and Behnam 2017 ). Peaks at 2927 cm⁻¹, 2850 cm⁻¹ and 1412 cm − 1 were assigned to stretching vibrations of -CH group. After biosorption, the peak at 2927 cm⁻¹ shifted to 2925 cm⁻¹, the peak at 2850 cm⁻¹ shifted to 2854 cm⁻¹, 2856 cm⁻¹, and the peak at 1412 cm − 1 shifted to 1408 cm − 1 under 100 mg/L and 1500 mg/L Pb 2+ stress, implied the involvement of -CH functional groups in Pb 2+ binding. Critical amide-related bands were observed at 1743 cm⁻¹ and 1653 cm⁻¹ (Acylamino I: C = O stretching), 1547 cm⁻¹ (Acylamino II: C–N stretching and N–H bending), and 1244 cm⁻¹ (Acylamino III: C–N stretching and N–H bending) (Chen et al. 2020 ; Dai et al. 2009 ). A shift of 2 cm⁻¹ at 1743 cm⁻¹, 1653 cm⁻¹ and 1244 cm⁻¹ at Pb 2+ exposure indicated the involvement of Acylamino I and Acylamino III in the adsorption of Pb 2+ , further confirming the pivotal role of proteins in Pb 2+ biosorption. The minor shifts observed at 1078 cm⁻¹ and 1034 cm⁻¹ indicated that both C–N and P = O functional groups participated in metal-binding process. The C–S stretching vibration band at 579 cm⁻¹ exhibited a rightward shift of 2 cm⁻¹, suggesting the potential involvement of this functional group in Pb 2+ biosorption. Chemical modification of functional groups To further assess the contribution of different functional groups to Pb 2+ binding, a series of chemical modifications were performed. As illustrated in Fig. 5 , The adsorption capacity of QN-3 biomass for Pb 2+ decreased by 45.20%, 55.81%, 43.37%, and 10.42% following treatment with acetic anhydride, methyl iodide, formaldehyde-formic acid, and triethyl phosphite-nitromethane, respectively. Acetylation with acetic anhydride effectively blocks hydroxyl groups (Fieser and Fieser 1961 ), preventing their participation in subsequent reactions. The formaldehyde-formic acid treatment provides an effective approach for methylating and thereby blocking primary and secondary amines (Davis et al. 1973 ), rendering them unavailable for further interactions. The observed reduction in Pb 2+ binding following the two treatments suggested that both hydroxyl groups and amine groups played a significant role in the uptake of Pb 2+ by QN-3. As a potent alkylating agent, methyl iodide primarily targets the carboxyl groups of the biomass, converting them into methyl ester derivatives by displacing the hydrogen atoms of the carboxyl groups (Panda et al. 2006 ). This modification effectively alters the functional groups, reducing their availability for adsorption processes. The significant reduction in Pb 2+ uptake by 55.81% indicated the critical role of carboxyl groups as the primary metal-binding sites in the adsorption process by QN-3. Moreover, the contribution of carboxyl groups was higher than those of hydroxyl and amine groups. Phosphate groups of orthophosphoric acid (Markowska et al. 1975 ), as well as those present in mono- or diester forms can be esterified by triethyl phosphite-nitromethane. Consequently, this modification is expected to exclude a portion of the phosphate groups in biomass that are involved in Pb 2+ adsorption. Reduction in Pb 2+ binding to a small extent supported the limited but measurable impact of phosphate group in the adsorption process. Zeta potential of Cystobasidium oligophagum QN-3 Zeta potential measurements revealed variations in the surface charge of Cystobasidium oligophagum QN-3 cells under different conditions. In natural environments, where pH is typically near-neutral, yeast surfaces generally carry a negative charge. As shown in Fig. 6 A, the zeta potential of QN-3 cells shifted from positive (at pH ≤ 3) to negative (at pH ≥ 4), with the isoelectric point (IEP) determined to be approximately 4.0. Pb 2+ binding by QN-3 was strongly influenced by surface charge. Within the experimental pH range of 4–6, the negative surface charge increased, facilitating Pb 2+ binding, Consequently, Pb 2+ adsorption increased significantly and reached maximum efficiency at pH 6.0, as demonstrated by the pH-dependent adsorption results. Further analysis of zeta potential at different Pb 2+ concentrations (Fig. 6 B) demonstrated that increasing Pb 2+ concentrations led to a gradual rise in zeta potential, indicating a reduction in net negative surface charge due to Pb 2+ adsorption. These results suggested that electrostatic interactions played a significant role, particularly in the initial rapid adsorption phase of Pb 2+ accumulation by QN-3. Pb 2+ adsorption ability of different cellular components at different Pb 2+ concentrations The distribution of Pb 2+ in cells after incubation with varying initial concentrations of Pb 2+ is detailed in Table 2 . The results indicated a concentration-dependent increase in Pb 2+ accumulation across different cellular components. At an initial concentration of 50 mg/L, the cell-surface bound Pb 2+ was 9.89 mg/g, which significantly increased to 24.94 mg/g at 200 mg/L. Thus, higher Pb 2+ concentrations enhanced surface binding capacity. Cytoplasmic water-soluble Pb 2+ levels remained relatively stable across different concentrations, with values of 6.73 mg/g at 50 mg/L and 6.75 mg/g at 200 mg/L. This stability indicated a potential saturation point for cytoplasmic accumulation under the tested conditions. Cellular soluble protein-bound Pb 2+ showed a slight increase from 0.84 mg/g at 50 mg/L to 1.54 ± 0.08 mg/g at 200 mg/L, suggesting a limited role of intracellular proteins in Pb 2+ binding at higher concentrations. Total Pb 2+ accumulation significantly increased from 16.35 mg/g at 50 mg/L to 32.39 mg/g at 200 mg/L, highlighting a direct correlation between initial Pb 2+ concentration and total cellular accumulation. These findings showed that the majority of Pb 2+ accumulation in yeast cells was localized to cell-surface structures, suggesting the potential foundational roles of the cytomembrane and cytoderm in Pb 2+ binding. However, a fraction of Pb 2+ might traverse the cell membrane, resulting in intracellular accumulation, cytoplasmic Pb-binding compounds might participate in cellular metabolic processes. Table 2 Distribution of Pb 2+ after incubation with different initial concentrations of Pb 2+ Pb 2+ (mg/L) Cell-surface bound Pb 2+ (mg/g) Cytoplasmic water-soluble Pb 2+ (mg/g) Cellular soluble protein bound Pb 2+ (mg/g) Total Pb 2+ (mg/g) 50 9.89 ± 0.58Bc 6.73 ± 0.43Ca 0.84 ± 0.09Db 16.35 ± 0.50Ac 100 15.93 ± 0.09Bb 7.00 ± 0.24Ca 1.43 ± 0.08Da 24.40 ± 0.60Ab 200 24.94 ± 1.22Ba 6.75 ± 0.16Ca 1.54 ± 0.08Da 32.39 ± 0.80Aa Values are presented as means ± SD (n = 3), and means in the same column with different lower case letters were significantly different (p < 0.05), means in the same row with different upper case letters were significantly different by Duncan's multiple range test (p < 0.05). Time course of Pb 2+ uptake in the presence of metabolic inhibitor Figure 7 shows the adsorption Pb 2+ amount in QN-3 biomass with or without metabolic inhibitor N,N’-dicyclohexyl carbodiimide (DCC). During the first 30 min, the Pb 2+ adsorbed by QN-3 biomass both increased rapidly whether in the presence of DCC or not. After 30 min, the Pb 2+ binding activity increased more slowly and until equilibrium at about 150 min. During the whole binding process, the Pb 2+ adsorbed by QN-3 biomass was inhibited in the presence of metabolic inhibitor DCC. The Pb 2+ adsorption capacity of the strain decreased by 36% in the presence of DCC at 150 min, indicating its dependence on ATPase activity. Effect of pretreatment on Pb 2+ adsorption ability of QN-3 biomass The cells of QN-3 were treated with ultrasonication, boiling, and NaOH to evaluate the impact of various pretreatment methods on the Pb 2+ adsorption capacity of strain QN-3. As illustrated in Fig. 8 , Pb 2+ adsorbed by QN-3 biomass for untreated, ultrasonication-treated, boiling-treated, and NaOH-treated cells were 27.84, 33.08, 31.42, and 41.15 mg/g, respectively. All the three pretreatment methods resulted in a statistically significant enhancement in adsorption efficiency compared to untreated cells (p < 0.05). Discussion Lead pollution poses a significant environmental threat caused by various industrial processes. Microbial-based remediation has emerged as a promising strategy for reducing this contamination. In this work, tolerant ability of Cystobasidium oligophagum QN-3 to Pb 2+ was determined. Compared to previously published studies, the tolerance of QN-3 to Pb 2+ was notably higher than that of some reported yeast strains. For instance, El-Sayed ( 2013 ) reported that Saccharomyces cerevisiae showed high tolerance to Pb 2+ concentration up to 600 mg/L, whereas QN-3 maintained optimal growth under the same conditions. Additionally, Banwo et al. (2020) demonstrated that four Candida sp. and Saccharomyces sp. strains completely ceased growth at Pb 2+ concentrations exceeding 1050 mg/L, while QN-3 still showed relatively good growth at 4000 mg/L. These comparisons suggested that QN-3 possessed adsorption conditions to lead and high potential for application in environments contaminated with heavy metals. The biological mechanisms underlying yeast cell tolerance involve several processes, including extracellular precipitation, crystallization and complexation, biosorption to cell walls, transformation of metal species, intracellular chelation through the production of phytochelatins and metallothioneins, as well as the localization and sequestration of metals within vacuoles (Liu et al. 2002 ; Staszak and Regel-Rosocka 2024). The high tolerance of QN-3 to elevated Pb 2+ concentrations made it a promising candidate for bioremediation. To the best of our knowledge, this study represents the first report on the lead resistance of Cystobasidium oligophagum , thereby expanding the pool of yeast species known for their lead-adsorption capabilities. Pb 2+ adsorption capacity of QN-3 were influenced by initial Pb 2+ concentration, temperature, pH, and incubation time. The results of adsorption capacity at initial Pb 2+ concentration aligned with the findings reported by El-Sayed ( 2013 ) and Dhankhar et al. ( 2011 ) on biosorption of Pb 2+ and uranium by isolates of Saccharomyces cerevisiae . The maximum Pb 2+ uptake capacity of wet and dry cells was determined as 22 and 30 mg g − 1 at 300 mg L − 1 initial Pb 2+ concentration. This phenomenon could be attributed to the relationship between metal ion binding capacity and the availability of active sites on the cell wall, as highlighted by Dalali and Hagghi ( 2016 ). At lower initial metal ions concentrations, the ratio of metal ions to the available surface area is relatively low, resulting in fractional sorption that remains largely independent of the initial metal ions concentration. However, at higher concentrations, the number of available sorption sites becomes insufficient relative to the metal ions present, and, hence, the efficiency of metal ions removal is significantly influenced by the initial concentration of the metal ions. Moreover, the proportion of metal ions bound to these sites diminishes relative to the free ions in the solution. This led to a reduction in uptake capacity as the initial concentration increased, which is likely attributed to the saturation of binding sites on the biosorbent. Temperature variations impact the stability, configuration, and ionization of cell wall components, as well as the chemical functional groups involved in the adsorption process (Hassouna et al. 2018 ). QN-3 showed maximum removal rate and adsorption amount of Pb 2+ at 30 ℃. Similarly, Faryal et al. ( 2006 ) found that the fungal strains Aspergillus fumigatus RH05 and Aspergillus flavus RH07 achieved their highest Zn adsorption capacity at 28°C. However, further elevation in temperature resulted in a gradual decrease in Zn removal efficiency. The pH of a solution is a critical parameter that significantly impacts the adsorption process, as it affects the toxicity, chemical behavior, and speciation of metal ions. Additionally, pH influences hydrolysis and complexation properties by altering the ionic forms of metals in the solution (Vallejo Aguilar et al. 2021 ). At lower pH levels, the protons associated with functional groups, such as carboxyl, phosphate, and amino groups in the cell wall, exhibit limited dissociation, thereby reducing the interaction between metal ions and the cells. (Say 2001). As the initial pH rises, the deprotonation of these functional groups enhances the negative charge density on the biomass surface, creating additional metal adsorption sites and thereby improving adsorption efficiency. Elevated pH levels can lead to the formation of metal hydroxide precipitates, thereby reducing the solubility of metals and limiting their availability for binding to the functional groups located within or on the cell wall, resulting in a reduction in binding capacity (Hlihor et al. 2015 ), as demonstrated in this study. As for the influence of time on lead adsorption, at the beginning of Pb 2+ adsorption, as all active sites remain unoccupied and readily accessible, metal ions biosorption occurs rapidly. However, over time, active uptake stage, the biosorption efficiency declines as the saturation level of metal ions in the solution increases (Göksungur et al. 2005 ). Comparable trends have been reported in studies involving Saccharomyces cerevesiae , Aspergillus fumigatus , and Penicillium sp. biomass during Pb 2+ or other metal ions adsorption (Acosta et al. 2023 ; Khamesy et al. 2016 ; Say et al. 2001 ). Cell surface characteristics plays a critical role in mediating metal-microorganism interactions. Heavy metal accumulation can alter cell surface ultrastructure, charge, hydrophobicity, and influencing metal tolerance and adsorption mechanisms (Kordialik-Bogacka 2011 ). The observed surface roughness and crystalline deposits on cells under Pb 2+ stress likely resulted from the aggregation of metal complexes into granular forms, highlighting the significance of the cell wall in the adsorption process. These findings aligned with those reported by Angeles de Paz et al. ( 2023 ), Limin et al. ( 2009 ), and Lin et al. ( 2010 ). The morphological deformities induced by Pb 2+ stress might stem from heavy metal toxicity, which could alter membrane permeability and cause oxidative damage (Gadd 1992 ). The SEM analysis also revealed elongation and high aggregation of cells. This observation was in accordance with the morphological changes reported in a cidophilic bacteria under heavy metal stress (Kordialik–Bogacka 2011). Such alterations could be attributed to a self-protective mechanism employed by the cells to mitigate the toxic impact of environmental stressors (Chakravarty and Banerjee 2008 ). Therefore, alterations in cell ultrastructure under varying Pb 2+ stress conditions may provide partial insight into the mechanisms underlying Pb 2+ adsorption and tolerance. The yeast cell wall, composed of proteins, glucan, mannan, lipids, chitin, chitosan, and inorganic ions (Brady et al. 1994 ), provides abundant functional groups capable of coordinating with heavy metals. FTIR analysis comparing the biomass before and after Pb 2+ binding revealed distinct spectral shifts corresponding to multifunctional interactions between Pb 2+ and biomolecular components on the cell wall. The FTIR data combined with chemical modification results demonstrated the adsorption of Pb 2+ by QN-3 biomass was facilitated by electrostatic interactions as well as complexation mechanisms involving functional groups, including–NH 2 , -OH, C = O, COO-, P = O, C-N and -CH groups etc., in the adsorption process by Cystobasidium oligophagum QN-3 biomass. These findings aligned with prior studies (Das et al. 2007 ; El-Sayed 2013 ; Chen et al. 2021 ; Ibrahim et al. 2022 ) identifying functional groups on the cell walls such as amine, hydroxyl, phosphate, carboxyl groups as primary contributors to heavy metal biosorption, though the relative changes of these groups could vary within yeast species. Furthermore, the elements of O, N, P, and S in polysaccharide, and protein on the cell surface were responsible for Pb 2+ adsorption due to their strong electronegativity through electrostatic interaction (Geesey et al. 2000 ). In conclusion, functional groups and electrostatic attraction of cell walls explained the robust Pb 2+ adsorption capacity observed in QN-3. The results corroborated established biosorption mechanism, emphasizing the structural and electrochemical properties of microbial cell walls in heavy metal binding. Besides surface biosorption, Pb 2+ binding mechanism in Cystobasidium oligophagum QN-3 also involved intracellular bioaccumulation processes, as confirmed in this study. The findings were consistent with previously published reports (Li and Tao 2013 ; Sun and Shao 2007 ; Sheng et al. 2016 ). For example, Sheng et al. ( 2016 ) reported that most of the Cd 2+ in Lactococcus lactis subsp. lactis cells could be found in the cell surface, moreover, the lactis presented intracellular Cd 2+ biosorption capacity, intracellular metabolism might be elevated in response to Cd 2+ stresses. Following initial surface interactions, a secondary metabolic-dependent process facilitates the transmembrane transport of metal ions into the intracellular compartment. This bioaccumulation mechanism enables the internalization and subsequent distribution of metal ion within cellular metabolic pathways (Khan et al. 2019 ; Dhankhar and Hooda 2011 ). Under normal physiological conditions, yeast cells in their growth and living state are capable of generating sufficient ATP to support active sequestration and influx processes (Prasenjit and Sumathi 2005 ). This Pb 2+ uptake biphasic pattern enabled QN-3 biomass to accumulate significantly higher quantities of metals. Pretreatments on Pb 2+ adsorption of Cystobasidium oligophagum QN-3 gave different Pb 2+ adsorbed values. The enhanced Pb 2+ adsorption efficiency observed in ultrasonication-treated cells might be attributed to the disruption of cellular integrity caused by ultrasonication. This process released intracellular components and exposed additional binding sites, thereby improving the capacity for Pb 2+ adsorption. The mechanism of increased adsorption efficiency of boiling-treated cells might be explained by the heat-induced denaturation of soluble proteins in the cell wall, which facilitated the formation of stable complexes with metal ions (Yin et al. 1999 ). Li et al. (2006) also found heat-treated Rhodotorula sp. cells showed higher lead removal capacity when compared with untreated yeast cells, and concluded that boiling treatment could cause the removal of some impurities such as lipids and proteins of the cell wall thereby expose additional binding sites. The maximum Pb 2+ uptake was achieved with NaOH-treated yeast cells, which might due to the removal of protein groups from the cell wall. These proteins typically form non-adsorbable complexes with Pb 2+ ions, and their elimination exposed more accessible metal binding sites and enhancing the overall negative charge on the cell surface, therefore enhanced the metal adsorption capacity (Gksungur et al. 2005). These results indicated that to enhance microbial adsorption capacity for heavy metals, various pretreatment methods-including thermal treatment, ultrasonication, and alkaline (e.g., NaOH) processing-could be applied to modify cellular structures. These approaches optimized the exposure of functional groups and binding sites, thereby unlocking greater application potential of microbial biomass for efficient metal removal. Conclusion The yeast strain Cystobasidium oligophagum QN-3 exhibited high Pb 2+ resistance and efficient Pb 2+ adsorption capacity in aqueous solution. This adsorption capacity was influenced by Pb 2+ concentration, temperature, pH, and incubation time. After Pb 2+ binding, the surface morphology of the yeast cells changed. During binding, the majority of Pb 2+ particles were adsorbed onto the cell surface, functional groups, such as –NH 2 , -OH, C = O, COO-, P = O, C-N, -CH groups etc. were involved in the adsorption of Pb 2+ . Functional group blocking experiments and zeta potential measurements demonstrated that Pb 2+ adsorption occurred through electrostatic interactions and complexation reactions. In addition to cell surface biosorption, intracellular Pb 2+ bioaccumulation was observed. The energy inhibitor DCC reduced Pb 2+ binding capacity, suggesting the involvement of ATP-dependent uptake mechanisms following initial surface biosorption. Pretreatments such as ultrasonication, boiling, and NaOH treatment significantly enhanced the strain’s Pb 2+ adsorption efficiency. These findings demonstrate that Cystobasidium oligophagum QN-3 holds promise for bioremediation of Pb²⁺-contaminated aqueous and solid matrices. Further research should explore its practical applications using native or pretreated cells in real environmental settings. Abbreviations SEM scanning electron microscopy FTIR fourier transform-infrared spectroscopy PDA potato dextrose agar PBS phosphate-buffered saline DCC N,N’-dicyclohexyl carbodiimide IEP isoelectric point Declarations Author contribution Wen Li: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. Tao Wang: Writing- review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition. Conceptualization. Funding This study was co-funded by Six Talent Peaks Project in Jiangsu Province (No. SWYY-222), the "343" Industrial Development Project Served by Universities in Xuzhou (gx2024021), and Jiangsu Province Policy Guidance Program (North Jiangsu Science and Technology Special Project) (XZ-SZ202131). Availability of data and materials Data will be made available on request. Ethics approval Not applicable. Conflict of interest The authors declare no competing interests. References Acosta I, Rodríguez A, Cárdenas JF, Martínez VM, Contreras D (2023) Lead removal from aqueous solutions using different biosorbents. In: Kumar, N., Jha, A.K. (eds) Lead toxicity: challenges and solution. Environmental Science and Engineering. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6901159","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":489241725,"identity":"750c8a2c-7f20-43c4-a368-5886cb34d6b6","order_by":0,"name":"Wen Li","email":"","orcid":"","institution":"Xuzhou University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Li","suffix":""},{"id":489241726,"identity":"82300efe-ab16-4a8d-ae70-2c85010a63b8","order_by":1,"name":"Tao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBCDBPb2xsYHH4hWfwCohefM4WbDGaRpuZHeJs1BjGqD42cPv/5QY5PHI/mwQZqBwU5Ot4GQljN5aRYHjqUV80gnNhgXMCQbmx0goMXsQI6ZwQG2w4n7gVqSZzAcSNxGUMv5N0At/w4n9kgebDjMQ5SWGznGDw62AbVIMDY2E6XF/sYbM4azfWmJPTyJzYwzDIjwi2R/jvGHim82iT3sx5//+FBhJ0dQCxCwSSDYBoSVgwAz8clkFIyCUTAKRiYAANRsSxYr+g3bAAAAAElFTkSuQmCC","orcid":"","institution":"Xuzhou University of Technology","correspondingAuthor":true,"prefix":"","firstName":"Tao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-06-16 03:23:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6901159/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6901159/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87491519,"identity":"35740e58-227a-45de-851c-b2acb88b10f8","added_by":"auto","created_at":"2025-07-24 11:59:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6364,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of strain QN-3 in PDA liquid medium with different concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e. Pb\u003csup\u003e2+ \u003c/sup\u003econcentrations ranged from 0 to 26000 mg/L.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/6a21fae7953ac952d276a190.png"},{"id":87491748,"identity":"89e168ee-f43e-4975-9974-34bf1ab0557a","added_by":"auto","created_at":"2025-07-24 12:07:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":19340,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration (A), temperature (B), pH (C), and incubation time (D) on Pb\u003csup\u003e2+ \u003c/sup\u003eadsorption capacity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/2be88f34497fa421de0be996.png"},{"id":87491749,"identity":"1216ace7-6acc-47fb-84b5-7cf0eea10a5d","added_by":"auto","created_at":"2025-07-24 12:07:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":199267,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopic (SEM) analysis. A, B, C were results of SEM detection under 0, 500, 1500 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e stresses, respectively.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/d3247c9de28986622726e17e.png"},{"id":87491521,"identity":"0dd0da0a-b9ad-42e4-8db9-84028f07aedb","added_by":"auto","created_at":"2025-07-24 11:59:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":17807,"visible":true,"origin":"","legend":"\u003cp\u003eFourier transform infrared spectroscopy (FTIR) spectra of yeast cells under 0 (black line), 500 (red line), and 1500 mg/L (blue line) Pb\u003csup\u003e2+\u003c/sup\u003e stresses.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/ec3715807082fa38143371d5.png"},{"id":87491750,"identity":"db6251e4-9ef4-4324-bad7-b161081758b9","added_by":"auto","created_at":"2025-07-24 12:07:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11036,"visible":true,"origin":"","legend":"\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity by various modified QN-3 biomass. Different superscript letters above the columns denote a significant difference at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/0cabefe67952175ac1e55674.png"},{"id":87491752,"identity":"17084cb3-7d6e-4f15-aa83-bd2189d4fb25","added_by":"auto","created_at":"2025-07-24 12:07:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":15177,"visible":true,"origin":"","legend":"\u003cp\u003eZeta potential of the yeast cells at different pH values (A) and concentrations of Pb\u003csup\u003e2+ \u003c/sup\u003e(B).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/a4af82d442612d034c67de2e.png"},{"id":87494139,"identity":"3cac6785-2863-4643-a44f-26b46758f950","added_by":"auto","created_at":"2025-07-24 12:31:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9579,"visible":true,"origin":"","legend":"\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity by DCC-treated and untreated strain. Different superscript letters above the columns denote a significant difference at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/b915e6bccf862f8a23af6e63.png"},{"id":87491526,"identity":"5ba3775f-ba12-4d78-9908-2747249f1b2b","added_by":"auto","created_at":"2025-07-24 11:59:53","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":8627,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different pretreatments on Pb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity of QN-3. Different superscript letters above the columns denote a significant difference at P \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/79b3e0164eea0b00f34fafc0.png"},{"id":89843882,"identity":"3c79c500-8e30-4cb1-a6bf-88c6453025f9","added_by":"auto","created_at":"2025-08-25 15:47:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1414566,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6901159/v1/5f91dd8a-0dae-4aae-94f9-a72a8f97c181.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lead (II) adsorption efficiency and mechanisms by a heavy metal-tolerant yeast Cystobasidium oligophagum QN-3","fulltext":[{"header":"Key points","content":"\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity of\u003cem\u003e\u0026nbsp;Cystobasidium oligophagum\u003c/em\u003e QN-3 was affected by environmental factors.\u003c/p\u003e\n\u003cp\u003ePb\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003ebinding induced morphological alterations and was mediated with groups on the cell surface.\u003c/p\u003e\n\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e adsorption involved ATP-dependent bioaccumulation following initial surface biosorption.\u0026nbsp;\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eHeavy metal pollution remains a critical environmental issue due to its persistence, toxicity, and detrimental effects on ecosystems and human health (Zaynab et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Lead (Pb), as the second most toxic metal, is extensively utilized in industries such as battery production, mining, automobiles, and electronics, leading to its widespread release into soil, air and water systems (Kshyanaprava and Das 2020). It can enter the human body through water, food, and direct contact with the materials that contain this element, it might cause anemia, abdominal pain, damage to the kidneys and nervous system, brain damage, respiratory syndromes, and cardiovascular disorders (Li et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Narjala 2020).\u003c/p\u003e\u003cp\u003eTo remove heavy metals from the environment, conventional remediation techniques, including chemical precipitation, membrane filtration, flocculation, ion exchange, electrodeposition, and electrochemical techniques are often limited by high costs, secondary pollution, and inefficiency in treating low-concentration contaminants (Atkovska et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Burakov et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Chwastowski et al. 2022; Le et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pei et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, bioremediation, which employs microorganisms to adsorb and immobilize heavy metals, has emerged as a sustainable, cost-effective, environment friendly alternative (Heidari et al. 2020). Among various microorganisms, yeast has gained significant attention due to its high adsorption capacity, safety, rapid growth, larger amounts of biomass from fermentation, and ease of cultivation (Massoud et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSo far, numerous studies have demonstrated the remarkable adsorption capabilities of various yeast strains for heavy metal removal from aqueous solutions, such as lead (Pb), cadmium (Cd), chromium (Cr), copper (Cu), mercury (Hg), nickel (Ni), zinc (Zn), gold (Au), arsenic (As)and so on (Aibeche et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bakkaloglu et al. 1998; Hadiani et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; \u0026Ouml;zer \u0026amp; \u0026Ouml;zer 2004; Roy et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2018\u003c/span\u003e;). Among these strains, \u003cem\u003eSaccharomyces\u003c/em\u003e, \u003cem\u003eCandida, Rhodotorula, Wickerhamomyces\u003c/em\u003e and \u003cem\u003ePichia\u003c/em\u003e species (Aibeche et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Banwo et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vidal and Wong D\u0026aacute;vila 2022) have been reported to be used as particularly effective biosorbents for lead contaminants. These microorganisms have shown significant potential for bioremediation applications due to their efficient metal-binding properties and capacity to withstand extreme conditions (e.g., acidity, osmotic stress) (Wang and Hu 2018). Besides biosorbent attributes, the effectiveness of adsorption performance can depend on environmental conditions such as pH, temperature, adsorption time and so on. For example, \u003cem\u003eSaccharomyces Cerevisiae\u003c/em\u003e is the most widely utilized type of yeast in water treatment. The optimum pH, contact time, and temperature for heavy metal adsorption by \u003cem\u003eSaccharomyces Cerevisiae\u003c/em\u003e as found from literatures are 5.0 to 7.0, 60 min, and 20\u0026deg;C to 30\u0026deg;C respectively. (Jena et al. 2022). Rapid uptake of metal ions by yeast cells has stimulated a growing interest in metal-yeast interaction and applicability of this phenomenon. To our best knowledge, the adsorption properties, mechanism of \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e toward lead have been rarely studied.\u003c/p\u003e\u003cp\u003eThe microorganisms can detoxify and remove Pb\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e by various complex mechanisms, such as biosorption, bioaccumulation, and so on (Shan et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Biosorption is a biochemical process of reversible and rapid binding of metal ions to the functional groups present on the surface of microbial biomass. It is an autonomous metabolism-independent process. Cell wall composition of yeast is rich in polysaccharides, proteins, and functional groups such as carboxyl, hydroxyl, and phosphate (Kordialik-Bogacka \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These functional groups facilitate the biosorption of metal ions through mechanisms such as ionic interaction, micro-precipitation, and covalent bonding (Torres \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Bioaccumulation is an active metabolism-dependent process in which microorganism intakes metal particles, which then accumulate within the cells. The metal adsorption performance of yeast exhibits significant interspecies and intraspecies variability, as adsorption capacity, specificity, and adsorption mechanisms are strongly influenced by properties of metal solution, types of yeast, and environmental parameters.\u003c/p\u003e\u003cp\u003eThis study aims to explore the lead tolerance and adsorption capacity of a novel yeast strain, \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3, isolated from contaminated environments, evaluate the effects\u003c/p\u003e\u003cp\u003eof environmental factors on adsorption performance, and elucidate the underlying mechanisms driving the process. Pb\u003csup\u003e2+\u003c/sup\u003e-induced morphological changes were analyzed by scanning electron microscopy (SEM), functional groups associated with Pb\u003csup\u003e2+\u003c/sup\u003e adsorption were characterized using Fourier transform-infrared spectroscopy (FTIR) combined with chemical modification. Zeta potential measurements were conducted under varying pH and Pb\u003csup\u003e2+\u003c/sup\u003e concentrations to assess surface charge dynamics. Pb\u003csup\u003e2+\u003c/sup\u003e accumulation in different cellular parts at different Pb\u003csup\u003e2+\u003c/sup\u003e concentrations was determined. Furthermore, the impacts of energy inhibitor, and various pretreatment methods on Pb\u003csup\u003e2+\u003c/sup\u003e binding capacity were evaluated.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eMaterials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePb (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e were of analytical grade and purchased from Fuchen Chemical Reagent Co.Ltd. (Tianjin, China). Sodium dodecyl sulfate (SDS) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 2.5% glutaraldehyde were of analytical grade and purchased from Shanghai Gefan Biotechnology Co., Ltd. (Shanghai, China). All the other chemical agents were of analytical grade and purchased from Sinopharm Chemical Reagent Co.Ltd. (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrain and pre-culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStrain \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 was\u0026nbsp;isolated from\u0026nbsp;soil sample\u0026nbsp;in Xuzhou, China,\u0026nbsp;and maintained\u0026nbsp;at 40% glycerinum at -20\u0026nbsp;\u0026deg;C. The sequence of \u003cem\u003eC. oligophagum\u003c/em\u003e QN-3 strain was deposited in NCBI Genbank under the accession number\u0026nbsp;MK417801. The strain is currently deposited in Jiangsu Province Engineering Research Center for Food Biological Transformation and Safety Detection. It\u0026nbsp;showed high tolerance of cadmium, and could remove Cd\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003efrom aqueous solution effectively (Li and Wang 2020).\u0026nbsp;QN-3 was pre-cultured\u0026nbsp;through two successive transfers\u0026nbsp;in\u0026nbsp;potato dextrose agar (PDA) liquid medium (containing 1% potato and 2% dextrose) in 250 mL Erlenmeyer Flasks at 30\u0026nbsp;\u0026deg;C\u0026nbsp;at 120 r/min\u0026nbsp;for 72 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePb\u003csup\u003e2+\u003c/sup\u003e tolerance assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pre-cultured yeast cells were inoculated on potato dextrose agar (PDA, containing 1% potato, 2% dextrose and 2% agar) plates with 0, 500, 1000, 1500, 2000, 4000, 6000, 8000 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e by ten-fold serial dilutions with NaCl solution, which were also cultivated in PDA liquid medium with the addition of 0, 500, 1000, 2000, 4000, 6000, 8000, 12000, 16000, 20000, 24000, 26000 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e. To access the amount of the yeast biomass in the cultured liquid medium, the value of OD\u003csub\u003e600\u003c/sub\u003e was determined by a microplate reader (Thermo Varioskan Flash, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePb\u003csup\u003e2+\u003c/sup\u003e binding by QN-3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e binding assay was performed following the method of Halttunen et al. (2008) with minor modifications. Pre-cultured \u003cem\u003eC. oligophagum\u003c/em\u003e QN-3 cells were collected through centrifugation (8000 g, 15 min, Sigma 3-30 Krefrigerated Centrifuge, Germany) and washed twice with ultrapure water before use. 0.1 g of wet biomass was added into 20 mL of Pb\u003csup\u003e2+\u003c/sup\u003e solutions (100 mg/L). The mixed sample was shaked at 120 r/min at a constant temperature of 30\u0026deg;C for 2.5 h. Following the Pb\u003csup\u003e2+\u003c/sup\u003e binding, yeast cells were separated (8000 g, 15 min) to obtain the supernatant. The residual Pb\u003csup\u003e2+\u003c/sup\u003e concentration in the supernatant was determined by flame atomic biosorption spectrophotometry (A3, AFG-13, Beijing Purkinje General Instrument Company, Beijing). The yeast cells were dried by vacuum freezing and weighted. The percentage of Pb\u003csup\u003e2+\u003c/sup\u003e removed by yeast biomass and amount of Pb\u003csup\u003e2+\u003c/sup\u003e adsorbed by 1 g of yeast biomass (dry weigh) were calculated using the following equations:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAQIAAABxCAYAAAA+jq2LAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAA1ZSURBVHhe7Z09cvI+F8WP37XYFAwrkFdAnoaKNp1ckoaOko5GLqFLS0WDtQJYAUOBtBe9hW2whflIAuQfOL8Zz8SSJcsfOtK9VyaBc86BEPLS/M9PIIS8HhQCQgiFgBBCISCEUAgIIaAQEEJAISCEgEJACAGFgBACCgEhBBQCQggoBIQQUAgIIaAQEEJAISCEgEJACAGFgBACCgEhBBQCQggoBIQQUAgIIaAQvAZWp4jjAEEQIAhipNY/4mfcu35yfygEz45OEL3N0f90cM5AiQ5aoX/QD7h3/eQhBPy/Bs+MRRpHmPcNVoNzvVMjCd4w85MrCNVUx7X1k/86FIJnxqaIoy1Gboqun3cLTtafC8QHFMxqAErEfx+aBs+M2WIt2oj89Ftxsv4Qg08F0WlRBP4IFIJnJmpDrLdYWgCw0GlyW0feufrNFmjvkMR0IP4JHHlqMikcAAcIJzPjZ/+YU/VnEg4yc6b4W2a1YuQ/BmcEt8BqpEmMRH992LNpjDjR+HrJ6+hOV3DOwbkVpt3bT9Sb67fYbSSyaZemwR/hMUJgU8RBGWc+xJu/03F+C5vGCIIAsT/HtSni9wVaw0NHKI8N4uOpuE3jWh3hYIXVcIf3OL2bGDwcu8Qcue/A6gRvG4XhHbyVVieIE+0nAwB0UjyDIEbiP4Qr8l8Of4pwNzLpIJTLJ4/GZUo4QDh1+9nq3cgknKg12DglRH3aa5QTxXWaTDrUMjMnhXRNs2SjhBPPMn/OpBOiMBmEdHewSJxRRf0N98wocXjXjHICddPkUv4r8ktC4PJO8ccewJEQHF1TkVZeVEUU8ixPNGpkTqJZJJ4Zo2Sz7yKTnugeyJR0yhQd+uiGGqdE/TmV/orr8l+Tx5gGV6MLL3O+7ad9OsnTEg2bJrmZESfQ1SlenKI+SWyqyyIt04ry+2l8kO/DaiTxYdqYNs88AQB2twHOhcjCFjrl3zbFeNM/M0WO0BYzLM6c7xkJBz3gLUJthq8TxGNgdGKRUncwxYksAAbbNdCpLG+M2gLY7ArT63T+K/NLQmChkzFmomo7WqTxGzZ9kzufjEJn9pa/IN0pjBLAZoHlvylWzkBhhrc4AYYrOJdBrj8qnehUXSEGqwwSgBzli2DCwSeUkMjcFF1oJNEY7c8VnDPI5BofZ3qm2a4h2l4UPWpDlC+dXmBTCIWezNEZnVtcE6LVATa7JntVIznysdS3I9/Fn6GLqcmAcZw/a5vmIvDpL1K6ErvDBgL+Y9lzKf9FeawQrD8QBQGCIMIYo/qqM7vEfC0Po0A4wFAJzKodsdMrRoK804j+ELl/LkIu6kVnOFtXFz2JQ712iXmnV7x0XUzdan+OqC3yY75COMCoP8+vcwyMpl1AJxhjhPzPgyPxtMT4dDHNzbiTW9MSX18sfns7SdjF9LOPzThAEM3R/5wWz5U8iscKgVAwzsFlEuvZGybX94Sb0u3J/VTRLufoV+brVqdI4hhxECD6WNfKXUt3UITUVlN0YZGON/k5dII3jGCcg+lvML7zKO6LxW9vF/ne7a4TttDBGlvjZxRcyn9RHisEJd0pMgnMxr8UMuv2INdzLK3GZNvHv3L0Kb6ka48+8elcbo6cIWoLrC+9UXqCeWeEQZj7FEQ7QgggbHW8sha5y6FpKHxm06Dwy7zP0TcOzvQxf//JSkRvdliYcAdfzun8V+Z3hABAd6gg1h+HWUH4D30xwzgtFtdYjcnHGrKXj9Zm+4Xh4kJduXmwxvx9DPQO5ondbQDRx79uiNBqLLeoOJmOCXOj/mQ+oJGU5sFFDLbrU7br90yDv4FG8j4GRoVJFg6wGnXwEX3FdKoS4l9fYD2fFM7fFOOZgNrP+i7lvyh+GOEuFLFaP+57FAs2yklRHAfhZBniyWSRVoQbvf19PdV1CafqKjHKiaNwXbYvI2SWrwNAHmo6nKMa8mxYR1DBKHEcbiyW3fp5zaGw58eoE/cvk8fPrMAo6cT+2ebPt74Gwzh1Yunzdfmvx2OE4JkxyonGRTNNi4cqL2B1/UFZR+1YQh4Hf4/gFliNdDLGtvf55fX8No3xvh3hk+vyyS9CISAnsTrF+/gD6zUACChThlbJs/FrzkLyH4e/RfhScEZAGuBvEb4aFAJyzMnfIiTPCk0DcszJ3yIkzwqFgBxz7rcIyVNC04A0opMYb7M1AAGZfT0sSv4WFAJCCE0DQgiFgBBCISCEgEJACAGFgBACCgEhBBQCQggoBIQQUAgIIaAQEEJAISCEgEJACAGFgBACCgEhBBQCQggoBIQQUAgIIaAQEEJAISCEgEJACAGFgBACCgEhBBQCQggoBIQQUAgIIaAQEEJAISCEgEJAforVKZJU4xH/LNmmCRL+W+a7QCEg30cneN/9w3TQRfm/km2aIA4CBEGAIE6vEohry4SDKaatSUUMNJKkfrxOEmgL2DTO60s0AIs0zutPdOVgcsAR8i2MU0K6rJqihINQzjjnnMuclNXcZr5exjuvUU7KzBnnnFHSHYpnTqJyXCYdLtb9ulAIyDfJnNx34GK/2vGu4lQZ4zIlncoOtVfJpHCqmpVJJ4RwopZYFYzMSVltK/GhaUC+h91hU93XC8xkD91qGgDY9DDtr2xxak+XQYgIG2z3BkedqA1sjZ8KdPwEbLCzFmk8Rns4QAhApwkS+hqO+EUh0EiusNlKWy++w4PTye3sxrPttBppEiPRDXk3I7eDL12LTWPEyQ2ce2HruONtdnm9ViMtbfdwgFU+86xtq0HRyZvKADBbAIsUaRIf3VOzBdpRsWNTJIseVqsVhphUrj9EqwNsJxNsRysMwtynMcYQ08EQ7fkEF27VS/F7QqAX2AiB2eL84wgHK2TST70N3amBEn7q9zjZTpsifl+gNVxh2m0e4W6CXWIOheHx8FojHKywGu7wfsYpdx0R2thiPzB3h1CdOaIgQBC9YY7WifG8wskyFrtNB73pAIOeLzd5XisEAI10Agyn+UWHgyl6iwSlbkRtYNMeosiuEKLV2WD3sxvwXPi2wmMo7LdMOjTaiHUyCc/+uxXGKYGKg+lnHLfTOCXEzeo/h1G+jXweo4QTP21YJr90zqsxat82o7z7l0knv3vOSnszefm9eyV+Z0Zgl5h3euh2e5BoUmaNpAj3BEGMcc0Y9cJNQVyZDlbLBYjLDJ3sQ0nNU3iLNCnCTXHiTRkrdcaH0eYor6Gd0BN8oF8fpWttKa6jOKfetyE9tEFXr7Vshz+aa0w+gP6/yhh8oVw4GKEzW/xsetyd4rO1vP06ArPFerOD1gkm29F+RLdpgmQ3xLQ0K75Kt4fOdgKtEyzQ5Jt4YXxleARVlW8eRStpJnMS1X3lBGThNTYuk+WI7pdTTuIw2udhKukyUx0ZijJS5el+Hd6+yWQl1OWfz2tnec6GUbdsS3kNSuDQNpfXkxfLnIRwsvCe10NtFWrtcleW82dD+XlxZrvL6E/+E/yCENTjwEcvadHR67PB6ktYdhTvpWwoV+2IzZ3S7wxepzqqsxLuOsrz23m8X+K3pX5cRWD8c/j7BQcxLPCP8/cLTrXvGnyR+Msb+Q3TwC4xX8/wVkxXo481sK44nS7SxdQoYBzlZkGTl/6mHNoaBG+YNZoydyJsoYMZFkW0wS7nWIs2Sod5nphiPJPoVee515T7IX4U4C9v5BeiBnY5RyerPogMEjNcCB7UCQeYrhyc6QMf0cWQ2c+QyGovThGKeghdDDOJ2VuUi+a8g+wzj4eX2OUc66NY/OVyx+Th3JpPwdv8MB55Hh4sBBbLeSUGDADooidxCCMWo9m4eOmsTrGoOuF0kjvaimNRhv/Cf+iLGcal48pqTD7WkMVQabbrSiV1ZovS2WWRjmcQ/X95p9nXWXSAWnz8QjsBRG2BdcPKl3NtqaMxGbdhShFaTVGPQOb3Ux3FDC+VA/IwHNDJ43D5TKthtKxu+9g/eT58W+GeHJxRno+gTD94EJ0AHCCcVLlDEKUDLVNOiIN9J4p15kVlTu7z8rJlffvjazZx7q9QqjyfV5/z6tw79ArOtbPIP3LSVdqSX099/3A/hFOmcCRW7NmDk7Gw/f3684zz5Zwr/B3eUl3ysjxUCF6PH64jyORRrD+TB0fjSWffhXKuwWFJXpsHmwavRojBZx+bcf5p7Fexuw3W5RLcPAW7zRqiHQHQWMxEfe1AedTZcrmD8X3eQXa85O4OWOiGZcKHtSDVdSDfST9eF9KURi7gKwO5AyZzSh7i+teTOSUqphOEE6owXc5+Vnu6XLmi8Kst+R7GZSpz5mgFoheGrX6G/KX0PE/Jygyo+BLxyCQjZ6EQkPvjC0FNxCqd/KvpRZ7Kyk+i898zOGkykZPQNCB/Gr0AWt38A6hJskBvGmG3aTaZyGkoBOTxRG2I2SL3m9gdNuVip6+mQxffDIRodWZAb4quXWKOPqgDX8SfIhByO/wwZlPYuB5V+Up6Hq6thkAP5zvpPiGNBI5rLAl5eWgaEEIoBIQQCgEhhEJACAGFgBACCgEhBBQCQggoBIQQUAgIIaAQEEJAISCEgEJACAGFgBACAP8Hs4wW4mWns8sAAAAASUVORK5CYII=\"\u003e\u003c/p\u003e\n\u003cp\u003ewhere C\u0026nbsp;is the initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration, and C\u003csub\u003eb\u003c/sub\u003e is the final Pb\u003csup\u003e2+\u003c/sup\u003e concentration after binding. M is the wight of yeast cells and V\u003csub\u003e0\u003c/sub\u003e is the volume of the Pb\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003esolution.\u003c/p\u003e\n\u003cp\u003eLead calibration curve was obtained based on six levels (0, 2, 4, 6, 8, 10, and 12 mg/L) of lead standard solution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration, temperature, pH, and incubation time on Pb\u003csup\u003e2+\u003c/sup\u003e binding capacity of QN-3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the effect of Pb\u003csup\u003e2+\u003c/sup\u003e concentration on Pb\u003csup\u003e2+\u003c/sup\u003e binding, the yeast biomass was added with 30, 50, 100, 300, 500, 700, 900 and 1500 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e solutions while the other conditions remained similar to those in the Pb\u003csup\u003e2+\u003c/sup\u003e binding assay. The Pb\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003econcentration\u003csup\u003e\u0026nbsp;\u003c/sup\u003eof samples before and after binding were then detected by flame atomic biosorption spectrophotometry.\u003c/p\u003e\n\u003cp\u003eThe influence of incubation temperature on Pb\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003ebinding was performed in Pb\u003csup\u003e2+\u003c/sup\u003e solution incubated at 26 \u0026deg;C, 30 \u0026deg;C, 34 \u0026deg;C, 38 \u0026deg;C, and 42 \u0026deg;C, respectively. Then the samples were analyzed.\u003c/p\u003e\n\u003cp\u003eThe effect of pH on binding ability was conducted in Pb\u003csup\u003e2+\u003c/sup\u003esolution with pH levels of 4.0, 5.0, 6.0, 7.0 and 8.0 adjusted by 1.0 M HCl or 1.0 M NaOH. Samples were collected by centrifugation and determined.\u003c/p\u003e\n\u003cp\u003eThe effect of incubation time was studied during 180 min. The samples were collected by centrifugation after incubation for 0, 30, 60, 90, 120, 150, and 180 min and then analyzed by flame atomic biosorption spectrophotometry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscope (SEM) analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples for SEM were prepared as described earlier with slight modifications\u0026nbsp;(Chakravarty and Banerjee 2008). After the Pb\u003csup\u003e2+\u003c/sup\u003e binding assay, the yeast cells incubated in 0, 500, and 1500 mg/L of Pb\u003csup\u003e2+\u003c/sup\u003e solutions were collected by centrifugation at 3,000 g, 4℃ for 15 min, washed with phosphate buffer (0.1 M, pH 7.2), and then fixed with 2.5% glutaraldehyde in the same phosphate buffer for 8 h. The cells were then washed with the phosphate buffer thrice for 10 min each, and dehydrated with series of ethanol (gradient concentration of \u0026nbsp; 50%,70%,80%,90%, and 100%). After that, tert-butyl alcohol was added to displace the alcohols three times for 30 min each; Then the samples were freeze-dried. Finally, lyophilized samples were vacuum gold coated and viewed under a SU8010 scanning electron microscope (Hitachi, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfrared spectra of pristine and Pb\u003csup\u003e2+\u003c/sup\u003e binding yeast cells were recorded on a FTIR spectrometer (Nicolet is10, Thermo, USA) in the region 4000 to 400 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003e to characterize the surface functional groups. Biosorbent before and after Pb\u003csup\u003e2+\u003c/sup\u003e binding with 0, 500 and 1500 mg/L of Pb\u003csup\u003e2+\u003c/sup\u003e concentrations were collected by centrifugation for 15 min at 8000 g, 4\u0026deg;C. After washing three times by ultrapure water, the yeast cells were dried by vacuum freezing. The dried samples were pressed into spectroscopic quality KBr micro slide with a sample/KBr ratio about 1/100. They were scanned by FTIR spectrometer after dried under infrared light.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical modification\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe pre-cultured yeast cells were subjected to chemical treatments for modifications of functional groups as described below according to the procedure reported by Das et al. (2007) and Panda et al. (2006). Hydroxyl groups: 1 g of biomass was stirred for 3 min at room temperature (30 ℃) by acetylation with 0.1% acetic anhydride dissolved in ultra dry petroleum ether. Carboxyl groups: 1 g of biomass was stirred for 4h with 200 ml of methyl iodide. Amino groups:1 g of biomass was refluxed for 4 h with 30 ml of formic acid and 15 mL of formaldehyde. Phosphate groups: 1 g of biomass was refluxed with 20 ml of nitromethane and 25 mL of triethyl phosphite for 6 h. The modified biomass was washed twice with deionized water and used for Pb\u003csup\u003e2+\u003c/sup\u003e binding assay with Pb\u003csup\u003e2+\u003c/sup\u003e concentration of 100 mg/L as described in Section 2.4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZeta potential measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurface charge properties of yeast biomass were carried out by zeta potential measurement according to the protocol of Das et al. (2007) with some modifications. Pre-cultured yeast cells were washed with potassium phosphate (10 mM ) buffer to remove the liquid medium, and then the yeast biomass were suspended in distilled water, adjusted to pH 1.0 to 7.0 and\u0026nbsp;0 mg/L to 700 mg/L of Pb\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003esolutions (pH 6.0). OD\u003csub\u003e600\u003c/sub\u003e of above yeast suspension was adjusted to 0.5, and zeta potential was measured by Zetasizer (Beijing Puchan General Instrument Co., LTD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of Pb\u003csup\u003e2+\u003c/sup\u003e in different cellular parts of yeast cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e binding assay was performed in different concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e solutions (50, 100, 200\u003c/p\u003e\n\u003cp\u003emg/L) as described in Section 2.4.\u003cem\u003e\u0026nbsp;\u003c/em\u003eYeast Cells were collected and dried by vacuum freezing. Then\u003c/p\u003e\n\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e accumulation in different cellular parts was extracted by the following procedures as previous\u003c/p\u003e\n\u003cp\u003ereport (Sheng et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(i) \u0026nbsp;Cytoplasmic water-soluble Pb\u003csup\u003e2+\u003c/sup\u003e: 300 mg of dried cells were mixed with 5 mL of Tris-HCl (10 mM, pH 8.0), after treated by ultrasonic vibration, the supernatant was obtained by centrifuged at 8000 g for 10 min, then the Pb\u003csup\u003e2+\u003c/sup\u003e concentration in the supernatant was determined by flame atomic biosorption spectrophotometry;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(ii) Cellular soluble protein bound Pb\u003csup\u003e2+\u003c/sup\u003e: the precipitate of procedure i was incubated with SDS solution (10 mM, pH 8.0) at 90 ℃ for 60 min, then centrifuged at 8000 g for 10 min, Pb\u003csup\u003e2+\u003c/sup\u003e concentration in the supernatant was determined;\u003c/p\u003e\n\u003cp\u003e(iii) Cell-surface bound Pb\u003csup\u003e2+\u003c/sup\u003e: the precipitate of procedure ii was digested in 10 mL of 65% nitric acid solution, then the Pb\u003csup\u003e2+\u003c/sup\u003e concentration was detected;\u003c/p\u003e\n\u003cp\u003e(iv) Total Pb\u003csup\u003e2+\u003c/sup\u003e: 300 mg of dried cells were digested in HNO\u003csub\u003e3\u003c/sub\u003e/HClO\u003csub\u003e4\u003c/sub\u003e (4:1, n/n) solution, then the total Pb\u003csup\u003e2+\u003c/sup\u003e accumulation were detected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTime course of Pb\u003csup\u003e2+\u003c/sup\u003e binding under metabolic inhibitors N,N\u0026rsquo;-dicyclohexyl carbodiimide (DCC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e binding course in the presence of metabolic inhibitors DCC was monitored by the procedure of Li et al. (2008) with minor modifications. The pre-cultured yeast cells were grown in PDA liquid medium. At the mid-exponential growth phase,\u0026nbsp;DCC (400mM)\u0026nbsp;was added to the liquid medium. After 15 min of incubation, the cells were\u0026nbsp;collected, washed twice, and used for Pb\u003csup\u003e2+\u003c/sup\u003e binding assay in 100 mg/L of Pb\u003csup\u003e2+\u003c/sup\u003e solution, then the Pb\u003csup\u003e2+\u003c/sup\u003e concentrations before and after binding assay were determined respectively as described in Section 2.4. The DCC-untreated yeast cells were used as control sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysical and chemical pretreatment of yeast cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrecultured yeast cells were collected and washed as described in Section 2.4. They were pretreated in three ways as follows: Heat-treated cells were prepared by mixing 2 g of yeast cells with 20 mL of distilled water and heating at 100 \u0026deg;C for 60 min. After that, yeast cells were collected by centrifugation at 8000 g for 10 min. Ultrasonication-treated cells were prepared by suspending 2 g of yeast cells with 20 mL of distilled water and treated by ultrasonic vibration at 600W using a JYD-650 sonifier (Zhixin, Shanghai, China). Then the treated cells were collected by centrifugation at 8000\u0026times;g for 10 min. NaOH-treated cells were prepared by mixing 2 g of yeast cells in 20 mL of 0.1M NaOH solution for 60 min. Then, yeast cells were collected by centrifugation at 8000 g for 10 min and washed three times with distilled water to natural pH. After pretreatment, the yeast cells were resuspended in Pb\u003csup\u003e2+\u003c/sup\u003e solution for Pb\u003csup\u003e2+\u003c/sup\u003e binding assay as described in Section 2.4. The untreated yeast biomass was used as control sample.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach sample was subjected to three independent experimental trials, and the mean values were determined. The statistical significance of the results was assessed using One-way ANOVA with SPSS 17.0 Statistics. A p-value of less than 0.05 was deemed to indicate statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003etolerant ability of\u003c/b\u003e \u003cb\u003eCystobasidium oligophagum\u003c/b\u003e \u003cb\u003eQN-3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e presents the growth of QN-3 on PDA plates with varying concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e. As shown in the table, QN-3 exhibited robust growth (+++) at Pb\u003csup\u003e2+\u003c/sup\u003e concentrations up to 1500 mg/L, indicating a strong tolerance of the strain to high levels of Pb\u003csup\u003e2+\u003c/sup\u003e. When the Pb\u003csup\u003e2+\u003c/sup\u003e concentration increased to 2000 mg/L and 4000 mg/L, the growth of QN-3 slightly declined but remained relatively good (++). However, at a Pb\u003csup\u003e2+\u003c/sup\u003e concentration of 6000 mg/L, the growth of QN-3 significantly weakened (+), and at 8000 mg/L, QN-3 ceased to grow entirely (-). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates the growth of QN-3 under varying concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e, ranging from 0 to 26,000 mg/L. The growth of QN-3 remained relatively stable at lower concentrations, with a slight decline observed as the Pb\u003csup\u003e2+\u003c/sup\u003e concentration increased. Notably, at concentrations above 1,000 mg/L, the growth of QN-3 showed a more pronounced decrease, indicating a threshold beyond which Pb\u003csup\u003e2+\u003c/sup\u003e significantly inhibited microbial activity. The results demonstrated that yeast cells exhibited significant tolerance to Pb\u003csup\u003e2+\u003c/sup\u003e, withstanding concentrations as high as 26000 mg/L which is 52,000 times greater than the allowable limit of 0.5 mg/l for Pb\u003csup\u003e2+\u003c/sup\u003e in industrial wastewater.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGrowth of QN-3 on PDA plates with different concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e (mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGrowth\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e500\u003c/p\u003e\u003cp\u003e1000\u003c/p\u003e\u003cp\u003e1500\u003c/p\u003e\u003cp\u003e2000\u003c/p\u003e\u003cp\u003e4000\u003c/p\u003e\u003cp\u003e6000\u003c/p\u003e\u003cp\u003e8000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003cp\u003e+++\u003c/p\u003e\u003cp\u003e+++\u003c/p\u003e\u003cp\u003e++\u003c/p\u003e\u003cp\u003e++\u003c/p\u003e\u003cp\u003e+\u003c/p\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"2\"\u003e+++ indicated growth best; ++ indicated growth better; + indicated growth; - indicated no growth.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of Pb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003econcentration, temperature, pH, and incubation time on Pb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eadsorption ability of QN-3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, QN-3 exhibited a remarkable capacity for Pb\u0026sup2;⁺ adsorption, particularly at lower concentrations. The removal efficiency approached 100% when the Pb\u003csup\u003e2+\u003c/sup\u003e concentration was 10 and 50 mg/L, and it remained above 60% for concentrations below 300 mg/L. However, a general trend of declining adsorption capacity was observed as the Pb\u003csup\u003e2+\u003c/sup\u003e concentration increased. It was also observed that with the Pb\u003csup\u003e2+\u003c/sup\u003e concentration increasing from 30 to 300 mg/L, the adsorption amount enhanced from 7.28 mg/g to 36.39 mg/g rapidly. When the Pb\u003csup\u003e2+\u003c/sup\u003e concentration exceeded 300 mg/L, the adsorption amount increased slightly, and reached a saturation value of 39.82 mg/g at 700 mg /L concentration of Pb\u003csup\u003e2+\u003c/sup\u003e. The uptake capacity was reduced when Pb\u003csup\u003e2+\u003c/sup\u003e concentration was higher than 700 mg/L. The temperature is a critical factor influencing the energy-dependent mechanisms involved in biosorbent-mediated metal removal. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the adsorption capacity exhibited an enhancement within the temperature range of 26\u0026deg;C to 30\u0026deg;C, with maximum removal rate and adsorption amount of Pb\u003csup\u003e2+\u003c/sup\u003e 68.53% and 25.50 mg/g at 30\u0026deg;C, respectively, and then decreased when the temperature was higher than 30\u0026deg;C. The influence of pH on Pb\u003csup\u003e2+\u003c/sup\u003e adsorption by QN-3 biomass is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. As the pH increased from 4.0 to 8.0, the removal efficiency and adsorption amount both initially rose and subsequently declined, with the maximum adsorption observed at pH 6.0. Metal ion binding occurs rapidly during the initial phase. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, within the first 30min, Pb\u003csup\u003e2+\u003c/sup\u003e were rapidly adsorbed by the yeast biomass, but after 30 min, the Pb\u003csup\u003e2+\u003c/sup\u003e binding activity increased more slowly and reached equilibrium at about 150 min.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eScanning Electron microscopy analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eScanning electron microscopy (SEM) was utilized to examine the surface morphology of the yeast cells before and after the binding of Pb\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The Pb\u003csup\u003e2+\u003c/sup\u003e binding biomass exhibited significant morphological changes compared to the original biomass. In the absence of Pb\u003csup\u003e2+\u003c/sup\u003e, the cells appeared as single, oval-shaped structures with smooth surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, under 500 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e stress, some cells retained their original shape, while others exhibited rough, wrinkled, and sunken surfaces (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At a higher concentration of 1500 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e, most cells lost their original morphology, displaying sunken and distorted structures with rough surfaces, some cells were elongated. Additionally, numerous white crystalline aggregates adhered to the cell surfaces, and high levels of cell aggregation and overlapping were evident (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eFTIR analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe FTIR spectrum is a powerful tool for identifying functional groups capable of binding to Pb\u003csup\u003e2+\u003c/sup\u003e. Each functional group exhibits a distinct absorption peak. Upon interaction with Pb\u003csup\u003e2+\u003c/sup\u003e, the absorption peaks of these functional groups may shift to either higher or lower wavenumbers (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The FTIR spectrum of QN-3 biomass without or with different Pb\u003csup\u003e2+\u003c/sup\u003e concentrations are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. A prominent absorption band at 3377 cm⁻\u0026sup1;, corresponding to -OH or -NH stretching vibrations. Under Pb\u003csup\u003e2+\u003c/sup\u003e stress conditions of 500 mg/L and 1500 mg/L, the peak at 3377 cm⁻\u0026sup1; exhibited a rightward shift of 20 cm⁻\u0026sup1; and leftward shift of 5 cm⁻\u0026sup1;, respectively, compared to the control group. This shift suggested the participation of -OH and -NH groups from polysaccharides, fatty acids, and proteins in the biosorption process (Pouya and Behnam \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Peaks at 2927 cm⁻\u0026sup1;, 2850 cm⁻\u0026sup1; and 1412 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were assigned to stretching vibrations of -CH group. After biosorption, the peak at 2927 cm⁻\u0026sup1; shifted to 2925 cm⁻\u0026sup1;, the peak at 2850 cm⁻\u0026sup1; shifted to 2854 cm⁻\u0026sup1;, 2856 cm⁻\u0026sup1;, and the peak at 1412 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e shifted to 1408 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under 100 mg/L and 1500 mg/L Pb\u003csup\u003e2+\u003c/sup\u003e stress, implied the involvement of -CH functional groups in Pb\u003csup\u003e2+\u003c/sup\u003e binding. Critical amide-related bands were observed at 1743 cm⁻\u0026sup1; and 1653 cm⁻\u0026sup1; (Acylamino I: C\u0026thinsp;=\u0026thinsp;O stretching), 1547 cm⁻\u0026sup1; (Acylamino II: C\u0026ndash;N stretching and N\u0026ndash;H bending), and 1244 cm⁻\u0026sup1; (Acylamino III: C\u0026ndash;N stretching and N\u0026ndash;H bending) (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). A shift of 2 cm⁻\u0026sup1; at 1743 cm⁻\u0026sup1;, 1653 cm⁻\u0026sup1; and 1244 cm⁻\u0026sup1; at Pb\u003csup\u003e2+\u003c/sup\u003e exposure indicated the involvement of Acylamino I and Acylamino III in the adsorption of Pb\u003csup\u003e2+\u003c/sup\u003e, further confirming the pivotal role of proteins in Pb\u003csup\u003e2+\u003c/sup\u003e biosorption. The minor shifts observed at 1078 cm⁻\u0026sup1; and 1034 cm⁻\u0026sup1; indicated that both C\u0026ndash;N and P\u0026thinsp;=\u0026thinsp;O functional groups participated in metal-binding process. The C\u0026ndash;S stretching vibration band at 579 cm⁻\u0026sup1; exhibited a rightward shift of 2 cm⁻\u0026sup1;, suggesting the potential involvement of this functional group in Pb\u003csup\u003e2+\u003c/sup\u003e biosorption.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChemical modification of functional groups\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo further assess the contribution of different functional groups to Pb\u003csup\u003e2+\u003c/sup\u003e binding, a series of chemical modifications were performed. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, The adsorption capacity of QN-3 biomass for Pb\u003csup\u003e2+\u003c/sup\u003e decreased by 45.20%, 55.81%, 43.37%, and 10.42% following treatment with acetic anhydride, methyl iodide, formaldehyde-formic acid, and triethyl phosphite-nitromethane, respectively. Acetylation with acetic anhydride effectively blocks hydroxyl groups (Fieser and Fieser \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1961\u003c/span\u003e), preventing their participation in subsequent reactions. The formaldehyde-formic acid treatment provides an effective approach for methylating and thereby blocking primary and secondary amines (Davis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1973\u003c/span\u003e), rendering them unavailable for further interactions. The observed reduction in Pb\u003csup\u003e2+\u003c/sup\u003e binding following the two treatments suggested that both hydroxyl groups and amine groups played a significant role in the uptake of Pb\u003csup\u003e2+\u003c/sup\u003e by QN-3. As a potent alkylating agent, methyl iodide primarily targets the carboxyl groups of the biomass, converting them into methyl ester derivatives by displacing the hydrogen atoms of the carboxyl groups (Panda et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This modification effectively alters the functional groups, reducing their availability for adsorption processes. The significant reduction in Pb\u003csup\u003e2+\u003c/sup\u003e uptake by 55.81% indicated the critical role of carboxyl groups as the primary metal-binding sites in the adsorption process by QN-3. Moreover, the contribution of carboxyl groups was higher than those of hydroxyl and amine groups. Phosphate groups of orthophosphoric acid (Markowska et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), as well as those present in mono- or diester forms can be esterified by triethyl phosphite-nitromethane. Consequently, this modification is expected to exclude a portion of the phosphate groups in biomass that are involved in Pb\u003csup\u003e2+\u003c/sup\u003e adsorption. Reduction in Pb\u003csup\u003e2+\u003c/sup\u003e binding to a small extent supported the limited but measurable impact of phosphate group in the adsorption process.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eZeta potential of Cystobasidium oligophagum QN-3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eZeta potential measurements revealed variations in the surface charge of \u003cem\u003eCystobasidium oligophagum QN-3\u003c/em\u003e cells under different conditions. In natural environments, where pH is typically near-neutral, yeast surfaces generally carry a negative charge. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, the zeta potential of QN-3 cells shifted from positive (at pH\u0026thinsp;\u0026le;\u0026thinsp;3) to negative (at pH\u0026thinsp;\u0026ge;\u0026thinsp;4), with the isoelectric point (IEP) determined to be approximately 4.0. Pb\u003csup\u003e2+\u003c/sup\u003e binding by QN-3 was strongly influenced by surface charge. Within the experimental pH range of 4\u0026ndash;6, the negative surface charge increased, facilitating Pb\u003csup\u003e2+\u003c/sup\u003e binding, Consequently, Pb\u003csup\u003e2+\u003c/sup\u003e adsorption increased significantly and reached maximum efficiency at pH 6.0, as demonstrated by the pH-dependent adsorption results. Further analysis of zeta potential at different Pb\u003csup\u003e2+\u003c/sup\u003e concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) demonstrated that increasing Pb\u003csup\u003e2+\u003c/sup\u003e concentrations led to a gradual rise in zeta potential, indicating a reduction in net negative surface charge due to Pb\u003csup\u003e2+\u003c/sup\u003e adsorption. These results suggested that electrostatic interactions played a significant role, particularly in the initial rapid adsorption phase of Pb\u003csup\u003e2+\u003c/sup\u003e accumulation by QN-3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eadsorption ability of different cellular components at different Pb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003econcentrations\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe distribution of Pb\u003csup\u003e2+\u003c/sup\u003e in cells after incubation with varying initial concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e is detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The results indicated a concentration-dependent increase in Pb\u003csup\u003e2+\u003c/sup\u003e accumulation across different cellular components. At an initial concentration of 50 mg/L, the cell-surface bound Pb\u003csup\u003e2+\u003c/sup\u003e was 9.89 mg/g, which significantly increased to 24.94 mg/g at 200 mg/L. Thus, higher Pb\u003csup\u003e2+\u003c/sup\u003e concentrations enhanced surface binding capacity. Cytoplasmic water-soluble Pb\u003csup\u003e2+\u003c/sup\u003e levels remained relatively stable across different concentrations, with values of 6.73 mg/g at 50 mg/L and 6.75 mg/g at 200 mg/L. This stability indicated a potential saturation point for cytoplasmic accumulation under the tested conditions. Cellular soluble protein-bound Pb\u003csup\u003e2+\u003c/sup\u003e showed a slight increase from 0.84 mg/g at 50 mg/L to 1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 mg/g at 200 mg/L, suggesting a limited role of intracellular proteins in Pb\u003csup\u003e2+\u003c/sup\u003e binding at higher concentrations. Total Pb\u003csup\u003e2+\u003c/sup\u003e accumulation significantly increased from 16.35 mg/g at 50 mg/L to 32.39 mg/g at 200 mg/L, highlighting a direct correlation between initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration and total cellular accumulation. These findings showed that the majority of Pb\u003csup\u003e2+\u003c/sup\u003e accumulation in yeast cells was localized to cell-surface structures, suggesting the potential foundational roles of the cytomembrane and cytoderm in Pb\u003csup\u003e2+\u003c/sup\u003e binding. However, a fraction of Pb\u003csup\u003e2+\u003c/sup\u003e might traverse the cell membrane, resulting in intracellular accumulation, cytoplasmic Pb-binding compounds might participate in cellular metabolic processes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of Pb\u003csup\u003e2+\u003c/sup\u003e after incubation with different initial concentrations of Pb\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mg/L)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCell-surface bound Pb\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCytoplasmic water-soluble Pb\u003csup\u003e2+\u003c/sup\u003e(mg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCellular soluble protein bound Pb\u003csup\u003e2+\u003c/sup\u003e(mg/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal Pb\u003csup\u003e2+\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e(mg/g)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58Bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09Db\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50Ac\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09Bb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08Da\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e24.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60Ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.94\u0026thinsp;\u0026plusmn;\u0026thinsp;1.22Ba\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16Ca\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08Da\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e32.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80Aa\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eValues are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3), and means in the same column with different lower case letters were significantly different (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), means in the same row with different upper case letters were significantly different by Duncan's multiple range test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTime course of Pb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003euptake in the presence of metabolic inhibitor\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the adsorption Pb\u003csup\u003e2+\u003c/sup\u003e amount in QN-3 biomass with or without metabolic inhibitor N,N\u0026rsquo;-dicyclohexyl carbodiimide (DCC). During the first 30 min, the Pb\u003csup\u003e2+\u003c/sup\u003e adsorbed by QN-3 biomass both increased rapidly whether in the presence of DCC or not. After 30 min, the Pb\u003csup\u003e2+\u003c/sup\u003e binding activity increased more slowly and until equilibrium at about 150 min. During the whole binding process, the Pb\u003csup\u003e2+\u003c/sup\u003e adsorbed by QN-3 biomass was inhibited in the presence of metabolic inhibitor DCC. The Pb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity of the strain decreased by 36% in the presence of DCC at 150 min, indicating its dependence on ATPase activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of pretreatment on Pb\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eadsorption ability of QN-3 biomass\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe cells of QN-3 were treated with ultrasonication, boiling, and NaOH to evaluate the impact of various pretreatment methods on the Pb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity of strain QN-3. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e adsorbed by QN-3 biomass for untreated, ultrasonication-treated, boiling-treated, and NaOH-treated cells were 27.84, 33.08, 31.42, and 41.15 mg/g, respectively. All the three pretreatment methods resulted in a statistically significant enhancement in adsorption efficiency compared to untreated cells (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLead pollution poses a significant environmental threat caused by various industrial processes. Microbial-based remediation has emerged as a promising strategy for reducing this contamination. In this work, tolerant ability of \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 to Pb\u003csup\u003e2+\u003c/sup\u003e was determined. Compared to previously published studies, the tolerance of QN-3 to Pb\u003csup\u003e2+\u003c/sup\u003e was notably higher than that of some reported yeast strains. For instance, El-Sayed (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e showed high tolerance to Pb\u003csup\u003e2+\u003c/sup\u003e concentration up to 600 mg/L, whereas QN-3 maintained optimal growth under the same conditions. Additionally, Banwo et al. (2020) demonstrated that four \u003cem\u003eCandida sp.\u003c/em\u003e and \u003cem\u003eSaccharomyces sp.\u003c/em\u003e strains completely ceased growth at Pb\u003csup\u003e2+\u003c/sup\u003e concentrations exceeding 1050 mg/L, while QN-3 still showed relatively good growth at 4000 mg/L. These comparisons suggested that QN-3 possessed adsorption conditions to lead and high potential for application in environments contaminated with heavy metals. The biological mechanisms underlying yeast cell tolerance involve several processes, including extracellular precipitation, crystallization and complexation, biosorption to cell walls, transformation of metal species, intracellular chelation through the production of phytochelatins and metallothioneins, as well as the localization and sequestration of metals within vacuoles (Liu et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Staszak and Regel-Rosocka 2024). The high tolerance of QN-3 to elevated Pb\u003csup\u003e2+\u003c/sup\u003e concentrations made it a promising candidate for bioremediation. To the best of our knowledge, this study represents the first report on the lead resistance of \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e, thereby expanding the pool of yeast species known for their lead-adsorption capabilities.\u003c/p\u003e\u003cp\u003ePb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity of QN-3 were influenced by initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration, temperature, pH, and incubation time. The results of adsorption capacity at initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration aligned with the findings reported by El-Sayed (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and Dhankhar et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) on biosorption of Pb\u003csup\u003e2+\u003c/sup\u003e and uranium by isolates of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. The maximum Pb\u003csup\u003e2+\u003c/sup\u003e uptake capacity of wet and dry cells was determined as 22 and 30 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 300 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e initial Pb\u003csup\u003e2+\u003c/sup\u003e concentration. This phenomenon could be attributed to the relationship between metal ion binding capacity and the availability of active sites on the cell wall, as highlighted by Dalali and Hagghi (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). At lower initial metal ions concentrations, the ratio of metal ions to the available surface area is relatively low, resulting in fractional sorption that remains largely independent of the initial metal ions concentration. However, at higher concentrations, the number of available sorption sites becomes insufficient relative to the metal ions present, and, hence, the efficiency of metal ions removal is significantly influenced by the initial concentration of the metal ions. Moreover, the proportion of metal ions bound to these sites diminishes relative to the free ions in the solution. This led to a reduction in uptake capacity as the initial concentration increased, which is likely attributed to the saturation of binding sites on the biosorbent. Temperature variations impact the stability, configuration, and ionization of cell wall components, as well as the chemical functional groups involved in the adsorption process (Hassouna et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). QN-3 showed maximum removal rate and adsorption amount of Pb\u003csup\u003e2+\u003c/sup\u003e at 30 ℃. Similarly, Faryal et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) found that the fungal strains \u003cem\u003eAspergillus fumigatus\u003c/em\u003e RH05 and \u003cem\u003eAspergillus flavus\u003c/em\u003e RH07 achieved their highest Zn adsorption capacity at 28\u0026deg;C. However, further elevation in temperature resulted in a gradual decrease in Zn removal efficiency. The pH of a solution is a critical parameter that significantly impacts the adsorption process, as it affects the toxicity, chemical behavior, and speciation of metal ions. Additionally, pH influences hydrolysis and complexation properties by altering the ionic forms of metals in the solution (Vallejo Aguilar et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). At lower pH levels, the protons associated with functional groups, such as carboxyl, phosphate, and amino groups in the cell wall, exhibit limited dissociation, thereby reducing the interaction between metal ions and the cells. (Say 2001). As the initial pH rises, the deprotonation of these functional groups enhances the negative charge density on the biomass surface, creating additional metal adsorption sites and thereby improving adsorption efficiency. Elevated pH levels can lead to the formation of metal hydroxide precipitates, thereby reducing the solubility of metals and limiting their availability for binding to the functional groups located within or on the cell wall, resulting in a reduction in binding capacity (Hlihor et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), as demonstrated in this study. As for the influence of time on lead adsorption, at the beginning of Pb\u003csup\u003e2+\u003c/sup\u003e adsorption, as all active sites remain unoccupied and readily accessible, metal ions biosorption occurs rapidly. However, over time, active uptake stage, the biosorption efficiency declines as the saturation level of metal ions in the solution increases (G\u0026ouml;ksungur et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Comparable trends have been reported in studies involving \u003cem\u003eSaccharomyces cerevesiae\u003c/em\u003e, \u003cem\u003eAspergillus fumigatus\u003c/em\u003e, and \u003cem\u003ePenicillium\u003c/em\u003e sp. biomass during Pb\u003csup\u003e2+\u003c/sup\u003e or other metal ions adsorption (Acosta et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Khamesy et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Say et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCell surface characteristics plays a critical role in mediating metal-microorganism interactions. Heavy metal accumulation can alter cell surface ultrastructure, charge, hydrophobicity, and influencing metal tolerance and adsorption mechanisms (Kordialik-Bogacka \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The observed surface roughness and crystalline deposits on cells under Pb\u003csup\u003e2+\u003c/sup\u003e stress likely resulted from the aggregation of metal complexes into granular forms, highlighting the significance of the cell wall in the adsorption process. These findings aligned with those reported by Angeles de Paz et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Limin et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and Lin et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The morphological deformities induced by Pb\u003csup\u003e2+\u003c/sup\u003e stress might stem from heavy metal toxicity, which could alter membrane permeability and cause oxidative damage (Gadd \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The SEM analysis also revealed elongation and high aggregation of cells. This observation was in accordance with the morphological changes reported in a\u003cem\u003ecidophilic bacteria\u003c/em\u003e under heavy metal stress (Kordialik\u0026ndash;Bogacka 2011). Such alterations could be attributed to a self-protective mechanism employed by the cells to mitigate the toxic impact of environmental stressors (Chakravarty and Banerjee \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, alterations in cell ultrastructure under varying Pb\u003csup\u003e2+\u003c/sup\u003e stress conditions may provide partial insight into the mechanisms underlying Pb\u003csup\u003e2+\u003c/sup\u003e adsorption and tolerance.\u003c/p\u003e\u003cp\u003eThe yeast cell wall, composed of proteins, glucan, mannan, lipids, chitin, chitosan, and inorganic ions (Brady et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), provides abundant functional groups capable of coordinating with heavy metals. FTIR analysis comparing the biomass before and after Pb\u003csup\u003e2+\u003c/sup\u003e binding revealed distinct spectral shifts corresponding to multifunctional interactions between Pb\u003csup\u003e2+\u003c/sup\u003e and biomolecular components on the cell wall. The FTIR data combined with chemical modification results demonstrated the adsorption of Pb\u003csup\u003e2+\u003c/sup\u003e by QN-3 biomass was facilitated by electrostatic interactions as well as complexation mechanisms involving functional groups, including\u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e, -OH, C\u0026thinsp;=\u0026thinsp;O, COO-, P\u0026thinsp;=\u0026thinsp;O, C-N and -CH groups etc., in the adsorption process by \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 biomass. These findings aligned with prior studies (Das et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; El-Sayed \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ibrahim et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) identifying functional groups on the cell walls such as amine, hydroxyl, phosphate, carboxyl groups as primary contributors to heavy metal biosorption, though the relative changes of these groups could vary within yeast species. Furthermore, the elements of O, N, P, and S in polysaccharide, and protein on the cell surface were responsible for Pb\u003csup\u003e2+\u003c/sup\u003e adsorption due to their strong electronegativity through electrostatic interaction (Geesey et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In conclusion, functional groups and electrostatic attraction of cell walls explained the robust Pb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity observed in QN-3. The results corroborated established biosorption mechanism, emphasizing the structural and electrochemical properties of microbial cell walls in heavy metal binding.\u003c/p\u003e\u003cp\u003eBesides surface biosorption, Pb\u003csup\u003e2+\u003c/sup\u003e binding mechanism in \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 also involved intracellular bioaccumulation processes, as confirmed in this study. The findings were consistent with previously published reports (Li and Tao \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sun and Shao \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Sheng et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). For example, Sheng et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) reported that most of the Cd\u003csup\u003e2+\u003c/sup\u003e in \u003cem\u003eLactococcus lactis\u003c/em\u003e subsp. \u003cem\u003elactis\u003c/em\u003e cells could be found in the cell surface, moreover, the \u003cem\u003elactis\u003c/em\u003e presented intracellular Cd\u003csup\u003e2+\u003c/sup\u003e biosorption capacity, intracellular metabolism might be elevated in response to Cd\u003csup\u003e2+\u003c/sup\u003e stresses. Following initial surface interactions, a secondary metabolic-dependent process facilitates the transmembrane transport of metal ions into the intracellular compartment. This bioaccumulation mechanism enables the internalization and subsequent distribution of metal ion within cellular metabolic pathways (Khan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dhankhar and Hooda \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Under normal physiological conditions, yeast cells in their growth and living state are capable of generating sufficient ATP to support active sequestration and influx processes (Prasenjit and Sumathi \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). This Pb\u003csup\u003e2+\u003c/sup\u003e uptake biphasic pattern enabled QN-3 biomass to accumulate significantly higher quantities of metals.\u003c/p\u003e\u003cp\u003ePretreatments on Pb\u003csup\u003e2+\u003c/sup\u003e adsorption of \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 gave different Pb\u003csup\u003e2+\u003c/sup\u003e adsorbed values. The enhanced Pb\u003csup\u003e2+\u003c/sup\u003e adsorption efficiency observed in ultrasonication-treated cells might be attributed to the disruption of cellular integrity caused by ultrasonication. This process released intracellular components and exposed additional binding sites, thereby improving the capacity for Pb\u003csup\u003e2+\u003c/sup\u003e adsorption. The mechanism of increased adsorption efficiency of boiling-treated cells might be explained by the heat-induced denaturation of soluble proteins in the cell wall, which facilitated the formation of stable complexes with metal ions (Yin et al. \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Li et al. (2006) also found heat-treated \u003cem\u003eRhodotorula\u003c/em\u003e sp. cells showed higher lead removal capacity when compared with untreated yeast cells, and concluded that boiling treatment could cause the removal of some impurities such as lipids and proteins of the cell wall thereby expose additional binding sites. The maximum Pb\u003csup\u003e2+\u003c/sup\u003e uptake was achieved with NaOH-treated yeast cells, which might due to the removal of protein groups from the cell wall. These proteins typically form non-adsorbable complexes with Pb\u003csup\u003e2+\u003c/sup\u003e ions, and their elimination exposed more accessible metal binding sites and enhancing the overall negative charge on the cell surface, therefore enhanced the metal adsorption capacity (Gksungur et al. 2005). These results indicated that to enhance microbial adsorption capacity for heavy metals, various pretreatment methods-including thermal treatment, ultrasonication, and alkaline (e.g., NaOH) processing-could be applied to modify cellular structures. These approaches optimized the exposure of functional groups and binding sites, thereby unlocking greater application potential of microbial biomass for efficient metal removal.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe yeast strain \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 exhibited high Pb\u003csup\u003e2+\u003c/sup\u003e resistance and efficient Pb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity in aqueous solution. This adsorption capacity was influenced by Pb\u003csup\u003e2+\u003c/sup\u003e concentration, temperature, pH, and incubation time. After Pb\u003csup\u003e2+\u003c/sup\u003e binding, the surface morphology of the yeast cells changed. During binding, the majority of Pb\u003csup\u003e2+\u003c/sup\u003e particles were adsorbed onto the cell surface, functional groups, such as \u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e, -OH, C\u0026thinsp;=\u0026thinsp;O, COO-, P\u0026thinsp;=\u0026thinsp;O, C-N, -CH groups etc. were involved in the adsorption of Pb\u003csup\u003e2+\u003c/sup\u003e. Functional group blocking experiments and zeta potential measurements demonstrated that Pb\u003csup\u003e2+\u003c/sup\u003e adsorption occurred through electrostatic interactions and complexation reactions. In addition to cell surface biosorption, intracellular Pb\u003csup\u003e2+\u003c/sup\u003e bioaccumulation was observed. The energy inhibitor DCC reduced Pb\u003csup\u003e2+\u003c/sup\u003e binding capacity, suggesting the involvement of ATP-dependent uptake mechanisms following initial surface biosorption. Pretreatments such as ultrasonication, boiling, and NaOH treatment significantly enhanced the strain\u0026rsquo;s Pb\u003csup\u003e2+\u003c/sup\u003e adsorption efficiency. These findings demonstrate that \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 holds promise for bioremediation of Pb\u0026sup2;⁺-contaminated aqueous and solid matrices. Further research should explore its practical applications using native or pretreated cells in real environmental settings.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003escanning electron microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFTIR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003efourier transform-infrared spectroscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePDA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003epotato dextrose agar\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ephosphate-buffered saline\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eN,N\u0026rsquo;-dicyclohexyl carbodiimide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIEP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eisoelectric point\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWen Li: Writing \u0026ndash; original draft, Visualization, Validation, Methodology, Investigation, Formal analysis. Tao Wang: Writing- review \u0026amp; editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition. Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was co-funded by Six Talent Peaks Project in Jiangsu Province (No. SWYY-222), the \u0026quot;343\u0026quot; Industrial Development Project Served by Universities in Xuzhou (gx2024021), and Jiangsu Province Policy Guidance Program (North Jiangsu Science and Technology Special Project) (XZ-SZ202131).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAcosta I, Rodr\u0026iacute;guez A, C\u0026aacute;rdenas JF, Mart\u0026iacute;nez VM, Contreras D (2023) Lead removal from aqueous solutions using different biosorbents. 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P Kumm Rev Mex Cienc Agr\u0026iacute;c 12(2): 275-289. https://doi.org/10.29312/remexca.v12i2.2687\u003c/li\u003e\n \u003cli\u003eVidal NF, Jorge WD (2020) Lead and cadmium removal with native yeast from coastal wetlands.\u003cem\u003e\u0026nbsp;\u003c/em\u003eOpen Chem\u003cem\u003e\u0026nbsp;\u003c/em\u003e20: 1096-1109. https://doi.org/10.1515/chem-2022-0211\u003c/li\u003e\n \u003cli\u003eWang Y, Qiu L, Hu M (2018) Application of yeast in the wastewater treatment, E3S web of conferences, 53: 04025. EDP Sciences. https://doi.org/10.1051/e3sconf/20185304025\u003c/li\u003e\n \u003cli\u003eYin P, Yu Q, Jin B, Ling Z (1999) Biosorption removal of cadmium from aqueous solution by using pretreated fungal biomass cultured from starch wastewater. Water Res 33 (8): 1960-1963. https://doi.org/10.1016/S0043-1354(98)00400-X\u003c/li\u003e\n \u003cli\u003eZaynab M, Al-Yahyai R, Ameen A, Sharif Y, Ali L, Fatima M, Khan KA. Li S (2022) Health and environmental effects of heavy metals. 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Environ Sci Pollut R 25(4): 3745-3755. https://doi.org/10.1007/s11356-017-0785-5\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lead, Biosorbent, Cystobasidium oligophagum, Heavy metals, Bioremediation ","lastPublishedDoi":"10.21203/rs.3.rs-6901159/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6901159/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe lead resistance and adsorption capacity of \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e\u0026nbsp;QN-3 were studied. The strain exhibited high Pb\u003csup\u003e2+\u003c/sup\u003e resistance, withstanding concentrations up to 6,000 mg/L on PDA plates and 26,000 mg/L in liquid PDA medium. The Pb\u003csup\u003e2+\u003c/sup\u003e adsorption capacity of QN-3 was significantly affected by Pb\u003csup\u003e2+\u003c/sup\u003e concentration, temperature, pH, and incubation time. Scanning electron microscopy (SEM) revealed notable morphological changes after adsorption of Pb\u003csup\u003e2+\u003c/sup\u003e, including cell surface wrinkling and elongation. Fourier transform infrared spectroscopy (FTIR) identified key functional groups (–NH\u003csub\u003e2\u003c/sub\u003e, -OH, C=O, COO-, P=O, C-N and -CH) on cell surface participating in Pb\u003csup\u003e2+\u003c/sup\u003e adsorption. Chemical modification of functional groups combined with zeta potential measurements at varying pH and Pb\u003csup\u003e2+\u003c/sup\u003e concentrations confirmed that electrostatic interactions and complexation were the predominant Pb\u003csup\u003e2+\u003c/sup\u003e adsorption mechanisms. Pb\u003csup\u003e2+\u003c/sup\u003e was primarily bound to the cell wall, with minimal intracellular accumulation. The decreased Pb\u003csup\u003e2+\u003c/sup\u003e uptake observed in the presence of metabolic inhibitor DCC suggested an ATP-dependent transport process following initial surface biosorption. Pretreatments including ultrasonication, boiling and alkaline treatment enhanced Pb\u003csup\u003e2+\u003c/sup\u003e adsorption efficiency. The exceptional Pb\u003csup\u003e2+ \u003c/sup\u003etolerance and adsorption capacity of \u003cem\u003eCystobasidium oligophagum\u003c/em\u003e QN-3 highlighted its potential for bioremediation applications.\u003c/p\u003e","manuscriptTitle":"Lead (II) adsorption efficiency and mechanisms by a heavy metal-tolerant yeast Cystobasidium oligophagum QN-3","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 11:59:48","doi":"10.21203/rs.3.rs-6901159/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c084d99b-ad90-4419-bc2e-58344b1dee3b","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-25T15:39:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 11:59:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6901159","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6901159","identity":"rs-6901159","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}